技术领域technical field
本发明涉及一种基因重组方法制备的定点突变的硫酸软骨素酶ABC融合蛋白。其关键是硫酸软骨素酶ABC基因的定点改造、克隆、表达及产物的发酵表达和分离纯化制备。The invention relates to a site-directed mutation chondroitinase ABC fusion protein prepared by a gene recombination method. The key is the site-specific transformation, cloning, expression of the chondroitinase ABC gene, and the fermentation expression and separation and purification of the product.
背景技术Background technique
硫酸软骨素裂解酶(chondroitinase或chondroitin sulfateyase，简称为“ChSase”)是一类能将硫酸软骨素、软骨素、透明质酸等糖胺聚糖降解为不饱和二糖(ΔDi及寡糖)的裂解酶。Chondroitin sulfate lyase (chondroitinase or chondroitin sulfateyase, referred to as "ChSase") is a kind of glycosaminoglycans that can degrade chondroitin sulfate, chondroitin, hyaluronic acid and other glycosaminoglycans into unsaturated disaccharides (ΔDi and oligosaccharides) Lyase.
随着对ChSase的深入研究，发现硫酸软骨素酶ABC(简称为“ChSase ABC”)有着广泛的应用价值。科研人员利用ChSase ABC检测硫酸软骨素和生产低分子量硫酸软骨素。鲍伦军，杨建成等用ChSase ABC酶解-高效液相色谱的方法检测鱼翅中的透明质酸，采用ZORBAX糖分析柱，紫外检测波长为226nm，检测到鱼翅中透明质酸的质量分数为0.86-1.96％(鲍伦军等.鱼翅中硫酸软骨素的酶解-高效液相色谱法测定.色谱,2002,20(6):557-559)。朱昱宁，敖雷等同样用酶解高效液相色谱的方法检测CS的含量，采用UltimateXB-NH2柱，紫外检测波长为232nm，流动相为醋酸钠缓冲液(pH5.6)-乙腈(950:50)，流速为1.0mL/min，此色谱条件可将CS A，B，C分开(朱昱宁等.酶解高效液相色谱法测定硫酸软骨素的含量.中国生化药物杂志,2012,33(6):827-829)。With the in-depth research on ChSase, it is found that chondroitin sulfate ABC (referred to as "ChSase ABC") has a wide range of application value. Researchers use ChSase ABC to detect chondroitin sulfate and produce low molecular weight chondroitin sulfate. Bao Lunjun, Yang Jiancheng, etc. used ChSase ABC enzymatic hydrolysis-high performance liquid chromatography to detect hyaluronic acid in shark fins. Using ZORBAX sugar analysis column, the ultraviolet detection wavelength was 226nm, and the mass fraction of hyaluronic acid in shark fins was detected as 0.86-1.96% (Bao Lunjun et al. Enzymatic hydrolysis of chondroitin sulfate in shark fin - determination by high performance liquid chromatography. Chromatography, 2002,20(6):557-559). Zhu Yuning, Ao Lei, etc. also used enzymatic high-performance liquid chromatography to detect the content of CS, using UltimateXB-NH2 column, the ultraviolet detection wavelength was 232nm, and the mobile phase was sodium acetate buffer (pH5.6)-acetonitrile (950:50 ), the flow rate is 1.0mL/min, and this chromatographic condition can separate CS A, B, and C (Zhu Yuning et al. Determination of the content of chondroitin sulfate by enzymatic high-performance liquid chromatography. Chinese Journal of Biochemical Medicine, 2012,33(6) :827-829).
另外ChSase ABC在临床应用方面也有很大的作用。在缓解视网膜变性方面，可降解硫酸软骨素蛋白多糖(CSPGs)，CSPGs是中枢神经系统(CNS)损伤后胶质瘢痕的重要组分，可抑制神经轴突再生。CSPGs主要通过糖氨多糖链抑制神经再生，因此去除GAG或者干扰其合成均可使轴突再生。黄玉苗,高朋芬等通过玻璃体腔注射ChSase ABC酶，免疫荧光法观察CSPGs的表达，反转录-聚合酶链反应(RT-PCR)法检测多功能蛋白聚糖mRNA的表达，检测光感受器凋亡情况，结果表明Chase ABC能通过降解变性大鼠视网膜异常沉积的CSPGs，抑制光感受器细胞的凋亡，从而促进损伤网膜的修复(黄玉苗等.硫酸软骨素酶缓解视网膜变性大鼠光感受器细胞凋亡.扬州大学学报,2012,33(4):19-23.)。In addition, ChSase ABC also plays a great role in clinical application. In terms of alleviating retinal degeneration, it can degrade chondroitin sulfate proteoglycans (CSPGs), which are important components of glial scars after central nervous system (CNS) injury, and can inhibit axon regeneration. CSPGs mainly inhibit nerve regeneration through glycosaminoglycan chains, so removing GAG or interfering with its synthesis can regenerate axons. Huang Yumiao, Gao Pengfen et al injected ChSase ABC enzyme into the vitreous cavity, observed the expression of CSPGs by immunofluorescence, detected the expression of versican mRNA by reverse transcription-polymerase chain reaction (RT-PCR), and detected the apoptosis of photoreceptors The results show that Chase ABC can inhibit the apoptosis of photoreceptor cells by degrading abnormally deposited CSPGs in the retina of degenerated rats, thereby promoting the repair of damaged retina (Huang Yumiao et al. Apoptosis. Journal of Yangzhou University, 2012,33(4):19-23.).
