CN112824527B - Artificially designed lysyl endonuclease, coding sequence and fermentation method - Google Patents
Artificially designed lysyl endonuclease, coding sequence and fermentation methodDownload PDFInfo
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Abstract
Description
Translated fromChinese技术领域Technical Field
本发明属于生物技术领域，特别涉及一种人工设计的赖氨酰内切酶及编码序列和发酵方法。The invention belongs to the field of biotechnology, and particularly relates to an artificially designed lysyl endonuclease, a coding sequence and a fermentation method.
背景技术Background Art
赖氨酰特异性内切酶是一条由268个氨基酸残基构成的多肽，其中肽链内部含有三个二硫键(Cys6-Cys216，Cys12-Cys80，Cys36-Cys58)，由His57、Asp113和Ser194构成的三联体决定了该酶的催化活性，而Asp225决定了其对赖氨酸的特异选择性。Lysyl-specific endonuclease is a polypeptide composed of 268 amino acid residues, which contains three disulfide bonds (Cys6-Cys216, Cys12-Cys80, Cys36-Cys58) within the peptide chain. The triplet composed of His57, Asp113 and Ser194 determines the catalytic activity of the enzyme, while Asp225 determines its specific selectivity for lysine.
赖氨酰特异性内切酶与牛胰蛋白酶的氨基酸序列虽然只有20％的同源性，但是决定酶催化活性以及赖氨酸特异性相关的氨基酸序列和空间结构完全一致，因此赖氨酰特异性内切酶被归为胰蛋白酶家族。与牛胰蛋白酶相比，赖氨酰特异性内切酶对赖氨酸碳端的选择性更高，并且有10倍于胰蛋白酶的活性、较广的最适pH范围(pH8.5～10.5)及在4M的尿素或0.1％的SDS中依然正常的稳定性。这些特性使赖氨酰特异性内切酶成为生物医药生产中一种非常有用的工具酶。Although the amino acid sequence of lysyl-specific endonuclease and bovine trypsin has only 20% homology, the amino acid sequence and spatial structure that determine enzyme catalytic activity and lysine specificity are completely consistent, so lysyl-specific endonuclease is classified as trypsin family. Compared with bovine trypsin, lysyl-specific endonuclease has higher selectivity to lysine carbon end, and has 10 times the activity of trypsin, wider optimum pH range (pH8.5～10.5) and still normal stability in 4M urea or 0.1% SDS. These characteristics make lysyl-specific endonuclease become a kind of very useful tool enzyme in biopharmaceutical production.
1978年，日本茨城大学Masaki等人从无色杆菌Achromobacter lyticus M497-1发酵液上清中分离到一种分子量为30KDa的碱性蛋白酶-无色杆菌赖氨酰内切酶，该酶可特异性水解赖氨酰基，包括赖氨酰-脯氨酸基团[Masaki,T.,et al.,A new proteolyticenzyme from Achromobacter lyticus M497-1.Agricultural and BiologicalChemistry,1978.42.]；Tsunasawa等人通过测序获得无色杆菌赖氨酰内切酶的基因序列和蛋白序列，通过分析发现，赖氨酰内切酶属于丝氨酸蛋白酶，赖氨酰内切酶前体除了含有268个氨基酸残基的成熟肽肽段，还包括N端205个氨基酸残基的前肽和C端180个氨基酸残基的大肽段，其底物结合位点由His210-Trp211-Gly212组成，通过序列比对发现，赖氨酰内切酶蛋白序列与牛胰蛋白酶仅有20％同源性[Tsunasawa,S.,et al.,The primarystructure and structural characteristics of Achromobacter lyticus protease I,a lysine-specific serine protease.J Biol Chem,1989.264(7):p.3832-9.]。In 1978, Masaki et al. from Ibaraki University in Japan isolated a 30KDa alkaline protease - Achromobacter lysyl endonuclease from the fermentation supernatant of Achromobacter lyticus M497-1. The enzyme can specifically hydrolyze lysyl groups, including lysyl-proline groups [Masaki, T., et al., A new proteolytic enzyme from Achromobacter lyticus M497-1. Agricultural and Biological Chemistry, 1978. 42.]; Tsunasawa et al. obtained the gene sequence and protein sequence of Achromobacter lyticus lysyl endonuclease by sequencing. The analysis showed that lysyl endonuclease belongs to serine protease. The lysyl endonuclease precursor contains not only a mature peptide segment of 268 amino acid residues, but also a propeptide of 205 amino acid residues at the N-terminus and a large peptide segment of 180 amino acid residues at the C-terminus. Its substrate binding site is composed of His210-Trp211-Gly212. The sequence alignment showed that the lysyl endonuclease protein sequence has only 20% homology with bovine trypsin [Tsunasawa, S., et al., The primary structure and structural characteristics of Achromobacter lyticus protease I, a lysine-specific serine protease. J Biol Chem, 1989. 264 (7): p. 3832-9.].
由于无色杆菌赖氨酰内切酶具有特异性高，耐受pH范围广且稳定的特点，不仅用于生物药如胰岛素等蛋白的前导肽及C肽的切割，还可用于单克隆抗体等大分子蛋白的肽图质谱研究，由于无色杆菌赖氨酰内切酶应用广泛，商业价值高，而利用天然无色杆菌Achromobacter lyticus M497-1生产的赖氨酰内切酶表达量低，周期长，成本明显较高，导致赖氨酰内切酶价格居高不下。Because Achromobacter lysyl endonuclease has the characteristics of high specificity, wide pH tolerance and stability, it is not only used for the cleavage of leader peptides and C-peptides of biological drugs such as insulin and other proteins, but also can be used for peptide mapping mass spectrometry research of large molecular proteins such as monoclonal antibodies. Because Achromobacter lysyl endonuclease is widely used and has high commercial value, the lysyl endonuclease produced by natural Achromobacter lyticus M497-1 has low expression level, long cycle and significantly higher cost, resulting in high prices for lysyl endonuclease.
Ohara等人利用基因工程手段，将无色杆菌赖氨酰内切酶酶原基因克隆至重组质粒，于重组大肠杆菌中表达，最终在大肠杆菌周质空间收获有活性的赖氨酰内切酶，但发酵液上清中并未检测到蛋白酶的表达[Ohara,T.,et al.,Cloning,nucleotide sequence,and expression of Achromobacter protease I gene.J Biol Chem,1989.264(34):p.20625-31.]；诺和诺德公司采用基因扩增技术将不含有C端前导肽的无色杆菌赖氨酰内切酶酶原基因克隆后，转化至大肠杆菌中，最终在重组大肠杆菌发酵液上清中检测到赖氨酰内切酶酶活，但酶活高低不明[process for producing extracellular proteins inbacteria.US6171823 B1]。Ohara et al. cloned the proenzyme gene of Achromobacter lysyl endonuclease into a recombinant plasmid by means of genetic engineering, expressed it in recombinant Escherichia coli, and finally harvested active lysyl endonuclease in the periplasmic space of Escherichia coli, but the expression of protease was not detected in the supernatant of fermentation broth [Ohara, T., et al., Cloning, nucleotide sequence, and expression of Achromobacter protease I gene. J Biol Chem, 1989. 264 (34): p. 20625-31.]; Novo Nordisk cloned the proenzyme gene of Achromobacter lysyl endonuclease without C-terminal leader peptide by gene amplification technology, and transformed it into Escherichia coli, and finally detected lysyl endonuclease activity in the supernatant of recombinant Escherichia coli fermentation broth, but the level of enzyme activity was unknown [process for producing extracellular proteins in bacteria. US6171823 B1].
发明内容Summary of the invention
本发明的首要目的在于克服现有技术的缺点与不足，提供一种人工设计的赖氨酰内切酶。The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an artificially designed lysyl endonuclease.
本发明的另一目的在于提供编码上述人工设计的赖氨酰内切酶的核苷酸序列。Another object of the present invention is to provide a nucleotide sequence encoding the above-mentioned artificially designed lysyl endonuclease.
本发明的再一目的在于提供所述的人工设计的赖氨酰内切酶的发酵方法。Another object of the present invention is to provide a fermentation method for the artificially designed lysyl endonuclease.
本发明的目的通过下述技术方案实现：一种人工设计的赖氨酰内切酶，是将野生型赖氨酰内切酶中的第167位、第193位和第207位中的至少一个氨基酸进行如下突变得到：第167位的缬氨酸突变为亮氨酸或异亮氨酸；第193位的丝氨酸突变为丙氨酸或甘氨酸；第207位的甘氨酸突变为缬氨酸或亮氨酸。从而，人工设计的赖氨酰内切酶相对于野生型赖氨酰内切酶具有更高的酶活。The object of the present invention is achieved by the following technical scheme: an artificially designed lysyl endonuclease is obtained by mutating at least one of the amino acids at positions 167, 193 and 207 in the wild-type lysyl endonuclease as follows: the valine at position 167 is mutated to leucine or isoleucine; the serine at position 193 is mutated to alanine or glycine; and the glycine at position 207 is mutated to valine or leucine. Thus, the artificially designed lysyl endonuclease has higher enzyme activity than the wild-type lysyl endonuclease.
所述的人工设计的赖氨酰内切酶还包括如下情形：是将野生型赖氨酰内切酶中的第30位、第49位、第106位和第155位赖氨酸中一个或多个突变为精氨酸。从而，人工设计的赖氨酰内切酶相对于野生型赖氨酰内切酶具有更强的稳定性。The artificially designed lysyl endonuclease also includes the following situation: one or more of the 30th, 49th, 106th and 155th lysine in the wild-type lysyl endonuclease are mutated to arginine. Thereby, the artificially designed lysyl endonuclease has stronger stability relative to the wild-type lysyl endonuclease.
所述的人工设计的赖氨酰内切酶优选为将野生型赖氨酰内切酶中的第30位、第49位、第106位和第155位赖氨酸突变为精氨酸。The artificially designed lysyl endonuclease preferably mutates the lysine at positions 30, 49, 106 and 155 in the wild-type lysyl endonuclease into arginine.
所述的人工设计的赖氨酰内切酶中的第167位、第193位和第207位的突变组合优选如下：V167I、S193A、G207V；V167I、S193A、G207L；V167I、S193G、G207V；V167L、S193A、G207V；V167I、S193G、G207L；V167L、S193A、G207L；V167L、S193G、G207V；V167L、S193G；V167L；或V167L、S193G、G207L。The mutation combinations at positions 167, 193 and 207 in the artificially designed lysyl endonuclease are preferably as follows: V167I, S193A, G207V; V167I, S193A, G207L; V167I, S193G, G207V; V167L, S193A, G207V; V167I, S193G, G207L; V167L, S193A, G207L; V167L, S193G, G207V; V167L, S193G; V167L; or V167L, S193G, G207L.
所述的人工设计的赖氨酰内切酶优选为氨基酸序列如下所示的赖氨酰内切酶(SEQ ID NO:1)：The artificially designed lysyl endonuclease is preferably a lysyl endonuclease with the amino acid sequence shown below (SEQ ID NO: 1):
GVSGSCNIDVVCPEGDGRRDIIRAVGAYSRSGTLACTGSLVNNTANDRRMYFLTAHHCGMGTASTAASIVVYWNYQNSTCRAPNTPASGANGDGSMSQTQSGSTVRATYATSDFTLLELNNAANPAFNLFWAGWDRRDQNYPGAIAIHHPNVAERRISNSTSPTSFIAWGGGAGTTHLNVQWQPSGGVTEPGASGSPIYSPEKRVLVQLHGGPSSCSATGTNRSDQYGRVFTSWTGGGAAASRLSDWLDPASTGAQFIDGLDSGGGTP。GVSGSCNIDVVCPEGDGRRDIIRAVGAYSRSGTLACTGSLVNNTANDRRMYFLTAHHCGMGTAASTAASIVVYWNYQNSTCRAPNTPASGANGDGSMSQTQSGSTVRATYATSDFTLLELNNAANPAFNLFWAGWDRRDQNYPGAIAIHHPNVAERRISNSTSPTSFIAWGGGAGTTHLNVQWQPSGGVTEPGASGSPIYSPEKRVLVQLHGGPSSCSATGT NRSDQYGRVFTSWTGGGAAASRLSDWLDPASTGAQFIDGLDSGGGTP.
编码上述人工设计的赖氨酰内切酶的核苷酸序列，是依据密码子原则得到；优选为依据宿主细胞密码子偏好性得到的核苷酸序列；更优选如下所示(SEQ ID NO:2)：The nucleotide sequence encoding the artificially designed lysyl endonuclease is obtained according to the codon principle; preferably, it is a nucleotide sequence obtained according to the codon preference of the host cell; more preferably, it is as follows (SEQ ID NO: 2):
ggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctcgttctggtaccctggcttgcaccggttctctggttaacaacaccgctaacgaccgtcgtatgtacttcctgaccgctcaccactgcggtatgggtaccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaacggtgacggttctatgtctcagacccagtctggttctaccgttcgtgctacctacgctacctctgacttcaccctgctggaactgaacaacgctgctaacccggctttcaacctgttctgggctggttgggaccgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaacgttgctgaacgtcgtatctctaactctacctctccgacctctttcatcgcttggggtggtggtgctggtaccacccacctgaacgttcagtggcagccgtctggtggtgttaccgaaccgggtgcttctggttctccgatctactctccggaaaaacgtgttctggttcagctgcacggtggtccgtcttcttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgttttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctggactctggtggtggtaccccgtaatga。ggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctcgttctggtaccctggcttgcaccggttctctggttaacaacaccgctaacgaccgtcgtatgtacttcctgaccgctcaccactgcggtatggg taccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaacggtgacggttctatgtctcagacccagtctggttctaccgttcgtgctacctacgctacctctgacttcaccctgctggaactgaacaacgctgctaacccggct ttcaacctgttctgggctggttgggac cgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaacgttgctgaacgtcgtatctctaactctacctctccgacctctttcatcgcttggggtggtggtgctggtaccacccacctgaacgttcagtggcagccgtctggtggtgttaccgaaccgggtgcttctggttctccgatctctctccgg aaaaacgtgttctggttcagctgcacggtggtccgtcttcttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgtttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctgg actctggtggtggtaccccgtaatga.