在提高后肢运动能力方面，ChSase ABC可以修复脊柱损伤。脊髓损伤是脊柱骨折的常见并发症，可致肢体瘫痪，大小便失禁及呼吸肌瘫痪等一系列严重并发症，脊髓损伤后神经再生和功能修复，是当今医学领域中的一大难题，ChSase ABC可以降解CSPGs。孙永新，刘宁等利用ChSase ABC联合高压氧预处理对成年大鼠脊髓损伤后不同时期后肢运动功能及腓肠肌运动终板内乙酰胆碱酯酶(AChE)含量的影响，来检测ChSase ABC的作用。结果表明高压氧预处理可以改善大鼠患肢的运动功能和提高AChE的活性，联合ChSase ABC作用更强(孙永新华等.硫酸软骨素酶ABC联合高压氧预处理对脊髓损伤大鼠后肢运动功能的影响.解剖科学进展,2012,18(6):526-529.)。Nicole J.Tester等用ChSase ABC来治疗受伤的猫，ChSase ABC修复了猫的脊柱损伤，提高了猫的运动能力(参见：Nicole J.etal.Chondroitinase ABC improves basic and skilled locomotion in spinal cordinjured cats.Experimental Neurology,2008,209(2):483-496)。In terms of improving hindlimb mobility, ChSase ABC can repair spinal injuries. Spinal cord injury is a common complication of spinal fractures, which can lead to a series of serious complications such as limb paralysis, urinary incontinence and respiratory muscle paralysis. Nerve regeneration and functional restoration after spinal cord injury is a major problem in today's medical field. ChSase ABC Can degrade CSPGs. Sun Yongxin, Liu Ning et al. used ChSase ABC combined with hyperbaric oxygen preconditioning to detect the effect of ChSase ABC on the hindlimb motor function and the content of acetylcholinesterase (AChE) in the gastrocnemius muscle motor endplate at different periods after spinal cord injury in adult rats. The results show that hyperbaric oxygen preconditioning can improve the motor function of rats' affected limbs and increase the activity of AChE, and the combined effect of ChSase ABC is stronger (Sun Yongxinhua et al. Chondroitinase ABC combined with hyperbaric oxygen preconditioning can improve the hindlimb movement of rats with spinal cord injury Effects on function. Advances in Anatomical Science, 2012, 18(6):526-529.). Nicole J.Tester et al. used ChSase ABC to treat injured cats. ChSase ABC repaired the cat's spinal cord injury and improved the cat's exercise capacity (see: Nicole J.etal.Chondroitinase ABC improves basic and skilled locomotion in spinal cordinjured cats.Experimental Neurology, 2008, 209(2):483-496).
近年来，Mark R Brown等经研究发现，突出椎间盘内的主要组分为蛋白聚糖，而用ChSase ABC及ChSase AC则可特异性降解掉突出的椎管内的糖胺聚糖、降低椎管内压，同时对椎管外周组织无损害(Brown，M D.Method for treating intervertebral discdisplacement with enzymes[P]:US,4696816,1987-09-29)。2001年Denholm EM等发现ChSase AC、ChSase B能抑制黑色素瘤血管的形成及瘤细胞的转移与增生等(Denholm E M,Lin Y Q,Silver P J.Anti-tumor activities of chondroitinase AC andchondroitinase B:inhibition of angiogenesis,proliferation andinvasion.European Journal of Pharmacology,2001,416(3):213-221)。Lee MC等发现当软骨用ChSase ABC处理后，移植的软骨细胞与软骨创面的黏附能力大大增强；(Lee M C,Sung K L,Kurtis M S,et a1.Adhesive force of chondrocytes to cartilage effectsof chondroitinase ABC.Clin Orthop,2000,370(1):286-294)。In recent years, Mark R Brown et al. have found through research that the main component of the herniated intervertebral disc is proteoglycan, and the use of ChSase ABC and ChSase AC can specifically degrade the glycosaminoglycan in the herniated spinal canal and lower the spinal canal. internal pressure without damaging the peripheral tissues of the spinal canal (Brown, M D. Method for treating intervertebral disc displacement with enzymes [P]: US, 4696816, 1987-09-29). In 2001, Denholm EM found that ChSase AC and ChSase B can inhibit the formation of melanoma blood vessels and the metastasis and proliferation of tumor cells (Denholm EM, Lin Y Q, Silver P J. Anti-tumor activities of chondroitinase AC and chondroitinase B: inhibition of angiogenesis , proliferation and invasion. European Journal of Pharmacology, 2001, 416(3):213-221). Lee MC et al. found that when the cartilage was treated with ChSase ABC, the adhesion ability of transplanted chondrocytes to the cartilage wound surface was greatly enhanced; (Lee M C, Sung K L, Kurtis M S, et a1. Adhesive force of chondrocytes to cartilage effects of chondroitinase ABC. , 2000, 370(1):286-294).