一种人工设计的赖氨酰内切酶原，由信号肽、N端前导肽和上述人工设计的赖氨酰内切酶依次连接得到。An artificially designed lysyl endonuclease proenzyme is obtained by sequentially connecting a signal peptide, an N-terminal leader peptide and the artificially designed lysyl endonuclease.
所述的信号肽的氨基酸序列如下所示：MKKTAIAIAVALAGFATVAQA。The amino acid sequence of the signal peptide is as follows: MKKTAIAIAVALAGFATVAQA.
所述的N端前导肽的氨基酸序列如下所示：The amino acid sequence of the N-terminal leader peptide is as follows:
APASRPAAFDYANLSSVDKVALRTMPAVDVAKAKAEDLQRDKRGDIPRFALAIDVDMTPQNSGAWEYTADGQFAVWRQRVRSEKALSLNFGFTDYYMPAGGRLLVYPATQAPAGDRGLISQYDASNNNSARQLWTAVVPGAEAVIEAVIPRDKVGEFKLRLTKVNHDYVGFGPLARRLAAASGEK。APASRPAAFDYANLSSVDKVALRTMPAVDVAKAKAEDLQRDKRGDIPRFALAIDVDMTPQNSGAWEYTADGQFAVWRQRVRSEKALSLNFGFTDYYMPAGGRLLVYPATQAPAGDRGLISQYDASNNNSARQLWTAVVPGAEAVIEAVIPRDKVGEFKLRLTKVNHDYVGFGPLARRLAAASGEK.
所述的人工设计的赖氨酰内切酶原的氨基酸序列优选如下所示：The amino acid sequence of the artificially designed prolysyl endonuclease is preferably as follows:
MKKTAIAIAVALAGFATVAQAAPASRPAAFDYANLSSVDKVALRTMPAVDVAKAKAEDLQRDKRGDIPRFALAIDVDMTPQNSGAWEYTADGQFAVWRQRVRSEKALSLNFGFTDYYMPAGGRLLVYPATQAPAGDRGLISQYDASNNNSARQLWTAVVPGAEAVIEAVIPRDKVGEFKLRLTKVNHDYVGFGPLARRLAAASGEKGVSGSCNIDVVCPEGDGRRDIIRAVGAYSRSGTLACTGSLVNNTANDRRMYFLTAHHCGMGTASTAASIVVYWNYQNSTCRAPNTPASGANGDGSMSQTQSGSTVRATYATSDFTLLELNNAANPAFNLFWAGWDRRDQNYPGAIAIHHPNVAERRISNSTSPTSFIAWGGGAGTTHLNVQWQPSGGVTEPGASGSPIYSPEKRVLVQLHGGPSSCSATGTNRSDQYGRVFTSWTGGGAAASRLSDWLDPASTGAQFIDGLDSGGGTP。MKKTAIAIAVALAGFATVAQAAPASRPAAFDYANLSSVDKVALRTMPAVDVAKAKAEDLQRDKRGDIPRFALAIDVDMTPQNSGAWEYTADGQFAVWRQRVRSEKALSLNFGFTDYYMPAGGRLLVYPATQAPAGDRGLISQYDASNNNSARQLWTAVVPGAEAVIEAVIPRDKVGEFKLRLTKVNHDYVGFGPLARRLAAASGEKGVSGS CNIDVVCPEGDGRRDIIRAVGAYSRS GTLACTGSLVNNTANDRRMYFLTAHHCGMGTAASIVVYWNYQNSTCRAPNTPASGANGDGSMSQTQSGSTVRATYATSDFTLLELNNAANPAFNLFWAGWDRRDQNYPGAIAIHHPNVAERRISNSTSPTSFIAWGGGAGTTHLNVQWQPSGGVTEPGASGSPIYSPEKRVLVQLHGGPSSCSATGTNRSDQYGRVFTSWTGGGAAASRLSDWLD PASTGAQFIDGLDSGGGTP.
编码上述人工设计的赖氨酰内切酶原的核苷酸序列，是依据密码子原则得到；优选为依据宿主细胞密码子偏好性得到的核苷酸序列；更优选如下所示(SEQ ID NO:3)：The nucleotide sequence encoding the artificially designed prolysyl endonuclease is obtained according to the codon principle; preferably, it is a nucleotide sequence obtained according to the codon preference of the host cell; more preferably, it is as follows (SEQ ID NO: 3):
atgaaaaaaaccgctatcgctatcgctgttgctctggctggtttcgctaccgttgctcaggctgctccggcttctcgtccggctgctttcgactacgctaacctgtcttctgttgacaaagttgctctgcgtaccatgccggctgttgacgttgctaaagctaaagctgaagacctgcagcgtgacaaacgtggtgacatcccgcgtttcgctctggctatcgacgttgacatgaccccgcagaactctggtgcttgggaatacaccgctgacggtcagttcgctgtttggcgtcagcgtgttcgttctgaaaaagctctgtctctgaacttcggtttcaccgactactacatgccggctggtggtcgtctgctggtttacccggctacccaggctccggctggtgaccgtggtctgatctctcagtacgacgcttctaacaacaactctgctcgtcagctgtggaccgctgttgttccgggtgctgaagctgttatcgaagctgttatcccgcgtgacaaagttggtgaattcaaactgcgtctgaccaaagttaaccacgactacgttggtttcggtccgctggctcgtcgtctggctgctgcttctggtgaaaaaggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctcgttctggtaccctggcttgcaccggttctctggttaacaacaccgctaacgaccgtcgtatgtacttcctgaccgctcaccactgcggtatgggtaccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaacggtgacggttctatgtctcagacccagtctggttctaccgttcgtgctacctacgctacctctgacttcaccctgctggaactgaacaacgctgctaacccggctttcaacctgttctgggctggttgggaccgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaacgttgctgaacgtcgtatctctaactctacctctccgacctctttcatcgcttggggtggtggtgctggtaccacccacctgaacgttcagtggcagccgtctggtggtgttaccgaaccgggtgcttctggttctccgatctactctccggaaaaacgtgttctggttcagctgcacggtggtccgtcttcttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgttttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctggactctggtggtggtaccccgtaatga。atgaaaaaaaccgctatcgctatcgctgttgctctggctggtttcgctaccgttgctcaggctgctccggcttctcgtccggctgctttcgactacgctaacctgtcttctgttgacaaagttgctctgcgtaccatgccggctgttgacgttgctaaagctaaagctgaagacctgcag cgtgacaaacgtggtgacatcccgcgtttcgctctggctatcgacgttgacatgaccccgcagaactctggtgcttgggaatacaccgctgacggtcagttcgctgtttggcgtcagcgtgttcgttctgaaaaagctctgtctctgaacttcggtttcaccgactactacatgccg gctggtggtcgtctgctggtttacccggctacccaggctccggctggtgaccgtggtctgatctctcagtacgacgcttctaacaacaactctgctcgtcagctgtggaccgctgttgttccgggtgctgaagctgttatcgaagctgttatcccgcgtgacaaagttggtgaattcaaactgcgtctgac caaagttaaccacgactacgttggtttcggtccgctggctcgtcgtctggctgctgcttctggtgaaaaaggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctcgttctggt accctggcttgcaccggttctctggttaacaacaccgctaacgaccgtcgtatgtacttcctgaccgctcaccactgcggtatgggtaccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaacggtgacggttctatgtctc agacccagtctggttctaccgttcgtgctacctacgctacctctgacttcaccctgctggaactgaacaacgctgctaacccggctttcaacctgttctgggctggttgggaccgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaac gttgctgaacgtcgtatctctaactctacctctccgacctctttcatcgcttggggtggtggtgctggtaccacccacctgaacgttcagtggcagccgtctggtggtgttaccgaaccgggtgcttctggttctccgatctactctccggaaaaacgtgttctggttcagctgcacggtggtccgtctt cttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgttttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctggactctggtggtggtaccccgtaatga.
一种表达上述人工设计的赖氨酰内切酶原的重组载体，为将上述编码人工设计的赖氨酰内切酶原的核苷酸序列重组于表达载体上得到。A recombinant vector for expressing the artificially designed prolysyl endonuclease is obtained by recombining the nucleotide sequence encoding the artificially designed prolysyl endonuclease onto an expression vector.
所述的表达载体为pET9a、pET28a-c(+)、pET32a-c(+)、pET30a-c(+)或pET33b(+)；优选为pET9a或pET28a。The expression vector is pET9a, pET28a-c(+), pET32a-c(+), pET30a-c(+) or pET33b(+); preferably pET9a or pET28a.
所述的表达上述人工设计的赖氨酰内切酶原的重组载体，通过以下步骤得到：The recombinant vector expressing the artificially designed prolysyl endonuclease is obtained by the following steps:
(1)在上述编码人工设计的赖氨酰内切酶原的核苷酸序列的5'端加上NdeI酶切位点，在3'端加上BamHI酶切位点，得到含有NdeI和BamHI双酶切位点的序列；(1) adding an NdeI restriction site to the 5' end of the nucleotide sequence encoding the artificially designed pro-lysyl endonuclease and a BamHI restriction site to the 3' end to obtain a sequence containing both NdeI and BamHI restriction sites;
(2)利用NdeI和BamHI限制性内切酶分别双酶切步骤(1)最终得到的序列以及表达载体；双酶切后的序列与双酶切后的表达载体连接，得到表达人工设计的赖氨酰内切酶原的重组载体。(2) using NdeI and BamHI restriction enzymes to double-digest the sequence and expression vector finally obtained in step (1); connecting the double-digested sequence with the double-digested expression vector to obtain a recombinant vector expressing the artificially designed prolysyl endonuclease.
一种表达人工设计的赖氨酰内切酶原的菌株，为将上述人工设计的赖氨酰内切酶原的重组载体转化到宿主菌株得到。The invention discloses a strain expressing artificially designed prolysyl endonuclease, which is obtained by transforming the recombinant vector of the artificially designed prolysyl endonuclease into a host strain.
所述的宿主菌株为大肠杆菌(Escherichia coli)BL21(DE3)、BL21(DE3)pLysS、BL21star(DE3)或BL21star(DE3)pLysS；优选为大肠杆菌BL21(DE3)。The host strain is Escherichia coli BL21 (DE3), BL21 (DE3) pLysS, BL21star (DE3) or BL21star (DE3) pLysS; preferably Escherichia coli BL21 (DE3).
所述的表达人工设计的赖氨酰内切酶原的菌株的发酵方法，包括如下步骤：将上述表达人工设计的赖氨酰内切酶原的菌株接种到发酵培养基中，进行发酵。The fermentation method of the strain expressing the artificially designed prolysyl endonuclease comprises the following steps: inoculating the strain expressing the artificially designed prolysyl endonuclease into a fermentation medium for fermentation.
所述的发酵条件优选如下：温度控制在35℃～37℃之间，pH值控制在6.5～7.0之间，搅拌转速控制在150rpm～700rpm之间，空气流速控制在200L/h～1600L/h之间，溶解氧(DO)控制在10～50％的最大氧饱和度，诱导剂IPTG的添加量在0.1mmol/L～0.6mmol/L(在发酵体系中的终浓度)；当碳源耗尽时开始补料，采用指数补料，比生长速率μ控制在0.03-0.15h-1之间。The fermentation conditions are preferably as follows: the temperature is controlled between 35°C and 37°C, the pH value is controlled between 6.5 and 7.0, the stirring speed is controlled between 150rpm and 700rpm, the air flow rate is controlled between 200L/h and 1600L/h, the dissolved oxygen (DO) is controlled between 10% and 50% of the maximum oxygen saturation, and the addition amount of the inducer IPTG is 0.1mmol/L to 0.6mmol/L (final concentration in the fermentation system); when the carbon source is exhausted, feeding begins, exponential feeding is adopted, and the specific growth rate μ is controlled between 0.03-0.15h-1 .
所述的发酵培养基的组成如下：每升含有酵母提取物2～5g、蛋白胨3～8g、氯化钠1～2g、磷酸二氢钾2～5g、磷酸氢二钠2～5g、二水合氯化钙0.01～0.02g、硫酸镁1～2g、甘油4～7g、硫酸铵5～7g、微量元素0.875mL；用水定容至1L，pH6.5～7.0；优选如下：每升含有酵母提取物3g、蛋白胨5g、氯化钠1g、磷酸二氢钾3g、磷酸氢二钠3.25g、二水合氯化钙0.014g、硫酸镁1g、甘油4.125g、硫酸铵6g、微量元素0.875mL；用水定容至1L，pH6.5～7.0。The composition of the fermentation medium is as follows: per liter, it contains 2-5g yeast extract, 3-8g peptone, 1-2g sodium chloride, 2-5g potassium dihydrogen phosphate, 2-5g disodium hydrogen phosphate, 0.01-0.02g calcium chloride dihydrate, 1-2g magnesium sulfate, 4-7g glycerol, 5-7g ammonium sulfate, and 0.875mL trace elements; it is diluted to 1L with water, and the pH value is 6.5-7.0; preferably, it contains 3g yeast extract, 5g peptone, 1g sodium chloride, 3g potassium dihydrogen phosphate, 3.25g disodium hydrogen phosphate, 0.014g calcium chloride dihydrate, 1g magnesium sulfate, 4.125g glycerol, 6g ammonium sulfate, and 0.875mL trace elements; it is diluted to 1L with water, and the pH value is 6.5-7.0.
所述的微量元素的组成如下：每升含有四水合氯化亚铁20～30g、氯化锌1～3g、六水合氯化钴2～4g、二水合钼酸钠2～4g、二水合氯化钙1～2g、二水合氯化铜1～2g、硼酸0.4～0.6g、一水合硫酸锰2～3g、浓度为质量百分比37％的浓盐酸100mL，用水定容至1L；优选如下：每升含有四水合氯化亚铁22.87g、氯化锌1.31g、六水合氯化钴2g、二水合钼酸钠2g、二水合氯化钙1g、二水合氯化铜1.25g、硼酸0.5g、一水合硫酸锰2.17g、浓度为质量百分比37％的浓盐酸100mL，用水定容至1L。The composition of the trace elements is as follows: per liter, it contains 20-30g of ferrous chloride tetrahydrate, 1-3g of zinc chloride, 2-4g of cobalt chloride hexahydrate, 2-4g of sodium molybdate dihydrate, 1-2g of calcium chloride dihydrate, 1-2g of cupric chloride dihydrate, 0.4-0.6g of boric acid, 2-3g of manganese sulfate monohydrate, 100mL of concentrated hydrochloric acid with a concentration of 37% by mass, and the volume is adjusted to 1L with water; preferably, per liter, it contains 22.87g of ferrous chloride tetrahydrate, 1.31g of zinc chloride, 2g of cobalt chloride hexahydrate, 2g of sodium molybdate dihydrate, 1g of calcium chloride dihydrate, 1.25g of cupric chloride dihydrate, 0.5g of boric acid, 2.17g of manganese sulfate monohydrate, 100mL of concentrated hydrochloric acid with a concentration of 37% by mass, and the volume is adjusted to 1L with water.
所述的补料的组成如下：每升含有250～500g甘油，余量为水；优选如下：每升含有500g甘油，余量为水。The composition of the feed is as follows: 250-500g glycerol per liter, and the balance is water; preferably: 500g glycerol per liter, and the balance is water.
所述的IPTG的添加量优选为按终浓度计算，为0.3mmol/L。The amount of IPTG added is preferably 0.3 mmol/L calculated based on the final concentration.
所述的人工设计的赖氨酰内切酶的酶活达到8000U/L以上(酶活：在30℃下每分钟催化底物生成1μmol对硝基苯胺的酶量定义为1U)。The enzyme activity of the artificially designed lysyl endonuclease reaches more than 8000 U/L (enzyme activity: the amount of enzyme that catalyzes the substrate to generate 1 μmol of p-nitroaniline per minute at 30° C. is defined as 1 U).
本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
(1)本发明发明人结合赖氨酰内切酶的保守序列和底物活性中心氨基酸序列，在考虑催化活性、蛋白质结构稳定性等方面，提供一种人工设计的赖氨酰内切酶。该酶具有高酶活、且高稳定性的优点。(1) The inventors of the present invention provide an artificially designed lysyl endonuclease by combining the conserved sequence of lysyl endonuclease and the amino acid sequence of the substrate active center, taking into account the catalytic activity, protein structure stability, etc. The enzyme has the advantages of high enzymatic activity and high stability.
(2)本发明提供了一种编码人工设计的无色杆菌赖氨酰内切酶原的核苷酸序列。该核苷酸序列为经过优化得到，适于大肠杆菌中进行表达。(2) The present invention provides a nucleotide sequence encoding an artificially designed prolysyl endonuclease of Achromobacterium, which is optimized and suitable for expression in Escherichia coli.
(3)本发明提供了表达人工设计的无色杆菌赖氨酰内切酶原的菌株的发酵方法，其中的发酵培养基，由有机氮源、碳源和无机盐组成，该培养基适合产赖氨酰内切酶基因工程菌的表达。通过该发酵方法得到的赖氨酰内切酶的酶活达到了8000U/L以上。(3) The present invention provides a fermentation method for expressing a strain of an artificially designed Achromobacter lysyl endonuclease, wherein the fermentation medium is composed of an organic nitrogen source, a carbon source and an inorganic salt, and the medium is suitable for the expression of a lysyl endonuclease-producing genetically engineered bacterium. The enzymatic activity of the lysyl endonuclease obtained by the fermentation method reaches more than 8000U/L.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是人工设计的赖氨酰内切酶氨基酸与野生型无色杆菌赖氨酰内切酶氨基酸序列比对图。FIG. 1 is a comparison diagram of the amino acid sequence of the artificially designed lysyl endonuclease and the wild-type Achromobacter lysyl endonuclease.
图2是本发明中工程菌BL21(DE3)/pET28a-LyC发酵液上清分别在诱导前和诱导后的SDS-PAGE图；其中，泳道M为蛋白Marker，各条带大小：116.0KDa、66.2KDa、45.0KDa、35.0KDa、25.0KDa、18.0KDa、14.4Kda；泳道1为诱导前5h的样品，泳道2为诱导前2h的样品，泳道3为诱导0h的样品，泳道4为诱导6h的样品，泳道5为诱导12h的样品，箭头指的是成熟肽。Figure 2 is an SDS-PAGE image of the supernatant of the fermentation broth of the engineered bacteria BL21 (DE3) / pET28a-LyC in the present invention before and after induction; wherein, lane M is a protein marker, and the sizes of each band are: 116.0KDa, 66.2KDa, 45.0KDa, 35.0KDa, 25.0KDa, 18.0KDa, and 14.4Kda;
具体实施方式DETAILED DESCRIPTION
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.
本发明实验过程概述：The experimental process of the present invention is summarized as follows:
一、赖氨酰内切酶的人工设计和筛选1. Artificial design and screening of lysyl endonucleases
赖氨酰内切酶属于丝氨酸蛋白酶家族，该类酶在碱性条件下的酶活是中性条件下的5～6倍。在产赖氨酰内切酶的野生菌中，先以一种无活性的前体蛋白质被合成的。这种前体蛋白质称为赖氨酰内切酶酶原，包括了信号肽序列、N端前导肽、成熟肽及C端延伸肽，酶原经由信号肽牵引着整个肽链进入细胞膜后，信号肽被切割下来。赖氨酰内切酶通过自切割，将N端肽链和C端肽链切割下来，形成成熟的有活性的赖氨酰内切酶。Lysyl endonuclease belongs to the family of serine proteases. The enzymatic activity of this type of enzyme under alkaline conditions is 5 to 6 times that under neutral conditions. In wild bacteria that produce lysyl endonucleases, it is first synthesized as an inactive precursor protein. This precursor protein is called lysyl endonuclease proenzyme, which includes a signal peptide sequence, an N-terminal leader peptide, a mature peptide, and a C-terminal extended peptide. After the proenzyme pulls the entire peptide chain into the cell membrane through the signal peptide, the signal peptide is cut off. Lysyl endonuclease cuts off the N-terminal peptide chain and the C-terminal peptide chain through self-cleavage to form a mature active lysyl endonuclease.
在赖氨酰内切酶的自切割过程中，N端前导肽起着很重要的作用，其可促进赖氨酰内切酶成熟肽的正确折叠修饰。相对于N端前导肽，C端延伸肽的作用并不明确。本发明发明人通过筛选，得到优选的信号肽(MKKTAIAIAVALAGFATVAQA，如SEQ ID NO:4所示)，替换赖氨酰内切酶自身信号肽。In the self-cleavage process of lysyl endonuclease, N-terminal leader peptide plays a very important role, which can promote the correct folding modification of lysyl endonuclease mature peptide.Relative to N-terminal leader peptide, the effect of C-terminal extension peptide is not clear.The inventors of the present invention obtain preferred signal peptide (MKKTAIAIAVALAGFATVAQA, as shown in SEQ ID NO:4) by screening, replace lysyl endonuclease self signal peptide.
本发明的研究人员分析赖氨酰内切酶-底物结合催化机制，在考虑底物催化活性及底物进入赖氨酰内切酶活性口袋特性，通过将野生型赖氨酰内切酶(如SEQ ID NO:5所示)活性口袋附近的多个亲水性或疏水性较弱的氨基酸残基突变为具有较大疏水侧链的氨基酸残基，使得其能够协助赖氨酰内切酶更高效地结合底物并促进底物的旋转。通过实验分析，将野生型赖氨酰内切酶第167位Val、第193位Ser及第207位Gly中一个或多个突变为Val、Leu、IIe或Ala；优选突变为Val167 IIe、Ser193 Ala、Gly207 Val，得到的人工设计的赖氨酰内切酶的活性得以提高。本发明的研究人员综合考虑赖氨酰内切酶结构特性和酶催化机制，将第30位、第49位、第106位和第155位赖氨酸中一个或多个突变为精氨酸，得到的人工设计的赖氨酰内切酶的酶活稳定性得以进一步增强。筛选得到的酶活最高的人工设计的赖氨酰内切酶命名为LyC，其氨基酸序列如SEQ ID NO:1所示。The researchers of the present invention analyze the lysyl endonuclease-substrate binding catalytic mechanism, and consider the substrate catalytic activity and the substrate entering the active pocket characteristics of the lysyl endonuclease, by mutating multiple hydrophilic or hydrophobic amino acid residues near the active pocket of the wild-type lysyl endonuclease (as shown in SEQ ID NO: 5) to amino acid residues with larger hydrophobic side chains, so that it can assist the lysyl endonuclease to bind the substrate more efficiently and promote the rotation of the substrate. Through experimental analysis, one or more of the 167th Val, the 193rd Ser and the 207th Gly of the wild-type lysyl endonuclease are mutated to Val, Leu, IIe or Ala; preferably mutated to Val167 IIe, Ser193 Ala, Gly207 Val, and the activity of the artificially designed lysyl endonuclease obtained is improved. The researchers of the present invention comprehensively considered the structural characteristics and enzyme catalytic mechanism of lysyl endonuclease, and mutated one or more of the lysines at positions 30, 49, 106 and 155 to arginine, and the enzyme activity stability of the artificially designed lysyl endonuclease obtained was further enhanced. The artificially designed lysyl endonuclease with the highest enzyme activity obtained by screening was named LyC, and its amino acid sequence is shown in SEQ ID NO:1.
所述的野生型赖氨酰内切酶的序列如下所示：The sequence of the wild-type lysyl endonuclease is as follows:
本发明的人工设计的赖氨酰内切酶与无色杆菌赖氨酰内切酶的氨基酸序列具有97％的同源性(在线NCBI Blast分析)，其氨基酸序列对比见图1。The amino acid sequence of the artificially designed lysyl endonuclease of the present invention has 97% homology with that of the Achromobacter lysyl endonuclease (online NCBI Blast analysis), and the amino acid sequence comparison thereof is shown in FIG1 .
二、重组表达载体的构建2. Construction of recombinant expression vector
Novagen公司开发的pET系列载体，使用具有强启动能力的T7启动子，可以高效驱动目的基因的表达，现已成为最常用的原核表达载体之一。The pET series vectors developed by Novagen use the T7 promoter with strong initiation ability to efficiently drive the expression of the target gene and have now become one of the most commonly used prokaryotic expression vectors.
本发明选用的载体pET 28a(+)使用T7lac复合启动子，可以自由关闭和开启基因的表达，在诱导之前基因基本不表达，大大降低了宿主菌的负荷，诱导后可以迅速表达出大量目的蛋白，获得的重组表达载体命名为pET28a-LyC。The vector pET 28a(+) selected in the present invention uses the T7lac composite promoter, which can freely turn off and on the expression of genes. Before induction, the gene is basically not expressed, which greatly reduces the load of the host bacteria. After induction, a large amount of target protein can be quickly expressed. The obtained recombinant expression vector is named pET28a-LyC.
本发明还选用pET9a作为表达载体，它具有单独的T7启动子，可以在没有诱导剂存在的情况下，启动目的基因的表达。赖氨酰内切酶酶原基因在宿主菌内首先表达为赖氨酰内切酶酶原，然后通过一系列的转运、切割和修饰成为具有生物活性的成熟酶。由此可见，外源蛋白质的过快表达可能会形成包涵体，而不能加工出较多的成熟酶，同时，对于赖氨酰内切酶这类蛋白酶，其在大肠杆菌内过量积累，可能会严重影响大肠杆菌正常的代谢，抑制菌体的生长。因此，采用组成型表达模式可以调节蛋白质的合成，提高宿主菌的产酶能力。同样，使用限制性酶切位点NdeI和BamHI将LyC克隆至载体pET9a，可以实现上述目的，获得的重组表达载体命名为pET9a-LyC。The present invention also selects pET9a as expression vector, and it has a single T7 promoter, can start the expression of target gene when there is no inducer.Lysyl endonuclease proenzyme gene is first expressed as lysyl endonuclease proenzyme in host bacteria, then becomes a mature enzyme with biological activity through a series of transport, cutting and modification.It can be seen that the too fast expression of exogenous protein may form inclusion bodies, and more mature enzymes can not be processed, and at the same time, for lysyl endonuclease this type of protease, it accumulates excessively in Escherichia coli, may have a strong impact on the normal metabolism of Escherichia coli, inhibits the growth of bacterium.Therefore, adopting constitutive expression mode can regulate the synthesis of protein, improve the enzyme production ability of host bacteria.Equally, using restriction enzyme site NdeI and BamHI, LyC is cloned into carrier pET9a, can achieve the above object, the recombinant expression vector obtained is named after pET9a-LyC.