此外硫酸软骨素酶ABC还可用于酶解生产硫酸软骨素。对低分子量硫酸软骨素的制备常采用酸水解法、离子交换法和酶解法。前两种方法需要较复杂的实验设备并且会污染环境，比较而言，酶解法需要的条件不高、方法简便且容易控制。酶解法的关键在于获得大量的硫酸软骨素裂解酶。分离纯化硫酸软骨素裂解酶的方法非常复杂，通常需要经过多步的色谱纯化，收率很低。异源重组表达ChSase ABC是常用来提高ChSase ABC产量的手段之一。然而对ChSase ABC的异源重组表达研究非常有限，到目前为止国内还没有这方面的报道。只有Pojasek等在E.coil中表达了硫酸软骨素酶AC酶和B酶的基因(chsase基因)，并分离纯化到活性蛋白，在其研究中所用的载体为pET15b和pCRT7/NT，这两个载体中的融合标签均为His-tag。但是His-tag存在明显的缺点，即跟亲和载体的亲和力不强，不能增加与之结合蛋白质的可溶性，难以提高纯化效果和蛋白质可溶性比例等(参见:Kevin Pojasek,Zachary Shriver,Patrick Kiley,Ganesh Venkataraman and RamSasisekharan.Recombinant Expression,Purification and Kinetic Characterizationof Chondroitinase AC and Chondroitinase B from Flavobacteriumhparinum.Biochemical and Biophysical Research Communications,2001,286:343-351)。In addition, chondroitin sulfate ABC can also be used for enzymatic hydrolysis to produce chondroitin sulfate. The preparation of low molecular weight chondroitin sulfate often adopts acid hydrolysis, ion exchange and enzymatic hydrolysis. The first two methods require more complicated experimental equipment and will pollute the environment. In comparison, the enzymatic hydrolysis method requires less conditions, and the method is simple and easy to control. The key of the enzymatic hydrolysis method is to obtain a large amount of chondroitin sulfate lyase. The method for isolating and purifying chondroitin sulfate lyase is very complicated, usually requires multi-step chromatographic purification, and the yield is very low. Expression of ChSase ABC by heterologous recombination is one of the means commonly used to increase the yield of ChSase ABC. However, the research on heterologous recombination expression of ChSase ABC is very limited, so far there is no report in this area in China. Only Pojasek et al expressed the genes of chondroitinase AC enzyme and B enzyme (chsase gene) in E.coil, and isolated and purified the active protein. The vectors used in their research were pET15b and pCRT7/NT, the two The fusion tags in the vector are all His-tags. However, His-tag has obvious disadvantages, that is, the affinity with the affinity carrier is not strong, the solubility of the protein bound to it cannot be increased, and it is difficult to improve the purification effect and protein solubility ratio (see: Kevin Pojasek, Zachary Shriver, Patrick Kiley, Ganesh Venkataraman and Ram Sasisekharan. Recombinant Expression, Purification and Kinetic Characterization of Chondroitinase AC and Chondroitinase B from Flavobacterium hparinum. Biochemical and Biophysical Research Communications, 2001, 286:343-351).
专利申请CN103305496A公开了一种从微生物提取硫酸软骨素酶的方法，但是该方法提取步骤繁琐，成本较高。Patent application CN103305496A discloses a method for extracting chondroitin sulfate from microorganisms, but the extraction steps of this method are cumbersome and the cost is high.
发明内容Contents of the invention
为解决现有技术的不足，本发明采用融合表达和定点突变的方法，获得了大量可溶性的ChSase ABC突变体的融合蛋白，进一步地提高了ChSase ABC酶活；且采用一步纯化的方法，纯化方法简单，并且得到高纯度的ChSase ABC突变体。In order to solve the deficiencies in the prior art, the present invention adopts fusion expression and site-directed mutation methods to obtain a large amount of fusion proteins of soluble ChSase ABC mutants, which further improves the activity of ChSase ABC enzymes; and adopts a one-step purification method, the purification method Simple and highly pure ChSase ABC mutants.
本发明的主要目的在于提供一种硫酸软骨素ABC酶突变体融合蛋白，其中，所述硫酸软骨素ABC酶突变体融合蛋白包括硫酸软骨素ABC酶突变体和麦芽糖结合蛋白，其中，所述硫酸软骨素ABC酶突变体具有SEQ ID NO:1所示的氨基酸序列，所述麦芽糖结合蛋白通过肽段连接部分与所述硫酸软骨素ABC酶N端连接。本发明提供了一种定点突变的硫酸软骨素ABC酶融合蛋白，其特征是将Asn771位的氨基酸突变为Ala。The main purpose of the present invention is to provide a chondroitin sulfate ABC enzyme mutant fusion protein, wherein, the chondroitin sulfate ABC enzyme mutant fusion protein comprises chondroitin sulfate ABC enzyme mutant and maltose binding protein, wherein the sulfate The chondroitin ABC enzyme mutant has the amino acid sequence shown in SEQ ID NO: 1, and the maltose binding protein is connected to the N-terminal of the chondroitin sulfate ABC enzyme through a peptide linking part. The invention provides a site-directed mutation chondroitin sulfate ABC enzyme fusion protein, which is characterized in that the amino acid at position Asn771 is mutated into Ala.
优选地，本发明所述的硫酸软骨素ABC酶突变体融合蛋白中，所述麦芽糖结合蛋白具有SEQ ID NO:2所示的氨基酸序列。Preferably, in the chondroitin sulfate ABC enzyme mutant fusion protein of the present invention, the maltose binding protein has the amino acid sequence shown in SEQ ID NO:2.
优选地，本发明所述的硫酸软骨素ABC酶突变体融合蛋白中，所述硫酸软骨素ABC酶突变体融合蛋白具有SEQ ID NO:3所示的氨基酸序列。Preferably, in the chondroitin sulfate ABC enzyme mutant fusion protein of the present invention, the chondroitin sulfate ABC enzyme mutant fusion protein has the amino acid sequence shown in SEQ ID NO:3.
优选地，本发明所述的硫酸软骨素ABC酶突变体融合蛋白中，以硫酸软骨素A为底物，所述的硫酸软骨素ABC酶突变体融合蛋白的酶活和比酶活分别为24.96IU/mL enzyme和31.32IU/mg protein，以硫酸软骨素B为底物，所述的硫酸软骨素ABC酶突变体融合蛋白酶活和比酶活分别为21.58IU/mL enzyme和27.07IU/mg protein。Preferably, in the chondroitin sulfate ABC enzyme mutant fusion protein of the present invention, chondroitin sulfate A is used as a substrate, and the enzymatic activity and specific enzyme activity of the chondroitin sulfate ABC enzyme mutant fusion protein are respectively 24.96 IU/mL enzyme and 31.32IU/mg protein, with chondroitin sulfate B as substrate, the enzymatic activity and specific enzyme activity of the chondroitin sulfate ABC enzyme mutant fusion protein are 21.58IU/mL enzyme and 27.07IU/mg protein respectively .