三、赖氨酰内切酶重组菌的建立3. Establishment of recombinant bacteria producing lysyl endonuclease
用氯化钙法将重组表达载体导入到大肠杆菌表达宿主中，构建赖氨酰内切酶重组菌。The recombinant expression vector was introduced into the Escherichia coli expression host by the calcium chloride method to construct the lysyl endonuclease recombinant bacteria.
四、工程菌的发酵培养4. Fermentation culture of engineered bacteria
本发明公开了一种发酵培养基，由有机氮源、碳源和无机盐组成，该培养基适合产赖氨酰内切酶基因工程菌的表达。本发明经过实验证明，补料方式采用指数补料为宜，比生长速率μ控制在0.03-0.15h-1之间。The invention discloses a fermentation medium, which is composed of an organic nitrogen source, a carbon source and an inorganic salt. The medium is suitable for the expression of a genetic engineering bacterium producing lysyl endonuclease. The invention has been proved through experiments that exponential feeding is suitable for the feeding mode, and the specific growth rate μ is controlled between 0.03-0.15h-1 .
本发明中：In the present invention:
所用到的用于点突变的引物如表1所示：The primers used for point mutation are shown in Table 1:
表1用于点突变的引物序列Table 1 Primer sequences used for point mutation
发酵小试实验步骤：Fermentation test steps:
(1)取20μL重组菌株，接种至50ml抗性液体培养基，在28℃、250rpm的摇床中培养16小时，从而活化菌种。再将50ml活化过的菌种接种到400ml抗性液体培养基中，在28℃，250rpm的条件下继续培养3h，获得种子培养物，控制它的菌体浓度OD600在0.8～1.2之间。(1) Take 20 μL of the recombinant strain and inoculate it into 50 ml of resistance liquid culture medium, and culture it in a shaker at 28°C and 250 rpm for 16 hours to activate the strain. Then inoculate 50 ml of the activated strain into 400 ml of resistance liquid culture medium, and continue to culture it at 28°C and 250 rpm for 3 hours to obtain a seed culture, and control its bacterial cell concentration OD600 between 0.8 and 1.2.
(2)使用20L搅拌式发酵罐，按照表2和表3所示的发酵培养基配方进行投料，投料体积为8L。严格控制发酵条件：温度控制在35℃～37℃之间，pH控制在6.5～7.0之间，发酵转速控制在150rpm～700rpm(根据DO的变化调控)之间，空气流速控制在200L/h～1600L/h之间(根据DO的变化调控)，溶解氧(DO)控制在10～50％的最大氧饱和度之间。培养至碳源耗尽时开始补料(补料培养基每L含500g甘油，用水定容至1L)，当碳源耗尽时开始补料，采用指数补料，比生长速率μ控制在0.12h-1；当培养至菌体浓度OD600≈30时，开始添加IPTG至终浓度0.3mmol/L，开始诱导。(2) A 20L stirred fermenter was used, and the fermentation medium formula shown in Table 2 and Table 3 was used for feeding, and the feeding volume was 8L. The fermentation conditions were strictly controlled: the temperature was controlled between 35°C and 37°C, the pH was controlled between 6.5 and 7.0, the fermentation speed was controlled between 150rpm and 700rpm (regulated according to the change of DO), the air flow rate was controlled between 200L/h and 1600L/h (regulated according to the change of DO), and the dissolved oxygen (DO) was controlled between 10% and 50% of the maximum oxygen saturation. Feeding was started when the carbon source was exhausted (the feed medium contained 500g glycerol per L, and the volume was adjusted to 1L with water). When the carbon source was exhausted, feeding was started, and exponential feeding was used, and the specific growth rate μ was controlled at 0.12h-1 ; when the bacterial concentration OD600 was cultured to 30, IPTG was added to a final concentration of 0.3mmol/L to start induction.
表2发酵培养基配方表Table 2 Fermentation medium formula
表3微量元素配方表Table 3 Trace element formula
发酵诱导12h后，取1ml发酵液，12000rmp离心1min后取上清，检测发酵液上清中赖氨酰内切酶酶活。After 12 hours of fermentation induction, 1 ml of fermentation broth was taken, centrifuged at 12000 rpm for 1 minute, and the supernatant was taken to detect the activity of lysyl endonuclease in the supernatant of the fermentation broth.
赖氨酰内切酶酶活检测方法如下：The method for detecting the activity of lysyl endonuclease is as follows:
取1450μL底物溶液(180mmol/L Tris-HCl(pH9.2)，0.25mmol/L Bz-Lys-pNA(Na-苯甲酰-DL-赖氨酸-对硝基苯酰，如式I所示))。Take 1450 μL of substrate solution (180 mmol/L Tris-HCl (pH 9.2), 0.25 mmol/L Bz-Lys-pNA (Na-benzoyl-DL-lysine-p-nitrobenzoyl, as shown in Formula I)).
于30℃水浴锅中加热5min，然后再加入50μL经稀释合适倍数的发酵液上清，于30℃反应5min，立即加入500μL的45％(V/V)乙酸终止反应，用纯化水稀释3倍后，在405nm波长下测定反应液的吸光度。酶活定义为：在30℃下每分钟催化底物生成1μmol对硝基苯胺的酶量，定义为1U。Heat in a 30℃ water bath for 5 minutes, then add 50μL of fermentation supernatant diluted to an appropriate multiple, react at 30℃ for 5 minutes, immediately add 500μL of 45% (V/V) acetic acid to terminate the reaction, dilute 3 times with purified water, and measure the absorbance of the reaction solution at a wavelength of 405nm. Enzyme activity is defined as: the amount of enzyme that catalyzes the substrate to generate 1μmol of p-nitroaniline per minute at 30℃, which is defined as 1U.
LB液体培养基的组成为：蛋白胨10g/L，酵母粉5g/L，氯化钠5g/L，pH7.0～7.5；The composition of LB liquid medium is: peptone 10g/L, yeast powder 5g/L, sodium chloride 5g/L, pH 7.0-7.5;
LB固体培养基的组成为LB液体培养基+琼脂粉15g/L。The composition of LB solid medium is LB liquid medium + agar powder 15g/L.
抗性液体培养基：含50μg/ml卡那霉素的LB液体培养基。Resistance liquid culture medium: LB liquid culture medium containing 50 μg/ml kanamycin.
抗性固体培养基：含50μg/ml卡那霉素的LB固体培养基。Resistance solid medium: LB solid medium containing 50 μg/ml kanamycin.
实施例1赖氨酰内切酶比活性研究Example 1 Study on the specific activity of lysyl endonuclease
1、突变载体的构建1. Construction of mutation vector
1.1利用定点突变技术，设计定点突变引物，如表1所示。1.1 Using site-directed mutagenesis technology, design site-directed mutagenesis primers, as shown in Table 1.
1.2野生型赖氨酰内切酶酶原由优选的信号肽、赖氨酰内切酶N端前导肽及野生型成熟肽依次连接得到，通过对野生型赖氨酰内切酶酶原的核苷酸序列密码子进行优化，根据大肠杆菌的密码子使用数据库，获得了相应的核苷酸序列，优化后的野生型赖氨酰内切酶酶原的核酸序列如SEQ ID NO:6所示。为了方便基因与载体的连接，在该核苷酸序列的5’添加限制性酶切位点NdeI(CATATG)，3’末端添加限制性酶切位点BamHI(GGATCC)，命名为LyC0，将该序列提交到南京金斯瑞生物科技有限公司进行合成，合成的LyC0序列已经由南京金斯瑞生物科技有限公司连接到载体pMD18T，获得的重组载体命名为pMD18T-LyC0。1.2 The wild-type lysyl endonuclease proenzyme is obtained by sequentially connecting the preferred signal peptide, the N-terminal leader peptide of the lysyl endonuclease and the wild-type mature peptide. The nucleotide sequence codons of the wild-type lysyl endonuclease proenzyme are optimized, and the corresponding nucleotide sequence is obtained according to the codon usage database of Escherichia coli. The optimized nucleic acid sequence of the wild-type lysyl endonuclease proenzyme is shown in SEQ ID NO: 6. In order to facilitate the connection between the gene and the vector, the restriction enzyme site NdeI (CATATG) is added to the 5' of the nucleotide sequence, and the restriction enzyme site BamHI (GGATCC) is added to the 3' end. It is named LyC0, and the sequence is submitted to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. The synthesized LyC0 sequence has been connected to the vector pMD18T by Nanjing GenScript Biotechnology Co., Ltd., and the obtained recombinant vector is named pMD18T-LyC0.
优化后的野生型赖氨酰内切酶酶原的核酸序列：Optimized nucleic acid sequence of wild-type lysyl endonuclease proenzyme:
atgaaaaaaaccgctatcgctatcgctgttgctctggctggtttcgctaccgttgctcaggctgctccggcttctcgtccggctgctttcgactacgctaacctgtcttctgttgacaaagttgctctgcgtaccatgccggctgttgacgttgctaaagctaaagctgaagacctgcagcgtgacaaacgtggtgacatcccgcgtttcgctctggctatcgacgttgacatgaccccgcagaactctggtgcttgggaatacaccgctgacggtcagttcgctgtttggcgtcagcgtgttcgttctgaaaaagctctgtctctgaacttcggtttcaccgactactacatgccggctggtggtcgtctgctggtttacccggctacccaggctccggctggtgaccgtggtctgatctctcagtacgacgcttctaacaacaactctgctcgtcagctgtggaccgctgttgttccgggtgctgaagctgttatcgaagctgttatcccgcgtgacaaagttggtgaattcaaactgcgtctgaccaaagttaaccacgactacgttggtttcggtccgctggctcgtcgtctggctgctgcttctggtgaaaaaggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctaaatctggtaccctggcttgcaccggttctctggttaacaacaccgctaacgaccgtaaaatgtacttcctgaccgctcaccactgcggtatgggtaccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaacggtgacggttctatgtctcagacccagtctggttctaccgttaaagctacctacgctacctctgacttcaccctgctggaactgaacaacgctgctaacccggctttcaacctgttctgggctggttgggaccgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaacgttgctgaaaaacgtatctctaactctacctctccgacctctttcgttgcttggggtggtggtgctggtaccacccacctgaacgttcagtggcagccgtctggtggtgttaccgaaccgggttcttctggttctccgatctactctccggaaaaacgtgttctgggtcagctgcacggtggtccgtcttcttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgttttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctggactctggtggtggtaccccgtaatga。atgaaaaaaaccgctatcgctatcgctgttgctctggctggtttcgctaccgttgctcaggctgctccggcttctcgtccggctgctttcgactacgctaacctgtcttctgttgacaaagttgctctgcgtaccatgccggctgttgacgttgctaaagctaaagctgaagacctgcag cgtgacaaacgtggtgacatcccgcgtttcgctctggctatcgacgttgacatgaccccgcagaactctggtgcttgggaatacaccgctgacggtcagttcgctgtttggcgtcagcgtgttcgttctgaaaaagctctgtctctgaacttcggtttcaccgactactacatgccg gctggtggtcgtctgctggtttacccggctacccaggctccggctggtgaccgtggtctgatctctcagtacgacgcttctaacaacaactctgctcgtcagctgtggaccgctgttgttccgggtgctgaagctgttatcgaagctgttatcccgcgtgacaaagttggtgaattcaaactgcgtctgac caaagttaaccacgactacgttggtttcggtccgctggctcgtcgtctggctgctgcttctggtgaaaaaggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctaaatctggt accctggcttgcaccggttctctggttaacaacaccgctaacgaccgtaaaatgtacttcctgaccgctcaccactgcggtatgggtaccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaacggtgacggttctatgtctcaga cccagtctggttctaccgttaaagctacctacgctacctctgacttcaccctgctggaactgaacaacgctgctaacccggctttcaacctgttctgggctggttgggaccgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaac gttgctgaaaaacgtatctctaactctacctctccgacctctttcgttgcttggggtggtggtgctggtaccacccacctgaacgttcagtggcagccgtctggtggtgttaccgaaccgggttcttctggttctccgatctactctccggaaaaacgtgttctgggtcagctgcacggtggtccgt cttcttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgtttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctggactctggtggtggtaccccgtaatga.
使用TaKaRa公司的限制性内切酶NdeI和BamHI对重组载体pMD18T-LyC0进行双酶切，取5μL酶切产物用质量体积比1％琼脂糖凝胶电泳进行分析，确认酶切完全后，将全部酶切产物进行质量体积比0.8％琼脂糖电泳，割下含1.4kb的DNA片段的凝胶，使用TIANGEN公司的琼脂糖凝胶回收试剂盒将凝胶中的DNA片段纯化到30μL去离子水中，就得到带粘性末端的基因片段。同样，用NdeI和BamHI对质粒pET28a(+)进行双酶切，将酶切后的质粒片段纯化到20μL去离子水中。使用TaKaRa公司的DNA Ligation Kit 2.0将基因片段和质粒片段于16℃连接过夜。取10μL过夜连接的产物，加入到80μL用CaCl2法(美国冷泉港实验室出版的《分子克隆实验指南》第三版)制备的大肠杆菌Top10F’(购自invitrogen公司)感受态细胞中，42℃处理90s，迅速加入300μL经过37℃预热的LB液体培养基，在37℃摇床中低速振荡(150～200rpm)培养45min，然后取100μL培养物涂布抗性固体培养基。在37℃培养箱中倒置培养该平板约18h，直到长出单菌落，随机挑取部分单菌落进行菌液PCR鉴定，PCR反应的条件为：94℃5min；94℃30s、55℃30s、72℃30s，30个循环；72℃5min；引物为：T7上游引物5′-TAATACGACTCACTATAGGG-3′、T7下游引物5′-GCTAGTTATTGCTCAGCGG-3′。将初步筛选得到的阳性克隆抽提质粒，通过NdeI和BamHI进行酶切鉴定，得到1.4kb的基因片段和5.3kb的载体片段的克隆，即为含有LyC0片段的克隆。将鉴定含有LyC0片段的克隆送至Invitrogen公司进行测序。将测序证明没有碱基突变和读码框移位的单克隆，接种至50ml抗性液体培养基，37℃、250rpm培养18h，获得的培养物用TIANGEN公司的质粒小提中量试剂盒抽提质粒(按说明书进行操作)，获得的重组表达质粒命名为pET28a-LyC0。The recombinant vector pMD18T-LyC0 was double-digested with restriction endonucleases NdeI and BamHI from TaKaRa, and 5 μL of the digestion products were analyzed by
1.3突变质粒的构建1.3 Construction of mutant plasmids
(1)通过引物V167I-F、V167I-R、S193A-F、S193A-R、G207V-F、G207V-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为IIe，第193位Ser替换为Ala，第207位Gly替换为Val，所得到的基因命名为LyC1，所得到的突变质粒命名为pET28a-LyC1。(1) Using primers V167I-F, V167I-R, S193A-F, S193A-R, G207V-F, and G207V-R, the 167th position Val of the mature peptide gene of the prolysyl endonuclease on the recombinant plasmid pET28a-LyC0 was replaced with IIe, the 193th position Ser was replaced with Ala, and the 207th position Gly was replaced with Val. The resulting gene was named LyC1, and the resulting mutant plasmid was named pET28a-LyC1.