优选地，本发明所述的硫酸软骨素ABC酶突变体融合蛋白中，所述的硫酸软骨素ABC酶突变体融合蛋白的Vmax和kcat/Km，以硫酸软骨素A为底物时，分别为22.52μmol/L·s和50.25L/μmol·s；以硫酸软骨素B为底物时，其分别为4.59μmol/L·s和12.22L/μmol·s。Preferably, in the chondroitin sulfate ABC enzyme mutant fusion protein of the present invention, the Vmax and kcat /Km of the chondroitin sulfate ABC enzyme mutant fusion protein, when chondroitin sulfate A is used as a substrate , respectively 22.52μmol/L·s and 50.25L/μmol·s; when chondroitin sulfate B was used as substrate, they were 4.59μmol/L·s and 12.22L/μmol·s respectively.
本发明的另一目的在于提供一种DNA，所述DNA序列编码所述的硫酸软骨素ABC酶突变体融合蛋白。Another object of the present invention is to provide a DNA sequence encoding the chondroitin sulfate ABC enzyme mutant fusion protein.
本发明的再一目的在于提供一种重组载体，所述重组载体包括载体和所述的编码硫酸软骨素ABC酶突变体融合蛋白DNA，其中所述载体包括pMAL-c2x。Another object of the present invention is to provide a recombinant vector, which includes a vector and the DNA encoding the chondroitin sulfate ABC enzyme mutant fusion protein, wherein the vector includes pMAL-c2x.
本发明的又一目的在于提供一种转化体，所述转化体是将所述的重组载体导入宿主细胞而得到的转化体，其中，所述宿主细胞包括E.coli BL21(DE3)。Another object of the present invention is to provide a transformant obtained by introducing the recombinant vector into a host cell, wherein the host cell includes E. coli BL21(DE3).
本发明的还一目的在于提供一种制备所述硫酸软骨素ABC酶突变体融合蛋白的方法，所述制备方法包括：(1)使用PCR通过带有突变位点的引物获得所述的的pMAL-c2x-ChSase ABC I突变体的重组载体；(2)转化所述步骤(1)中的所述重组载体到所述宿主细胞，表达所述硫酸软骨素ABC酶突变体融合蛋白；以及(3)破碎所述转化体，使用麦芽糖结合蛋白亲和层析分离所述表达的硫酸软骨素ABC酶突变体融合蛋白。Another object of the present invention is to provide a method for preparing the chondroitin sulfate ABC enzyme mutant fusion protein, the preparation method comprising: (1) using PCR to obtain the pMAL by using primers with mutation sites -the recombinant vector of the c2x-ChSase ABC I mutant; (2) transforming the recombinant vector in the step (1) into the host cell to express the fusion protein of the chondroitin sulfate ABC enzyme mutant; and (3 ) crushing the transformant, and separating the expressed chondroitin sulfate ABC enzyme mutant fusion protein using maltose binding protein affinity chromatography.
本发明的还测定了突变后的硫酸软骨素ABC酶融合蛋白的最适温度为45度和最适pH为7.5。The present invention also determines that the optimum temperature of the mutated chondroitin sulfate ABC enzyme fusion protein is 45 degrees and the optimum pH is 7.5.
本发明同样测定了突变后的硫酸软骨素ABC酶融合蛋白的动力学常数，其中包括Vmax和kcat/Km，以硫酸软骨素A为底物时，其分别为22.52μmol/L·s和50.25L/μmol·s，以硫酸软骨素B为底物时，其分别为4.59μmol/L·s和12.22L/μmol·s。The present invention also measures the kinetic constants of the mutated chondroitin sulfate ABC enzyme fusion protein, including Vmax and kcat /Km , which are respectively 22.52 μmol/L·s when chondroitin sulfate A is used as the substrate and 50.25L/μmol·s, when chondroitin sulfate B was used as substrate, they were 4.59μmol/L·s and 12.22L/μmol·s, respectively.
本发明还同样测定了突变后的硫酸软骨素ABC酶融合蛋白的热稳定性，其中包括在45℃，40℃，35℃和30℃，在30℃和35℃下，210min后能保留90％的酶活，在40℃下，210min后能保留72％的酶活，在45℃下，210min能保留41％的酶活。The present invention also measures the thermostability of the mutated chondroitin sulfate ABC enzyme fusion protein, including 45°C, 40°C, 35°C and 30°C, at 30°C and 35°C, 90% can be retained after 210min At 40°C, 72% of the enzyme activity can be retained after 210 minutes, and at 45°C, 41% of the enzyme activity can be retained after 210 minutes.
本发明通过定点突变的方法将Asn771氨基酸突变成Ala，以硫酸软骨素A为底物，Asn771Ala的酶活和比酶活分别为24.96IU/mL enzyme和31.32IU/mg protein，以硫酸软骨素B为底物，酶活和比酶活分别为21.58IU/mL enzyme和27.07IU/mg protein。未突变的MBP-ChSase ABC I，以硫酸软骨素A为底物，酶活和比酶活分别为20.18IU/mL enzyme和22.52IU/mg protein，以硫酸软骨素B为底物，酶活和比酶活分别为13.08IU/mL enzyme和14.60IU/mg protein。In the present invention, the Asn771 amino acid is mutated into Ala by the method of site-directed mutation, and chondroitin sulfate A is used as a substrate. B is the substrate, the enzyme activity and specific enzyme activity are 21.58IU/mL enzyme and 27.07IU/mg protein respectively. Unmutated MBP-ChSase ABC I, with chondroitin sulfate A as substrate, the enzyme activity and specific enzyme activity were 20.18IU/mL enzyme and 22.52IU/mg protein, with chondroitin sulfate B as substrate, the enzyme activity and The specific enzyme activities are 13.08IU/mL enzyme and 14.60IU/mg protein respectively.