(2)通过引物G207L-F和G207L-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第207位Gly替换为Leu，所得到的基因命名为LyC2，所得到的突变质粒命名为pET28a-LyC2。(2) Using primers G207L-F and G207L-R, the Gly at position 207 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Leu. The resulting gene was named LyC2, and the resulting mutant plasmid was named pET28a-LyC2.
(3)通过引物S193G-F、S193G-R、G207L-F和G207L-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第193位Ser替换为Gly，第207位Gly替换为Leu，所得到的基因命名为LyC3，所得到的突变质粒命名为pET28a-LyC3。(3) Using primers S193G-F, S193G-R, G207L-F and G207L-R, the Ser at position 193 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Gly, and the Gly at position 207 was replaced with Leu. The resulting gene was named LyC3, and the resulting mutant plasmid was named pET28a-LyC3.
(4)通过引物V167L-F、V167L-R、G207L-F和G207L-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为Leu，第207位Gly替换为Leu所得到的基因命名为LyC4，所得到的突变质粒命名为pET28a-LyC4。(4) Using primers V167L-F, V167L-R, G207L-F and G207L-R, the Val at position 167 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Leu, and the Gly at position 207 was replaced with Leu. The resulting gene was named LyC4, and the resulting mutant plasmid was named pET28a-LyC4.
(5)通过引物V167L-F、V167L-R、S193G-F、S193G-R、G207L-F和G207L-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为Leu，第193位Ser替换为Gly，第207位Gly替换为Leu，所得到的基因命名为LyC5，所得到的突变质粒命名为pET28a-LyC5。(5) Using primers V167L-F, V167L-R, S193G-F, S193G-R, G207L-F and G207L-R, the 167th position Val of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Leu, the 193rd position Ser was replaced with Gly, and the 207th position Gly was replaced with Leu. The resulting gene was named LyC5, and the resulting mutant plasmid was named pET28a-LyC5.
(6)通过引物S193G-F、S193G-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第193位Ser替换为Gly，所得到的基因命名为LyC6，所得到的突变质粒命名为pET28a-LyC6。(6) Using primers S193G-F and S193G-R, the Ser at position 193 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Gly. The resulting gene was named LyC6, and the resulting mutant plasmid was named pET28a-LyC6.
(7)通过引物V167L-F、V167L-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为Leu，所得到的基因命名为LyC7，所得到的突变质粒命名为pET28a-LyC7。(7) Using primers V167L-F and V167L-R, the Val at position 167 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Leu. The resulting gene was named LyC7, and the resulting mutant plasmid was named pET28a-LyC7.
(8)通过引物V167L-F、V167L-R、S193G-F、S193G-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为Leu，第193位Ser替换为Gly，所得到的基因命名为LyC8，所得到的突变质粒命名为pET28a-LyC8。(8) Using primers V167L-F, V167L-R, S193G-F, and S193G-R, the Val at position 167 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Leu, and the Ser at position 193 was replaced with Gly. The resulting gene was named LyC8, and the resulting mutant plasmid was named pET28a-LyC8.
(9)通过引物G193V-F、G193V-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第193位Gly替换为Val，所得到的基因命名为LyC9，所得到的突变质粒命名为pET28a-LyC9。(9) Using primers G193V-F and G193V-R, the 193rd Gly of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Val. The resulting gene was named LyC9, and the resulting mutant plasmid was named pET28a-LyC9.
(10)通过引物V167L-F、V167L-R、S193G-F、S193G-R、G207V-F和G207V-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为Leu，第193位Ser替换为Gly，第207位Gly替换为Val，所得到的基因命名为LyC10，所得到的突变质粒命名为pET28a-LyC10。(10) Using primers V167L-F, V167L-R, S193G-F, S193G-R, G207V-F and G207V-R, the 167th position Val of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Leu, the 193th position Ser was replaced with Gly, and the 207th position Gly was replaced with Val. The resulting gene was named LyC10, and the resulting mutant plasmid was named pET28a-LyC10.
(11)通过引物S193A-F、S193A-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第193位Ser替换为Ala，所得到的基因命名为LyC11，所得到的突变质粒命名为pET28a-LyC11。(11) Using primers S193A-F and S193A-R, the Ser at position 193 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Ala. The resulting gene was named LyC11, and the resulting mutant plasmid was named pET28a-LyC11.
(12)通过引物V167L-F、V167L-R、S193A-F、S193A-R、G207L-F和G207L-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为Leu，第193位Ser替换为Ala，第207位Gly替换为Leu，所得到的基因命名为LyC12，所得到的突变质粒命名为pET28a-LyC12。(12) Using primers V167L-F, V167L-R, S193A-F, S193A-R, G207L-F and G207L-R, the Val at position 167 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Leu, the Ser at position 193 was replaced with Ala, and the Gly at position 207 was replaced with Leu. The resulting gene was named LyC12, and the resulting mutant plasmid was named pET28a-LyC12.
(13)通过引物V167I-F、V167I-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为IIe，所得到的基因命名为LyC13，所得到的突变质粒命名为pET28a-LyC13。(13) Using primers V167I-F and V167I-R, the 167th position Val of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with IIe. The resulting gene was named LyC13, and the resulting mutant plasmid was named pET28a-LyC13.
(14)通过引物V167I-F、V167I-R、S193G-F、S193G-R、G207L-F和G207L-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为IIe，第193位Ser替换为Gly，第207位Gly替换为Leu，所得到的基因命名为LyC14，所得到的突变质粒命名为pET28a-LyC14。(14) Using primers V167I-F, V167I-R, S193G-F, S193G-R, G207L-F and G207L-R, the 167th position Val of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with IIe, the 193th position Ser was replaced with Gly, and the 207th position Gly was replaced with Leu. The resulting gene was named LyC14, and the resulting mutant plasmid was named pET28a-LyC14.
(15)通过引物S193A-F、S193A-R、G207V-F和G207V-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第193位Ser替换为Ala，第207位Gly替换为Val，所得到的基因命名为LyC15，所得到的突变质粒命名为pET28a-LyC15。(15) Using primers S193A-F, S193A-R, G207V-F, and G207V-R, the Ser at position 193 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Ala, and the Gly at position 207 was replaced with Val. The resulting gene was named LyC15, and the resulting mutant plasmid was named pET28a-LyC15.
(16)通过引物V167L-F、V167L-R、S193A-F、S193A-R、G207V-F和G207V-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为Leu，第193位Ser替换为Ala，第207位Gly替换为Val，所得到的基因命名为LyC16，所得到的突变质粒命名为pET28a-LyC16。(16) Using primers V167L-F, V167L-R, S193A-F, S193A-R, G207V-F, and G207V-R, the Val at position 167 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with Leu, the Ser at position 193 was replaced with Ala, and the Gly at position 207 was replaced with Val. The resulting gene was named LyC16, and the resulting mutant plasmid was named pET28a-LyC16.
(17)通过引物V167I-F、V167I-R、G207V-F和G207V-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为IIe，第207位Gly替换为Val，所得到的基因命名为LyC17，所得到的突变质粒命名为pET28a-LyC17。(17) Using primers V167I-F, V167I-R, G207V-F, and G207V-R, the Val at position 167 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with IIe, and the Gly at position 207 was replaced with Val. The resulting gene was named LyC17, and the resulting mutant plasmid was named pET28a-LyC17.
(18)通过引物V167I-F、V167I-R、S193G-F、S193G-R、G207V-F和G207V-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为IIe，第193位Ser替换为Gly，第207位Gly替换为Val，所得到的基因命名为LyC18，所得到的突变质粒命名为pET28a-LyC18。(18) Using primers V167I-F, V167I-R, S193G-F, S193G-R, G207V-F and G207V-R, the 167th position Val of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with IIe, the 193th position Ser was replaced with Gly, and the 207th position Gly was replaced with Val. The resulting gene was named LyC18, and the resulting mutant plasmid was named pET28a-LyC18.
(19)通过引物V167I-F、V167I-R、S193A-F、S193A-R，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为IIe，第193位Ser替换为Ala，所得到的基因命名为LyC19，所得到的突变质粒命名为pET28a-LyC19。(19) Using primers V167I-F, V167I-R, S193A-F, and S193A-R, the 167th position Val of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced with IIe, and the 193rd position Ser was replaced with Ala. The resulting gene was named LyC19, and the resulting mutant plasmid was named pET28a-LyC19.
(20)通过引物V167I-F、V167I-R、S193A-F、S193A-R、G207L-F和G20LV-R，，将重组质粒pET28a-LyC0上的赖氨酰内切酶酶原成熟肽基因第167位Val替换为IIe，第193位Ser替换为Ala，第207位Gly替换为Leu，所得到的基因命名为LyC20，所得到的突变质粒命名为pET28a-LyC20。(20) Using primers V167I-F, V167I-R, S193A-F, S193A-R, G207L-F and G20LV-R, the 167th position Val of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC0 was replaced by IIe, the 193th position Ser was replaced by Ala, and the 207th position Gly was replaced by Leu. The resulting gene was named LyC20, and the resulting mutant plasmid was named pET28a-LyC20.
2、将所得的突变质粒通过测序确定，然后按照美国冷泉港实验室出版的《分子克隆实验指南》第三版提供的氯化钙法，将上述突变载体转化至感受态大肠杆菌BL21(DE3)，构建重组菌株。接着进行发酵小试实验，诱导12h后，检测发酵液上清中赖氨酰内切酶酶活，研究结果如表4所示。2. The obtained mutant plasmid was confirmed by sequencing, and then the above mutant vector was transformed into competent Escherichia coli BL21 (DE3) according to the calcium chloride method provided in the third edition of the Molecular Cloning Laboratory published by the Cold Spring Harbor Laboratory in the United States to construct a recombinant strain. Then, a small fermentation experiment was carried out. After 12 hours of induction, the lysyl endonuclease activity in the fermentation broth supernatant was detected. The research results are shown in Table 4.
表4不同突变位点赖氨酰内切酶相对酶活Table 4 Relative enzyme activity of lysyl endonucleases at different mutation sites
由表4可知，将野生型赖氨酰内切酶第167位氨基酸Val，第193位Ser及第207位Gly分别替换为Val、Leu、IIe、Gly或Ala，均可提高酶活，其中，最优组合为Val167 IIe、Ser193Ala、Gly207 Val。As shown in Table 4, replacing the amino acid Val at position 167, Ser at position 193 and Gly at position 207 of the wild-type lysyl endonuclease with Val, Leu, IIe, Gly or Ala, respectively, can improve the enzyme activity, among which the optimal combination is Val167 IIe, Ser193Ala, Gly207 Val.
实施例2不同突变位点的赖氨酰内切酶的稳定性研究Example 2 Stability study of lysyl endonuclease at different mutation sites
1、突变载体的构建1. Construction of mutation vector
1.1利用定点突变技术，设计定点突变引物，如表1所示。1.1 Using site-directed mutagenesis technology, design site-directed mutagenesis primers, as shown in Table 1.
1.2突变体的构建，按照TaKaRa公司的点突变试剂盒说明书操作，以pET28a-LyC1为模板：1.2 Construction of mutants: According to the instructions of TaKaRa's point mutation kit, pET28a-LyC1 was used as the template:
(1)通过引物Arg49-F、Arg49-R、Arg106-F、Arg106-R、Arg155-F、Arg155-R，将重组质粒pET28a-LyC1上的赖氨酰内切酶酶原成熟肽基因第49位Lys替换为Arg，第106位Lys替换为Arg，第155位Lys替换为Arg，所得到的基因命名为LyC21，所得到的突变质粒命名为pET28a-LyC21。(1) Using primers Arg49-F, Arg49-R, Arg106-F, Arg106-R, Arg155-F, and Arg155-R, the Lys at position 49 of the lysyl endonuclease proenzyme mature peptide gene on the recombinant plasmid pET28a-LyC1 was replaced with Arg, the Lys at position 106 was replaced with Arg, and the Lys at position 155 was replaced with Arg. The resulting gene was named LyC21, and the resulting mutant plasmid was named pET28a-LyC21.