优选地，本发明制备所述硫酸软骨素ABC酶突变体融合蛋白的方法中，所述步骤(3)中麦芽糖结合蛋白亲和层析包括使用直链淀粉树脂、马铃薯淀粉柱分离。Preferably, in the method for preparing the chondroitin sulfate ABC enzyme mutant fusion protein of the present invention, the maltose-binding protein affinity chromatography in the step (3) includes using amylose resin and potato starch column separation.
本发明还提供了一种生产低分子量硫酸软骨素的方法，所述生产方法包括：使用所述的硫酸软骨素ABC酶突变体融合蛋白生产低分子量硫酸软骨素。The present invention also provides a method for producing low-molecular-weight chondroitin sulfate, the production method comprising: using the chondroitin-sulfate ABC enzyme mutant fusion protein to produce low-molecular-weight chondroitin sulfate.
附图说明Description of drawings
图1为以未突变的pMAL-c2x-ChSase ABC I为模板，使用含有突变位点的primer经PCR扩增得到的突变的pMAL-c2x-ChSase ABC I电泳图谱，泳道M为：Marker，泳道1为60℃为退火温度的PCR产物。Figure 1 is the electrophoresis pattern of the mutant pMAL-c2x-ChSase ABC I obtained by PCR amplification using the primer containing the mutation site using the unmutated pMAL-c2x-ChSase ABC I as a template. Lane M is: Marker, lane 1 60 °C is the annealing temperature for the PCR products.
图2为表达的蛋白电泳图，其中，泳道M为marker(由上至下分子量依次均为250kDa、150kDa、100kDa、70kDa)，泳道1为空白对照、泳道2为E.coli BL21(DE3)/pMAL-ChSase ABC的可溶蛋白，箭头所指处为融合蛋白MBP-ChSase ABC I突变体(140kDa)。Figure 2 is the electrophoresis diagram of the expressed protein, in which, lane M is marker (molecular weights are 250kDa, 150kDa, 100kDa, 70kDa from top to bottom), lane 1 is blank control, lane 2 is E.coli BL21(DE3)/ The soluble protein of pMAL-ChSase ABC, the point indicated by the arrow is the fusion protein MBP-ChSase ABC I mutant (140kDa).
序列表中：In the sequence listing:
序列1为硫酸软骨素ABC酶突变体的氨基酸序列；Sequence 1 is the amino acid sequence of a chondroitin sulfate ABC enzyme mutant;
序列2为大肠杆菌(Escherichia coli)麦芽糖结合蛋白氨基酸序列；Sequence 2 is the amino acid sequence of Escherichia coli maltose binding protein;
序列3为本发明的硫酸软骨素ABC酶突变体融合蛋白氨基酸序列；Sequence 3 is the amino acid sequence of the chondroitin sulfate ABC enzyme mutant fusion protein of the present invention;
序列4为本发明的硫酸软骨素酶ABC酶突变体融合蛋白DNA序列；Sequence 4 is the fusion protein DNA sequence of the chondroitinase ABC enzyme mutant of the present invention;
序列5为引入突变位点的用于PCR扩增pMAL-c2x-ChSase ABC I突变体的上游引物核苷酸序列；Sequence 5 is the nucleotide sequence of the upstream primer used for PCR amplification of the pMAL-c2x-ChSase ABC I mutant introduced into the mutation site;
序列6为引入突变位点的用于PCR扩增pMAL-c2x-ChSase ABC I突变体的下游引物核苷酸序列。Sequence 6 is the nucleotide sequence of the downstream primer used for PCR amplification of the pMAL-c2x-ChSase ABC I mutant introduced into the mutation site.
具体实施方式detailed description
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.
以下，对本文的实施方式进行具体说明。Hereinafter, the embodiments herein will be specifically described.
硫酸软骨素ABC酶突变体融合蛋白Chondroitin Sulfate ABC Enzyme Mutant Fusion Protein
本文涉及的突变的硫酸软骨素ABC酶融合蛋白(以下，有时也简称为“MBP-ChSaseABC”)，其融合有突变的硫酸软骨素ABC酶和麦芽糖结合蛋白(以下，有时也简称为“MBP”)。The mutated chondroitin sulfate ABC enzyme fusion protein (hereinafter, sometimes referred to as "MBP-ChSaseABC") involved in this paper is fused with mutated chondroitin sulfate ABC enzyme and maltose binding protein (hereinafter, sometimes referred to as "MBP" for short). ).
本文涉及的硫酸软骨素ABC酶可以是任何硫酸软骨素酶，优选选自普通变形杆菌(Proteus vulgaris)、嗜水气单孢菌(Aeromonas liquefaciens)及肝素黄杆菌(Flavobacterium heparinum)来源的硫酸软骨素酶。根据本文的一个优选的实施方案，硫酸软骨素ABC酶是普通变形杆菌来源的硫酸软骨素酶；特别地，硫酸软骨素酶是去除了编码信号肽碱基的普通变形杆菌的硫酸软骨素ABC酶，并通过定点突变获得了突变的硫酸软骨素ABC酶。根据本文一个优选的实施方案，本文硫酸软骨素ABC酶突变体具有SEQ ID NO:1所示的氨基酸。The chondroitin sulfate ABC enzyme referred to herein may be any chondroitin sulfate enzyme, preferably selected from chondroitin sulfate derived from Proteus vulgaris, Aeromonas liquefaciens and Flavobacterium heparinum enzyme. According to a preferred embodiment herein, the chondroitin sulfate ABC enzyme is a chondroitin sulfate enzyme derived from Proteus vulgaris; in particular, the chondroitin sulfate enzyme is a chondroitin sulfate ABC enzyme of Proteus vulgaris from which the coding signal peptide base has been removed , and obtained a mutant chondroitin sulfate ABCase by site-directed mutagenesis. According to a preferred embodiment herein, the chondroitin sulfate ABC enzyme mutant herein has the amino acid shown in SEQ ID NO:1.