(2)通过引物Arg30-F、Arg30-R、Arg155-F和Arg155-R，将重组质粒pET28a-LyC1上的赖氨酰内切酶酶原成熟肽基因第30位Lys替换为Arg，第155位Lys替换为Arg，所得到的基因命名为LyC22，所得到的突变质粒命名为pET28a-LyC22。(2) Using primers Arg30-F, Arg30-R, Arg155-F and Arg155-R, the Lys at position 30 of the lysyl endonuclease proenzyme mature peptide gene on the recombinant plasmid pET28a-LyC1 was replaced with Arg, and the Lys at position 155 was replaced with Arg. The resulting gene was named LyC22, and the resulting mutant plasmid was named pET28a-LyC22.
(3)通过引物Arg30-F和Arg30-R，将重组质粒pET28a-LyC1上的赖氨酰内切酶酶原成熟肽基因第30位Lys替换为Arg，所得到的基因命名为LyC23，所得到的突变质粒命名为pET28a-LyC23。(3) Using primers Arg30-F and Arg30-R, the Lys at position 30 of the mature peptide gene of the lysyl endonuclease proenzyme on the recombinant plasmid pET28a-LyC1 was replaced with Arg. The resulting gene was named LyC23, and the resulting mutant plasmid was named pET28a-LyC23.
(4)通过引物Arg30-F、Arg30-R、Arg49-F、Arg49-R、Arg106-F、Arg106-R、Arg155-F、Arg155-R，将重组质粒pET28a-LyC1上的赖氨酰内切酶酶原成熟肽基因第30位Lys替换为Arg，第49位Lys替换为Arg，第106位Lys替换为Arg，第155位Lys替换为Arg，所得到的基因命名为LyC，所得到的突变质粒命名为pET28a-LyC。(4) Using primers Arg30-F, Arg30-R, Arg49-F, Arg49-R, Arg106-F, Arg106-R, Arg155-F, and Arg155-R, the Lys at position 30 of the lysyl endonuclease proenzyme mature peptide gene on the recombinant plasmid pET28a-LyC1 was replaced with Arg, the Lys at position 49 was replaced with Arg, the Lys at position 106 was replaced with Arg, and the Lys at position 155 was replaced with Arg. The resulting gene was named LyC, and the resulting mutant plasmid was named pET28a-LyC.
2、将所得的突变质粒通过测序确定，然后按照美国冷泉港实验室出版的《分子克隆实验指南》第三版提供的氯化钙法，将上述构建的重组质粒转化至感受态大肠杆菌BL21(DE3)，构建重组菌株。将获得的重组菌进行发酵小试实验。发酵(诱导12h，发酵周期30h)结束后，收集5ml发酵液上清，进行酶活稳定性研究实验，发酵液上清置于室温中，每隔24h取样检测酶活，研究结果如表5所示：2. The mutant plasmid obtained was confirmed by sequencing, and then the recombinant plasmid constructed above was transformed into competent Escherichia coli BL21 (DE3) according to the calcium chloride method provided in the third edition of the Molecular Cloning Experiment Guide published by the Cold Spring Harbor Laboratory in the United States to construct a recombinant strain. The obtained recombinant bacteria were subjected to a small fermentation experiment. After the fermentation (induction for 12 hours, fermentation cycle for 30 hours) was completed, 5 ml of the fermentation broth supernatant was collected for enzyme activity stability research experiments. The fermentation broth supernatant was placed at room temperature, and the enzyme activity was tested by sampling every 24 hours. The research results are shown in Table 5:
表5不同突变位点赖氨酰内切酶稳定性研究Table 5 Study on the stability of lysyl endonuclease at different mutation sites
由表5可知，研究表明K30R、K49R、K106R及K155R一个或多肽突变后，酶活无显著变化，但是酶稳定性有显著差异。将野生型赖氨酰内切酶中的赖氨酸替换为精氨酸，可明显提高赖氨酰内切酶稳定性，由于赖氨酰内切酶有特异性的赖氨酰残基切割活性，不仅可水解其他蛋白，还能水解自身，随着赖氨酰内切酶成熟肽赖氨酸残基的增加，其稳定性逐步下降。As shown in Table 5, studies have shown that after one or polypeptide mutations of K30R, K49R, K106R and K155R, the enzyme activity has no significant change, but the enzyme stability has significant differences. Lysine in the wild-type lysyl endonuclease is replaced with arginine, which can significantly improve the stability of the lysyl endonuclease. Because the lysyl endonuclease has a specific lysyl residue cleavage activity, it can not only hydrolyze other proteins, but also hydrolyze itself. With the increase of the lysine residues of the mature peptide of the lysyl endonuclease, its stability progressively decreases.
实施例3Example 3
参照《分子克隆实验指南》第三版的重组载体构建及感受态大肠杆菌制备及转化方法，获得重组菌株BL21(DE3)/pET28a-LyC，将赖氨酰内切酶基因工程菌BL21(DE3)/pET28a-LyC进行发酵小试。诱导时间为12h，发酵周期为30h。发酵结束后，发酵液上清中酶活达到8000U/L。Referring to the third edition of the Molecular Cloning Experiment Guide, the recombinant vector construction and competent Escherichia coli preparation and transformation methods were obtained to obtain the recombinant strain BL21 (DE3) / pET28a-LyC, and the lysyl endonuclease genetic engineering bacteria BL21 (DE3) / pET28a-LyC were fermented in a small test. The induction time was 12h and the fermentation cycle was 30h. After the fermentation, the enzyme activity in the supernatant of the fermentation broth reached 8000U/L.
赖氨酰内切酶的电泳分析：分别取5ml诱导前5h、诱导前2h、诱导前0h、诱导6h和诱导12h的发酵液12000rmp离心1min，取发酵液上清，用聚丙烯酰胺凝胶电泳分析(SDS-PAGE，分离胶浓度为12％，浓缩胶浓度为5％)，结果见图2。由此可见，本发明的赖氨酰内切酶分子量与理论大小相符，约为30KDa；赖氨酰内切酶基因工程菌BL21(DE3)/pET28a-LyC在诱导前无目的蛋白的表达，表明基因工程菌无本底表达，诱导6h和诱导12h，均在发酵液上清中检测目的蛋白，且目的蛋白的表达随着诱导时间的延长有明显提高。Electrophoresis analysis of lysyl endonuclease: take 5ml of fermentation liquid 12000rmp centrifugation 1min before 5h, before 2h, before 0h, 6h and 12h of induction respectively, take the fermentation liquid supernatant, use polyacrylamide gel electrophoresis analysis (SDS-PAGE, separation gel concentration is 12%, and concentrated gel concentration is 5%), the results are shown in Figure 2. It can be seen that the molecular weight of lysyl endonuclease of the present invention is consistent with the theoretical size, which is about 30KDa; Lysyl endonuclease genetic engineering bacteria BL21 (DE3) / pET28a-LyC has no expression of target protein before induction, indicating that genetic engineering bacteria have no background expression, induction 6h and induction 12h, all detect target protein in fermentation liquid supernatant, and the expression of target protein is significantly improved with the extension of induction time.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
<110> 珠海联邦制药股份有限公司<110> Zhuhai United Pharmaceutical Co., Ltd.
<120> 人工设计的赖氨酰内切酶及编码序列和发酵方法<120> Artificially designed lysyl endonuclease, coding sequence and fermentation method
<160> 30<160> 30
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 268<211> 268
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 赖氨酰内切酶的氨基酸序列<223> Amino acid sequence of lysyl endonuclease
<400> 1<400> 1
Gly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly AspGly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp
1 5 10 151 5 10 15
Gly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Arg Ser GlyGly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Arg Ser Gly
20 25 3020 25 30
Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp ArgThr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg
35 40 4535 40 45
Arg Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala SerArg Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser
50 55 6050 55 60
Thr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr CysThr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys
65 70 75 8065 70 75 80
Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser MetArg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met
85 90 9585 90 95
Ser Gln Thr Gln Ser Gly Ser Thr Val Arg Ala Thr Tyr Ala Thr SerSer Gln Thr Gln Ser Gly Ser Thr Val Arg Ala Thr Tyr Ala Thr Ser
100 105 110100 105 110
Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe AsnAsp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn
115 120 125115 120 125
Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly AlaLeu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala
130 135 140130 135 140
Ile Ala Ile His His Pro Asn Val Ala Glu Arg Arg Ile Ser Asn SerIle Ala Ile His His Pro Asn Val Ala Glu Arg Arg Ile Ser Asn Ser
145 150 155 160145 150 155 160
Thr Ser Pro Thr Ser Phe Ile Ala Trp Gly Gly Gly Ala Gly Thr ThrThr Ser Pro Thr Ser Phe Ile Ala Trp Gly Gly Gly Ala Gly Thr Thr
165 170 175165 170 175
His Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro GlyHis Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly
180 185 190180 185 190
Ala Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Val GlnAla Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Val Gln
195 200 205195 200 205
Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg SerLeu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser
210 215 220210 215 220
Asp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala AlaAsp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala
225 230 235 240225 230 235 240
Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala GlnAla Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln
245 250 255245 250 255
Phe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr ProPhe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro
260 265260 265
<210> 2<210> 2
<211> 810<211> 810
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 赖氨酰内切酶的核苷酸序列<223> Nucleotide sequence of lysyl endonuclease
<400> 2<400> 2
ggtgtttctg gttcttgcaa catcgacgtt gtttgcccgg aaggtgacgg tcgtcgtgac 60ggtgtttctg gttcttgcaa catcgacgtt gtttgcccgg aaggtgacgg tcgtcgtgac 60
atcatccgtg ctgttggtgc ttactctcgt tctggtaccc tggcttgcac cggttctctg 120atcatccgtg ctgttggtgc ttactctcgt tctggtaccc tggcttgcac cggttctctg 120
gttaacaaca ccgctaacga ccgtcgtatg tacttcctga ccgctcacca ctgcggtatg 180gttaacaaca ccgctaacga ccgtcgtatg tacttcctga ccgctcacca ctgcggtatg 180
ggtaccgctt ctaccgctgc ttctatcgtt gtttactgga actaccagaa ctctacctgc 240ggtaccgctt ctaccgctgc ttctatcgtt gtttactgga actaccagaa ctctacctgc 240
cgtgctccga acaccccggc ttctggtgct aacggtgacg gttctatgtc tcagacccag 300cgtgctccga acaccccggc ttctggtgct aacggtgacg gttctatgtc tcagacccag 300
tctggttcta ccgttcgtgc tacctacgct acctctgact tcaccctgct ggaactgaac 360tctggttcta ccgttcgtgc tacctacgct acctctgact tcaccctgct ggaactgaac 360
aacgctgcta acccggcttt caacctgttc tgggctggtt gggaccgtcg tgaccagaac 420aacgctgcta acccggcttt caacctgttc tgggctggtt gggacgtcg tgaccagaac 420
tacccgggtg ctatcgctat ccaccacccg aacgttgctg aacgtcgtat ctctaactct 480tacccgggtg ctatcgctat ccaccacccg aacgttgctg aacgtcgtat ctctaactct 480
acctctccga cctctttcat cgcttggggt ggtggtgctg gtaccaccca cctgaacgtt 540acctctccga cctctttcat cgcttggggt ggtggtgctg gtaccaccca cctgaacgtt 540
cagtggcagc cgtctggtgg tgttaccgaa ccgggtgctt ctggttctcc gatctactct 600cagtggcagc cgtctggtgg tgttaccgaa ccgggtgctt ctggttctcc gatctactct 600
ccggaaaaac gtgttctggt tcagctgcac ggtggtccgt cttcttgctc tgctaccggt 660ccggaaaaac gtgttctggt tcagctgcac ggtggtccgt cttcttgctc tgctaccggt 660
accaaccgtt ctgaccagta cggtcgtgtt ttcacctctt ggaccggtgg tggtgctgct 720accaaccgtt ctgaccagta cggtcgtgtt ttcacctctt ggaccggtgg tggtgctgct 720
gcttctcgtc tgtctgactg gctggacccg gcttctaccg gtgctcagtt catcgacggt 780gcttctcgtc tgtctgactg gctggacccg gcttctaccg gtgctcagtt catcgacggt 780
ctggactctg gtggtggtac cccgtaatga 810ctggactctg gtggtggtac cccgtaatga 810
<210> 3<210> 3
<211> 1428<211> 1428
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 赖氨酰内切酶原的核苷酸序列<223> Nucleotide sequence of pro-lysyl endonuclease
<400> 3<400> 3
atgaaaaaaa ccgctatcgc tatcgctgtt gctctggctg gtttcgctac cgttgctcag 60atgaaaaaaa ccgctatcgc tatcgctgtt gctctggctg gtttcgctac cgttgctcag 60
gctgctccgg cttctcgtcc ggctgctttc gactacgcta acctgtcttc tgttgacaaa 120gctgctccgg cttctcgtcc ggctgctttc gactacgcta acctgtcttc tgttgacaaa 120