硫酸软骨素ABC酶突变体融合蛋白中，硫酸软骨素ABC酶突变体直接与麦芽糖结合蛋白融合。In the chondroitin sulfate ABC enzyme mutant fusion protein, the chondroitin sulfate ABC enzyme mutant is directly fused with maltose binding protein.
本文涉及的麦芽糖结合蛋白可以是任何本领域普通技术人员能够得到的麦芽糖结合蛋白。优选来自于大肠杆菌的麦芽糖结合蛋白。来自大肠杆菌的天然的MBP能够与麦芽糖特异吸附，参与大肠杆菌对麦芽糖的转运和利用。MBP不但能与直链淀粉结合，实现亲和分离，也能与马铃薯淀粉实现亲和分离，从而可以大大降低酶的分离纯化成本，有利于实现工业化规模的分离纯化。在一个更优选的实施方案中，本文的麦芽糖结合蛋白具有SEQ IDNO:2所示的氨基酸。The maltose-binding protein referred to herein may be any maltose-binding protein available to those of ordinary skill in the art. Maltose binding protein from Escherichia coli is preferred. The natural MBP from Escherichia coli can specifically adsorb maltose and participate in the transport and utilization of maltose by Escherichia coli. MBP can not only combine with amylose to achieve affinity separation, but also achieve affinity separation with potato starch, which can greatly reduce the cost of enzyme separation and purification, and is conducive to the realization of separation and purification on an industrial scale. In a more preferred embodiment, the maltose binding protein herein has the amino acid shown in SEQ ID NO:2.
优选，本文的突变的硫酸软骨素ABC酶融合蛋白具有SEQ ID NO:3所示的氨基酸。Preferably, the mutated chondroitin sulfate ABC enzyme fusion protein herein has the amino acid shown in SEQ ID NO:3.
DNAdna
本文还涉及一种编码上述所有突变的硫酸软骨素ABC酶融合蛋白的DNA，其是该融合蛋白的编码序列。This paper also relates to a DNA encoding all the mutated chondroitin sulfate ABC enzyme fusion proteins, which is the coding sequence of the fusion protein.
用于本说明书的术语“编码序列”的意思是直接指定其蛋白产物的氨基酸序列的核苷酸序列。The term "coding sequence" used in this specification means a nucleotide sequence that directly specifies the amino acid sequence of its protein product.
编码序列的边界通常由开读框决定，所述开读框通常以ATG起始密码子或可供选择的起始密码子例如GTG和TTG开始，并且以终止密码子例如TAA、TAG和TGA结束。编码序列可以是DNA、cDNA或重组核苷酸序列。The boundaries of the coding sequence are usually determined by an open reading frame that usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG and TGA . A coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.
根据本文一个优选的实施方案，DNA具有SEQ ID NO:4所示的碱基序列。According to a preferred embodiment herein, the DNA has the base sequence shown in SEQ ID NO:4.
重组载体recombinant vector
本文还涉及突变载体，其包含编码上述硫酸软骨素ABC酶突变体融合蛋白。The present invention also relates to a mutant vector comprising a fusion protein encoding the aforementioned chondroitin sulfate ABC enzyme mutant.
本文涉及的重组载体包含编码上述所有的融合蛋白的DNA序列、启动子、转录和翻译终止信号。本文中所述的DNA序列可以和其它调控序列结合在一起，产生重组载体，该载体可以包括一个或多个(数个)方便的限制位点以允许在这些位点插入或取代编码肽段的DNA序列。备选的，可以通过在适当的用于表达的载体中插入包含所述氨基酸序列的DNA序列来表达本文的DNA序列。在制备重组载体的过程中，将编码序列导入载体中，从而将该编码序列与适当的表达调控序列可操作地连接。The recombinant vector involved in this paper contains the DNA sequences encoding all the fusion proteins mentioned above, promoters, transcription and translation termination signals. The DNA sequences described herein may be combined with other regulatory sequences to produce recombinant vectors which may include one or more (several) convenient restriction sites to allow insertion or substitution of peptide-encoding peptides at these sites. DNA sequence. Alternatively, the DNA sequence herein can be expressed by inserting a DNA sequence comprising said amino acid sequence in an appropriate vector for expression. In preparing a recombinant vector, a coding sequence is introduced into the vector so that the coding sequence is operably linked to appropriate expression control sequences.
启动子、转录信号、翻译终止信号以及其它调控序列是任何本领域普通技术人员能够可以根据常规选择而确定的。Promoters, transcription signals, translation termination signals and other regulatory sequences can be determined by any person of ordinary skill in the art according to routine selection.
重组载体可以是任何载体(例如，质粒或病毒)，其能够方便地进行重组DNA步骤，并且能够产生核苷酸序列的表达。载体的选择将通常依赖于载体与将引入该载体的宿主细胞的相容性。载体可以是线状或闭合环状质粒。A recombinant vector can be any vector (eg, a plasmid or virus) that facilitates recombinant DNA procedures and is capable of producing expression of a nucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which it will be introduced. Vectors can be linear or closed circular plasmids.