gttgctctgc gtaccatgcc ggctgttgac gttgctaaag ctaaagctga agacctgcag 180gttgctctgc gtaccatgcc ggctgttgac gttgctaaag ctaaagctga agacctgcag 180
cgtgacaaac gtggtgacat cccgcgtttc gctctggcta tcgacgttga catgaccccg 240cgtgacaaac gtggtgacat cccgcgtttc gctctggcta tcgacgttga catgaccccg 240
cagaactctg gtgcttggga atacaccgct gacggtcagt tcgctgtttg gcgtcagcgt 300cagaactctg gtgcttggga atacaccgct gacggtcagt tcgctgtttg gcgtcagcgt 300
gttcgttctg aaaaagctct gtctctgaac ttcggtttca ccgactacta catgccggct 360gttcgttctg aaaaagctct gtctctgaac ttcggtttca ccgactacta catgccggct 360
ggtggtcgtc tgctggttta cccggctacc caggctccgg ctggtgaccg tggtctgatc 420ggtggtcgtc tgctggttta cccggctacc caggctccgg ctggtgaccg tggtctgatc 420
tctcagtacg acgcttctaa caacaactct gctcgtcagc tgtggaccgc tgttgttccg 480tctcagtacg acgcttctaa caacaactct gctcgtcagc tgtggaccgc tgttgttccg 480
ggtgctgaag ctgttatcga agctgttatc ccgcgtgaca aagttggtga attcaaactg 540ggtgctgaag ctgttatcga agctgttatc ccgcgtgaca aagttggtga attcaaactg 540
cgtctgacca aagttaacca cgactacgtt ggtttcggtc cgctggctcg tcgtctggct 600cgtctgacca aagttaacca cgactacgtt ggtttcggtc cgctggctcg tcgtctggct 600
gctgcttctg gtgaaaaagg tgtttctggt tcttgcaaca tcgacgttgt ttgcccggaa 660gctgcttctg gtgaaaaagg tgtttctggt tcttgcaaca tcgacgttgt ttgcccggaa 660
ggtgacggtc gtcgtgacat catccgtgct gttggtgctt actctcgttc tggtaccctg 720ggtgacggtc gtcgtgacat catccgtgct gttggtgctt actctcgttc tggtaccctg 720
gcttgcaccg gttctctggt taacaacacc gctaacgacc gtcgtatgta cttcctgacc 780gcttgcaccg gttctctggt taacaacacc gctaacgacc gtcgtatgta cttcctgacc 780
gctcaccact gcggtatggg taccgcttct accgctgctt ctatcgttgt ttactggaac 840gctcaccact gcggtatggg taccgcttct accgctgctt ctatcgttgt ttactggaac 840
taccagaact ctacctgccg tgctccgaac accccggctt ctggtgctaa cggtgacggt 900taccagaact ctacctgccg tgctccgaac accccggctt ctggtgctaa cggtgacggt 900
tctatgtctc agacccagtc tggttctacc gttcgtgcta cctacgctac ctctgacttc 960tctatgtctc agacccagtc tggttctacc gttcgtgcta cctacgctac ctctgacttc 960
accctgctgg aactgaacaa cgctgctaac ccggctttca acctgttctg ggctggttgg 1020accctgctgg aactgaacaa cgctgctaac ccggctttca acctgttctg ggctggttgg 1020
gaccgtcgtg accagaacta cccgggtgct atcgctatcc accacccgaa cgttgctgaa 1080gaccgtcgtg accagaacta cccgggtgct atcgctatcc accacccgaa cgttgctgaa 1080
cgtcgtatct ctaactctac ctctccgacc tctttcatcg cttggggtgg tggtgctggt 1140cgtcgtatct ctaactctac ctctccgacc tctttcatcg cttggggtgg tggtgctggt 1140
accacccacc tgaacgttca gtggcagccg tctggtggtg ttaccgaacc gggtgcttct 1200accacccacc tgaacgttca gtggcagccg tctggtggtg ttaccgaacc gggtgcttct 1200
ggttctccga tctactctcc ggaaaaacgt gttctggttc agctgcacgg tggtccgtct 1260ggttctccga tctactctcc ggaaaaacgt gttctggttc agctgcacgg tggtccgtct 1260
tcttgctctg ctaccggtac caaccgttct gaccagtacg gtcgtgtttt cacctcttgg 1320tcttgctctg ctaccggtac caaccgttct gaccagtacg gtcgtgtttt cacctcttgg 1320
accggtggtg gtgctgctgc ttctcgtctg tctgactggc tggacccggc ttctaccggt 1380accggtggtg gtgctgctgc ttctcgtctg tctgactggc tggacccggc ttctaccggt 1380
gctcagttca tcgacggtct ggactctggt ggtggtaccc cgtaatga 1428gctcagttca tcgacggtct ggactctggt ggtggtaccc cgtaatga 1428
<210> 4<210> 4
<211> 21<211> 21
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 信号肽的氨基酸序列<223> Amino acid sequence of signal peptide
<400> 4<400> 4
Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe AlaMet Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala
1 5 10 151 5 10 15
Thr Val Ala Gln AlaThr Val Ala Gln Ala
2020
<210> 5<210> 5
<211> 268<211> 268
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 野生型赖氨酰内切酶的氨基酸序列<223> Amino acid sequence of wild-type lysyl endonuclease
<400> 5<400> 5
Gly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly AspGly Val Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp
1 5 10 151 5 10 15
Gly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Lys Ser GlyGly Arg Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Lys Ser Gly
20 25 3020 25 30
Thr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp ArgThr Leu Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg
35 40 4535 40 45
Lys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala SerLys Met Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser
50 55 6050 55 60
Thr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr CysThr Ala Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys
65 70 75 8065 70 75 80
Arg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser MetArg Ala Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met
85 90 9585 90 95
Ser Gln Thr Gln Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr SerSer Gln Thr Gln Ser Gly Ser Thr Val Lys Ala Thr Tyr Ala Thr Ser
100 105 110100 105 110
Asp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe AsnAsp Phe Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn
115 120 125115 120 125
Leu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly AlaLeu Phe Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala
130 135 140130 135 140
Ile Ala Ile His His Pro Asn Val Ala Glu Lys Arg Ile Ser Asn SerIle Ala Ile His His Pro Asn Val Ala Glu Lys Arg Ile Ser Asn Ser
145 150 155 160145 150 155 160
Thr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr ThrThr Ser Pro Thr Ser Phe Val Ala Trp Gly Gly Gly Ala Gly Thr Thr
165 170 175165 170 175
His Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro GlyHis Leu Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly
180 185 190180 185 190
Ser Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Gly GlnSer Ser Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Gly Gln
195 200 205195 200 205
Leu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg SerLeu His Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser
210 215 220210 215 220
Asp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala AlaAsp Gln Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala
225 230 235 240225 230 235 240
Ala Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala GlnAla Ser Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln
245 250 255245 250 255
Phe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr ProPhe Ile Asp Gly Leu Asp Ser Gly Gly Gly Thr Pro
260 265260 265
<210> 6<210> 6
<211> 1428<211> 1428
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 优化后的野生型赖氨酰内切酶酶原的核苷酸序列<223> Optimized nucleotide sequence of wild-type lysyl endonuclease proenzyme
<400> 6<400> 6
atgaaaaaaa ccgctatcgc tatcgctgtt gctctggctg gtttcgctac cgttgctcag 60atgaaaaaaa ccgctatcgc tatcgctgtt gctctggctg gtttcgctac cgttgctcag 60
gctgctccgg cttctcgtcc ggctgctttc gactacgcta acctgtcttc tgttgacaaa 120gctgctccgg cttctcgtcc ggctgctttc gactacgcta acctgtcttc tgttgacaaa 120
gttgctctgc gtaccatgcc ggctgttgac gttgctaaag ctaaagctga agacctgcag 180gttgctctgc gtaccatgcc ggctgttgac gttgctaaag ctaaagctga agacctgcag 180
cgtgacaaac gtggtgacat cccgcgtttc gctctggcta tcgacgttga catgaccccg 240cgtgacaaac gtggtgacat cccgcgtttc gctctggcta tcgacgttga catgaccccg 240
cagaactctg gtgcttggga atacaccgct gacggtcagt tcgctgtttg gcgtcagcgt 300cagaactctg gtgcttggga atacaccgct gacggtcagt tcgctgtttg gcgtcagcgt 300
gttcgttctg aaaaagctct gtctctgaac ttcggtttca ccgactacta catgccggct 360gttcgttctg aaaaagctct gtctctgaac ttcggtttca ccgactacta catgccggct 360
ggtggtcgtc tgctggttta cccggctacc caggctccgg ctggtgaccg tggtctgatc 420ggtggtcgtc tgctggttta cccggctacc caggctccgg ctggtgaccg tggtctgatc 420
tctcagtacg acgcttctaa caacaactct gctcgtcagc tgtggaccgc tgttgttccg 480tctcagtacg acgcttctaa caacaactct gctcgtcagc tgtggaccgc tgttgttccg 480
ggtgctgaag ctgttatcga agctgttatc ccgcgtgaca aagttggtga attcaaactg 540ggtgctgaag ctgttatcga agctgttatc ccgcgtgaca aagttggtga attcaaactg 540
cgtctgacca aagttaacca cgactacgtt ggtttcggtc cgctggctcg tcgtctggct 600cgtctgacca aagttaacca cgactacgtt ggtttcggtc cgctggctcg tcgtctggct 600
gctgcttctg gtgaaaaagg tgtttctggt tcttgcaaca tcgacgttgt ttgcccggaa 660gctgcttctg gtgaaaaagg tgtttctggt tcttgcaaca tcgacgttgt ttgcccggaa 660
ggtgacggtc gtcgtgacat catccgtgct gttggtgctt actctaaatc tggtaccctg 720ggtgacggtc gtcgtgacat catccgtgct gttggtgctt actctaaatc tggtaccctg 720
gcttgcaccg gttctctggt taacaacacc gctaacgacc gtaaaatgta cttcctgacc 780gcttgcaccg gttctctggt taacaacacc gctaacgacc gtaaaatgta cttcctgacc 780
gctcaccact gcggtatggg taccgcttct accgctgctt ctatcgttgt ttactggaac 840gctcaccact gcggtatggg taccgcttct accgctgctt ctatcgttgt ttactggaac 840
taccagaact ctacctgccg tgctccgaac accccggctt ctggtgctaa cggtgacggt 900taccagaact ctacctgccg tgctccgaac accccggctt ctggtgctaa cggtgacggt 900
tctatgtctc agacccagtc tggttctacc gttaaagcta cctacgctac ctctgacttc 960tctatgtctc agacccagtc tggttctacc gttaaagcta cctacgctac ctctgacttc 960
accctgctgg aactgaacaa cgctgctaac ccggctttca acctgttctg ggctggttgg 1020accctgctgg aactgaacaa cgctgctaac ccggctttca acctgttctg ggctggttgg 1020
gaccgtcgtg accagaacta cccgggtgct atcgctatcc accacccgaa cgttgctgaa 1080gaccgtcgtg accagaacta cccgggtgct atcgctatcc accacccgaa cgttgctgaa 1080
aaacgtatct ctaactctac ctctccgacc tctttcgttg cttggggtgg tggtgctggt 1140aaacgtatct ctaactctac ctctccgacc tctttcgttg cttggggtgg tggtgctggt 1140
accacccacc tgaacgttca gtggcagccg tctggtggtg ttaccgaacc gggttcttct 1200accacccacc tgaacgttca gtggcagccg tctggtggtg ttaccgaacc gggttcttct 1200
ggttctccga tctactctcc ggaaaaacgt gttctgggtc agctgcacgg tggtccgtct 1260ggttctccga tctactctcc ggaaaaacgt gttctgggtc agctgcacgg tggtccgtct 1260
tcttgctctg ctaccggtac caaccgttct gaccagtacg gtcgtgtttt cacctcttgg 1320tcttgctctg ctaccggtac caaccgttct gaccagtacg gtcgtgtttt cacctcttgg 1320
accggtggtg gtgctgctgc ttctcgtctg tctgactggc tggacccggc ttctaccggt 1380accggtggtg gtgctgctgc ttctcgtctg tctgactggc tggacccggc ttctaccggt 1380
gctcagttca tcgacggtct ggactctggt ggtggtaccc cgtaatga 1428gctcagttca tcgacggtct ggactctggt ggtggtaccc cgtaatga 1428
<210> 7<210> 7
<211> 474<211> 474
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> 赖氨酰内切酶原的氨基酸序列<223> Amino acid sequence of pro-lysyl endonuclease
<400> 7<400> 7
Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe AlaMet Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala
1 5 10 151 5 10 15
Thr Val Ala Gln Ala Ala Pro Ala Ser Arg Pro Ala Ala Phe Asp TyrThr Val Ala Gln Ala Ala Pro Ala Ser Arg Pro Ala Ala Phe Asp Tyr
20 25 3020 25 30
Ala Asn Leu Ser Ser Val Asp Lys Val Ala Leu Arg Thr Met Pro AlaAla Asn Leu Ser Ser Val Asp Lys Val Ala Leu Arg Thr Met Pro Ala
35 40 4535 40 45
Val Asp Val Ala Lys Ala Lys Ala Glu Asp Leu Gln Arg Asp Lys ArgVal Asp Val Ala Lys Ala Lys Ala Glu Asp Leu Gln Arg Asp Lys Arg
50 55 6050 55 60
Gly Asp Ile Pro Arg Phe Ala Leu Ala Ile Asp Val Asp Met Thr ProGly Asp Ile Pro Arg Phe Ala Leu Ala Ile Asp Val Asp Met Thr Pro
65 70 75 8065 70 75 80
Gln Asn Ser Gly Ala Trp Glu Tyr Thr Ala Asp Gly Gln Phe Ala ValGln Asn Ser Gly Ala Trp Glu Tyr Thr Ala Asp Gly Gln Phe Ala Val
85 90 9585 90 95
Trp Arg Gln Arg Val Arg Ser Glu Lys Ala Leu Ser Leu Asn Phe GlyTrp Arg Gln Arg Val Arg Ser Glu Lys Ala Leu Ser Leu Asn Phe Gly
100 105 110100 105 110
Phe Thr Asp Tyr Tyr Met Pro Ala Gly Gly Arg Leu Leu Val Tyr ProPhe Thr Asp Tyr Tyr Met Pro Ala Gly Gly Arg Leu Leu Val Tyr Pro
115 120 125115 120 125
Ala Thr Gln Ala Pro Ala Gly Asp Arg Gly Leu Ile Ser Gln Tyr AspAla Thr Gln Ala Pro Ala Gly Asp Arg Gly Leu Ile Ser Gln Tyr Asp
130 135 140130 135 140
Ala Ser Asn Asn Asn Ser Ala Arg Gln Leu Trp Thr Ala Val Val ProAla Ser Asn Asn Asn Ser Ala Arg Gln Leu Trp Thr Ala Val Val Pro
145 150 155 160145 150 155 160
Gly Ala Glu Ala Val Ile Glu Ala Val Ile Pro Arg Asp Lys Val GlyGly Ala Glu Ala Val Ile Glu Ala Val Ile Pro Arg Asp Lys Val Gly
165 170 175165 170 175
Glu Phe Lys Leu Arg Leu Thr Lys Val Asn His Asp Tyr Val Gly PheGlu Phe Lys Leu Arg Leu Thr Lys Val Asn His Asp Tyr Val Gly Phe
180 185 190180 185 190
Gly Pro Leu Ala Arg Arg Leu Ala Ala Ala Ser Gly Glu Lys Gly ValGly Pro Leu Ala Arg Arg Leu Ala Ala Ala Ser Gly Glu Lys Gly Val
195 200 205195 200 205
Ser Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp Gly ArgSer Gly Ser Cys Asn Ile Asp Val Val Cys Pro Glu Gly Asp Gly Arg
210 215 220210 215 220
Arg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Arg Ser Gly Thr LeuArg Asp Ile Ile Arg Ala Val Gly Ala Tyr Ser Arg Ser Gly Thr Leu
225 230 235 240225 230 235 240
Ala Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg Arg MetAla Cys Thr Gly Ser Leu Val Asn Asn Thr Ala Asn Asp Arg Arg Met
245 250 255245 250 255
Tyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser Thr AlaTyr Phe Leu Thr Ala His His Cys Gly Met Gly Thr Ala Ser Thr Ala
260 265 270260 265 270
Ala Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys Arg AlaAla Ser Ile Val Val Tyr Trp Asn Tyr Gln Asn Ser Thr Cys Arg Ala
275 280 285275 280 285
Pro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met Ser GlnPro Asn Thr Pro Ala Ser Gly Ala Asn Gly Asp Gly Ser Met Ser Gln
290 295 300290 295 300
Thr Gln Ser Gly Ser Thr Val Arg Ala Thr Tyr Ala Thr Ser Asp PheThr Gln Ser Gly Ser Thr Val Arg Ala Thr Tyr Ala Thr Ser Asp Phe
305 310 315 320305 310 315 320
Thr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn Leu PheThr Leu Leu Glu Leu Asn Asn Ala Ala Asn Pro Ala Phe Asn Leu Phe
325 330 335325 330 335
Trp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala Ile AlaTrp Ala Gly Trp Asp Arg Arg Asp Gln Asn Tyr Pro Gly Ala Ile Ala
340 345 350340 345 350
Ile His His Pro Asn Val Ala Glu Arg Arg Ile Ser Asn Ser Thr SerIle His His Pro Asn Val Ala Glu Arg Arg Ile Ser Asn Ser Thr Ser
355 360 365355 360 365
Pro Thr Ser Phe Ile Ala Trp Gly Gly Gly Ala Gly Thr Thr His LeuPro Thr Ser Phe Ile Ala Trp Gly Gly Gly Ala Gly Thr Thr His Leu
370 375 380370 375 380
Asn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly Ala SerAsn Val Gln Trp Gln Pro Ser Gly Gly Val Thr Glu Pro Gly Ala Ser
385 390 395 400385 390 395 400
Gly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Val Gln Leu HisGly Ser Pro Ile Tyr Ser Pro Glu Lys Arg Val Leu Val Gln Leu His
405 410 415405 410 415
Gly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser Asp GlnGly Gly Pro Ser Ser Cys Ser Ala Thr Gly Thr Asn Arg Ser Asp Gln
420 425 430420 425 430
Tyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala Ala SerTyr Gly Arg Val Phe Thr Ser Trp Thr Gly Gly Gly Ala Ala Ala Ser
435 440 445435 440 445
Arg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln Phe IleArg Leu Ser Asp Trp Leu Asp Pro Ala Ser Thr Gly Ala Gln Phe Ile
450 455 460450 455 460
Asp Gly Leu Asp Ser Gly Gly Gly Thr ProAsp Gly Leu Asp Ser Gly Gly Gly Thr Pro
465 470465 470
<210> 8<210> 8
<211> 185<211> 185
<212> PRT<212> PRT
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> N端前导肽的氨基酸序列<223> Amino acid sequence of N-terminal leader peptide
<400> 8<400> 8
Ala Pro Ala Ser Arg Pro Ala Ala Phe Asp Tyr Ala Asn Leu Ser SerAla Pro Ala Ser Arg Pro Ala Ala Phe Asp Tyr Ala Asn Leu Ser Ser
1 5 10 151 5 10 15
Val Asp Lys Val Ala Leu Arg Thr Met Pro Ala Val Asp Val Ala LysVal Asp Lys Val Ala Leu Arg Thr Met Pro Ala Val Asp Val Ala Lys
20 25 3020 25 30
Ala Lys Ala Glu Asp Leu Gln Arg Asp Lys Arg Gly Asp Ile Pro ArgAla Lys Ala Glu Asp Leu Gln Arg Asp Lys Arg Gly Asp Ile Pro Arg
35 40 4535 40 45
Phe Ala Leu Ala Ile Asp Val Asp Met Thr Pro Gln Asn Ser Gly AlaPhe Ala Leu Ala Ile Asp Val Asp Met Thr Pro Gln Asn Ser Gly Ala
50 55 6050 55 60
Trp Glu Tyr Thr Ala Asp Gly Gln Phe Ala Val Trp Arg Gln Arg ValTrp Glu Tyr Thr Ala Asp Gly Gln Phe Ala Val Trp Arg Gln Arg Val
65 70 75 8065 70 75 80
Arg Ser Glu Lys Ala Leu Ser Leu Asn Phe Gly Phe Thr Asp Tyr TyrArg Ser Glu Lys Ala Leu Ser Leu Asn Phe Gly Phe Thr Asp Tyr Tyr
85 90 9585 90 95
Met Pro Ala Gly Gly Arg Leu Leu Val Tyr Pro Ala Thr Gln Ala ProMet Pro Ala Gly Gly Arg Leu Leu Val Tyr Pro Ala Thr Gln Ala Pro
100 105 110100 105 110
Ala Gly Asp Arg Gly Leu Ile Ser Gln Tyr Asp Ala Ser Asn Asn AsnAla Gly Asp Arg Gly Leu Ile Ser Gln Tyr Asp Ala Ser Asn Asn Asn
115 120 125115 120 125
Ser Ala Arg Gln Leu Trp Thr Ala Val Val Pro Gly Ala Glu Ala ValSer Ala Arg Gln Leu Trp Thr Ala Val Val Pro Gly Ala Glu Ala Val
130 135 140130 135 140
Ile Glu Ala Val Ile Pro Arg Asp Lys Val Gly Glu Phe Lys Leu ArgIle Glu Ala Val Ile Pro Arg Asp Lys Val Gly Glu Phe Lys Leu Arg
145 150 155 160145 150 155 160
Leu Thr Lys Val Asn His Asp Tyr Val Gly Phe Gly Pro Leu Ala ArgLeu Thr Lys Val Asn His Asp Tyr Val Gly Phe Gly Pro Leu Ala Arg
165 170 175165 170 175
Arg Leu Ala Ala Ala Ser Gly Glu LysArg Leu Ala Ala Ala Ser Gly Glu Lys
180 185180 185
<210> 9<210> 9
<211> 29<211> 29
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> V167I-F<223> V167I-F
<400> 9<400> 9
ccaccacccc aagcgatgaa agaggtcgg 29ccaccacccc aagcgatgaa agaggtcgg 29
<210> 10<210> 10
<211> 29<211> 29
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> V167I-R<223> V167I-R
<400> 10<400> 10
ccgacctctt tcatcgcttg gggtggtgg 29ccgacctctttcatcgcttg gggtggtgg 29
<210> 11<210> 11
<211> 29<211> 29
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> V167L-F<223> V167L-F
<400> 11<400> 11
ccaccacccc aagccaggaa agaggtcgg 29ccaccacccc aagccaggaa agaggtcgg 29
<210> 12<210> 12
<211> 29<211> 29
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> V167L-R<223> V167L-R
<400> 12<400> 12
ccgacctctt tcctggcttg gggtggtgg 29ccgacctctttcctggcttg gggtggtgg 29
<210> 13<210> 13
<211> 30<211> 30
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> S193A-F<223> S193A-F
<400> 13<400> 13
gatcggagaa ccagaagcac ccggttcggt 30gatcggagaa ccagaagcac ccggttcggt 30
<210> 14<210> 14
<211> 30<211> 30
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> S193A-R<223> S193A-R
<400> 14<400> 14
accgaaccgg gtgcttctgg ttctccgatc 30accgaaccgg gtgcttctgg ttctccgatc 30
<210> 15<210> 15
<211> 30<211> 30
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> S193G-F<223> S193G-F
<400> 15<400> 15
gatcggagaa ccagaaccac ccggttcggt 30gatcggagaa ccagaaccac ccggttcggt 30
<210> 16<210> 16
<211> 30<211> 30
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> S193G-R<223> S193G-R
<400> 16<400> 16
accgaaccgg gtggttctgg ttctccgatc 30accgaaccgg gtggttctgg ttctccgatc 30
<210> 17<210> 17
<211> 27<211> 27
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> G207V-F<223> G207V-F
<400> 17<400> 17
accgtgcagc tgaaccagaa cacgttt 27accgtgcagc tgaaccagaa cacgttt 27
<210> 18<210> 18
<211> 27<211> 27
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> G207V-R<223> G207V-R
<400> 18<400> 18
aaacgtgttc tggttcagct gcacggt 27aaacgtgttc tggttcagct gcacggt 27
<210> 19<210> 19
<211> 27<211> 27
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> G207L-F<223> G207L-F
<400> 19<400> 19
accgtgcagc tgcagcagaa cacgttt 27accgtgcagc tgcagcagaa cacgttt 27
<210> 20<210> 20
<211> 27<211> 27
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> G207L-R<223> G207L-R
<400> 20<400> 20
aaacgtgttc tgctgcagct gcacggt 27aaacgtgttc tgctgcagct gcacggt 27
<210> 21<210> 21
<211> 34<211> 34
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Arg30-F<223> Arg30-F
<400> 21<400> 21
caagccaggg taccagattt agagtaagca ccaa 34caagccaggg taccagattt agagtaagca ccaa 34
<210> 22<210> 22
<211> 34<211> 34
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Arg30-R<223> Arg30-R
<400> 22<400> 22
ttggtgctta ctctaaatct ggtaccctgg cttg 34ttggtgctta ctctaaatct ggtaccctgg cttg 34
<210> 23<210> 23
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Arg49-F<223> Arg49-F
<400> 23<400> 23
cggtcaggaa gtacatttta cggtcgttag cggtgttgtt 40cggtcaggaa gtacatttta cggtcgttag cggtgttgtt 40
<210> 24<210> 24
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Arg49-R<223> Arg49-R
<400> 24<400> 24
aacaacaccg ctaacgaccg taaaatgtac ttcctgaccg 40aacaacaccg ctaacgaccg taaaatgtac ttcctgaccg 40
<210> 25<210> 25
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Arg106-F<223> Arg106-F
<400> 25<400> 25
aggtagcgta ggtagcttta acggtagaac cagactgggt 40aggtagcgta ggtagcttta acggtagaac cagactgggt 40
<210> 26<210> 26
<211> 40<211> 40
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Arg106-R<223> Arg106-R
<400> 26<400> 26
acccagtctg gttctaccgt taaagctacc tacgctacct 40acccagtctg gttctaccgt taaagctacc tacgctacct 40
<210> 27<210> 27
<211> 44<211> 44
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Arg155-F<223> Arg155-F
<400> 27<400> 27
gtagagttag agatacgttt ttcagcaacg ttcgggtggt ggat 44gtagagttag agatacgttt ttcagcaacg ttcgggtggt ggat 44
<210> 28<210> 28
<211> 44<211> 44
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> Arg155-R<223> Arg155-R
<400> 28<400> 28
atccaccacc cgaacgttgc tgaaaaacgt atctctaact ctac 44atccaccacc cgaacgttgc tgaaaaacgt atctctaact ctac 44
<210> 29<210> 29
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> T7上游引物<223> T7 upstream primer
<400> 29<400> 29
taatacgact cactataggg 20taatacgactcactataggg 20
<210> 30<210> 30
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<220><220>
<223> T7下游引物<223> T7 downstream primer
<400> 30<400> 30
gctagttatt gctcagcgg 19gctagttatt gctcagcgg 19
Claims (13)
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| CN201911139993.XACN112824527B (en) | 2019-11-20 | 2019-11-20 | Artificially designed lysyl endonuclease, coding sequence and fermentation method |
| CN202310357181.2ACN116334049B (en) | 2019-11-20 | 2019-11-20 | Artificially designed lysyl endonuclease, coding sequence and fermentation method |
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| CN115717137B (en)* | 2022-12-27 | 2024-01-26 | 北京惠之衡生物科技有限公司 | Lysyl specific endonuclease mutant and preparation method and application thereof |
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| CN109486800B (en)* | 2018-11-21 | 2019-12-13 | 珠海冀百康生物科技有限公司 | Novel lysyl endopeptidase and preparation method thereof |
| CN109439643B (en)* | 2018-11-21 | 2020-12-04 | 珠海冀百康生物科技有限公司 | Novel lysine specific endonuclease and preparation method thereof |
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