载体可以是自主复制载体，例如，质粒、染色体外元件、微型染色体或人工染色体。载体可以含有任何用于确保自复制的构建。或者，载体可以是一种当被引入宿主细胞中时，整合到基因组中并且与整合了该载体的染色体一起复制的载体。此外，可以使用单独的载体或质粒或两个或更多个载体或质粒，其共同含有待引入宿主细胞基因组的完整DNA，或可以使用转座子。A vector can be an autonomously replicating vector, eg, a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. Vectors may contain any constructs designed to ensure self-replication. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates together with the chromosome into which the vector has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids, which together contain the entire DNA to be introduced into the genome of the host cell, may be used, or a transposon may be used.
根据本文一个优选的实施方案，本文的重组载体是pMAL-c2x。优选，该突变载体是通过以下步骤构建：According to a preferred embodiment herein, the recombinant vector herein is pMAL-c2x. Preferably, the mutant vector is constructed through the following steps:
以未突变的pMAL-c2x-ChSase ABC I为模板，以SEQ ID NO:5和SEQ ID NO:6为引物，PCR获得pMAL-c2x-ChSase ABC I突变体。Using unmutated pMAL-c2x-ChSase ABC I as a template and using SEQ ID NO: 5 and SEQ ID NO: 6 as primers, the pMAL-c2x-ChSase ABC I mutant was obtained by PCR.
转化体Transformant
本文还涉及转化体，其是将包含编码上述硫酸软骨素ABC酶突变体融合蛋白DNA导入宿主细胞而得到的转化体。The present invention also relates to a transformant, which is a transformant obtained by introducing the fusion protein DNA encoding the aforementioned chondroitin sulfate ABCase mutant into host cells.
本文涉及的转化体可用于蛋白质的生产，通过将包含本文DNA序列的载体导入宿主细胞而得到该转化体，并在适当的培养基中培养该转化体，可以用来生产所需要的目标蛋白质。The transformant involved in this article can be used for protein production. The transformant obtained by introducing the vector containing the DNA sequence herein into the host cell, and culturing the transformant in an appropriate medium can be used to produce the desired target protein.
术语“宿主细胞”包括母体细胞的任何后代，其由于复制过程中发生的突变而不同于母体细胞。宿主细胞可以是在本文的肽段的重组产生中有用的任何细胞，例如，原核或真核细胞。原核宿主细胞可以是任何革兰氏阳性细菌或革兰氏阴性细菌。革兰氏阳性细菌包括但不限于，芽孢杆菌属、链球菌属、链霉菌属、葡萄球菌属、肠球菌属、乳酸菌属、乳球菌属、梭菌属、地芽孢杆菌属和海洋芽孢杆菌属。革兰氏阴性细菌包括但不限于，大肠杆菌、假单胞菌属、沙门氏菌属、弯曲杆菌属、螺杆菌属、黄杆菌属、梭杆菌属、泥杆菌属、奈瑟氏菌属和脲原体属。细菌宿主细胞可以是任何芽孢杆菌属细胞。在本文的实施中有用的芽孢杆菌属细胞包括但不限于，嗜碱芽孢杆菌、解淀粉芽孢杆菌、短芽孢杆菌、环状芽孢杆菌、克劳氏芽孢杆菌、凝结芽孢杆菌、坚强芽孢杆菌、灿烂芽孢杆菌、迟缓芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌、短小芽孢杆菌、嗜热脂肪芽孢杆菌、枯草芽孢杆菌和苏云金芽孢杆菌细胞。细菌宿主细胞还可以是任何链球菌属细胞。在本文的实施中有用的链球菌属细胞包括但不限于，似马链球菌、酿脓链球菌、乳房链球菌和马链球菌兽瘟亚种细胞。细菌宿主细胞还可以是任何链霉菌属细胞。在本文的实施中有用的链霉菌属细胞包括但不限于，不产色链霉菌、除虫链霉菌、天蓝链霉菌、灰色链霉菌和浅青紫链霉菌细胞。宿主细胞还可以是真核生物，如哺乳动物、昆虫、植物或真菌细胞。在一个具体的方面，宿主细胞是真菌细胞。在一个具体的方面，真菌宿主细胞是酵母细胞。在另一个具体的方面，真菌宿主细胞是丝状真菌细胞。The term "host cell" includes any progeny of a parental cell that differs from the parental cell due to mutations that occur during replication. A host cell can be any cell useful in the recombinant production of the peptides herein, eg, prokaryotic or eukaryotic cells. The prokaryotic host cell can be any gram-positive or gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Marine Bacillus . Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Geobacter, Neisseria, and Urea body. The bacterial host cell can be any Bacillus cell. Bacillus cells useful in the practice herein include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis cells. The bacterial host cell can also be any Streptococcus cell. Streptococcus cells useful in the practice herein include, but are not limited to, S. equisimilis, S. pyogenes, S. uberis, and S. equi subsp. zooepidemicus cells. The bacterial host cell can also be any Streptomyces cell. Streptomyces cells useful in the practice herein include, but are not limited to, S. achromogenes, S. avermitilis, S. coelicolor, S. griseus, and S. lividans cells. The host cell can also be a eukaryote, such as a mammalian, insect, plant or fungal cell. In a specific aspect, the host cell is a fungal cell. In a specific aspect, the fungal host cell is a yeast cell. In another specific aspect, the fungal host cell is a filamentous fungal cell.
根据本文一个优选的实施方案，细菌宿主细胞是大肠杆菌BL21(DE3)。According to a preferred embodiment herein, the bacterial host cell is E. coli BL21(DE3).
突变的硫酸软骨素ABC酶融合蛋白的方法Method for mutated chondroitin sulfate ABC enzyme fusion protein
本文还涉及一种制备硫酸软骨素ABC酶突变体融合蛋白的方法，包括This paper also relates to a method for preparing chondroitin sulfate ABC enzyme mutant fusion protein, comprising
(i).提供未突变的pMAL-c2x-ChSase ABC I质粒载体；(i). Provide unmutated pMAL-c2x-ChSase ABC I plasmid vector;
(ii).以SEQ ID NO:5和SEQ ID NO:6为引物，PCR获得突变后的pMAL-c2x-ChSaseABC I突变载体。(ii). Using SEQ ID NO:5 and SEQ ID NO:6 as primers, the mutated pMAL-c2x-ChSaseABC I mutant vector was obtained by PCR.
本文涉及的硫酸软骨素ABC酶突变体的N端通过肽段连接部分与麦芽糖结合蛋白结合。The N-terminus of the chondroitin sulfate ABC enzyme mutant involved in this paper combines with maltose binding protein through a peptide linker.
纯化硫酸软骨素ABC酶突变体融合蛋白的方法Method for Purifying Chondroitin Sulfate ABC Enzyme Mutant Fusion Protein
本文还涉及纯化硫酸软骨素ABC酶突变体融合蛋白的方法，其包括：This paper also relates to a method for purifying a chondroitin sulfate ABC enzyme mutant fusion protein, comprising:
a.利用直链淀粉树脂吸附含突变的硫酸软骨素ABC酶融合蛋白的粗产物的步骤，以及a. the step of using amylose resin to adsorb the crude product containing the mutated chondroitin sulfate ABC enzyme fusion protein, and
b.利用麦芽糖洗脱纯化硫酸软骨素ABC酶突变体融合蛋白的步骤。b. The step of purifying the chondroitin sulfate ABC enzyme mutant fusion protein by elution with maltose.
本文涉及的纯化硫酸软骨素ABC酶突变体融合蛋白的方法，在所述a和b之前，该方法还包括：The method for purifying chondroitin sulfate ABC enzyme mutant fusion protein involved herein, before said a and b, the method also includes:
提供未突变的pMAL-c2x-ChSase ABC I质粒载体；Provide unmutated pMAL-c2x-ChSase ABC I plasmid vector;
PCR获得突变的pMAL-c2x-ChSase ABC I突变载体；The mutant pMAL-c2x-ChSase ABC I mutant vector was obtained by PCR;
将重组载体导入宿主细胞而得到转化体；以及Introducing the recombinant vector into the host cell to obtain a transformant; and
利用培养基培养该转化体并获得所述含硫酸软骨素ABC酶突变体融合蛋白的粗产物。The transformant was cultured with a medium to obtain a crude product of the chondroitin sulfate-containing ABCase mutant fusion protein.
其中，转化体的培养可以使用包含碳源、氮源、无机盐、各种维生素等的常规营养培养基来进行，作为碳源，可以使用例如葡萄糖、蔗糖、果糖、麦芽糖等糖类，乙醇、甲醇等醇类，柠檬酸、苹果酸、琥珀酸、马来酸、富马酸等有机酸类，废糖蜜等。作为氮源，可以单独或混合使用例如氨气、硫酸铵、氯化铵、硝酸铵、尿素等。此外，作为无机盐，可以使用例如磷酸一氢钾、磷酸二氢钾、硫酸镁等。此外，培养基中可以添加蛋白胨、肉膏、酵母提取物、玉米浆、酪蛋白氨基酸、生物素等各种维生素等营养素。Among them, the cultivation of transformants can be carried out using a conventional nutrient medium containing carbon sources, nitrogen sources, inorganic salts, various vitamins, etc., as carbon sources, sugars such as glucose, sucrose, fructose, maltose, ethanol, Alcohols such as methanol, organic acids such as citric acid, malic acid, succinic acid, maleic acid, fumaric acid, waste molasses, etc. As the nitrogen source, for example, ammonia gas, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like can be used alone or in combination. Moreover, as an inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. can be used, for example. In addition, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acids, and biotin can be added to the medium.
培养通常在通气搅拌、振荡等需氧条件下进行。对培养温度没有特殊限制，只要是宿主微生物能够生长繁育的温度即可，此外，对培养过程中的pH也没有特殊限制，只要是宿主微生物能够生长繁育的pH即可。培养中的pH调整可以通过添加酸或碱来进行。Cultivation is usually carried out under aerobic conditions such as aeration, stirring and shaking. There is no particular limitation on the culture temperature, as long as it is a temperature at which the host microorganisms can grow and reproduce. In addition, there is no special restriction on the pH during the cultivation process, as long as it is a pH at which the host microorganisms can grow and reproduce. pH adjustment during culture can be performed by adding acid or alkali.
根据本文一个优选的实施方案，对上述的转化体培养是在诱导的条件下进行的培养，由此表达得到硫酸软骨素ABC酶突变体。诱导培养的条件是：0.5mM的IPTG。在一个进一步优选的实施方案中，上述诱导培养条件是：10-42℃诱导培养15-28小时。在一个更优选的方面，上述诱导培养条件是：16℃诱导培养20小时。According to a preferred embodiment herein, the above-mentioned transformants are cultured under inducing conditions, thereby expressing the chondroitin sulfate ABC enzyme mutant. The condition of induction culture is: 0.5mM IPTG. In a further preferred embodiment, the above induction culture conditions are: induction culture at 10-42°C for 15-28 hours. In a more preferred aspect, the above induction culture conditions are: induction culture at 16°C for 20 hours.
根据本文一个优选的实施方案，进行上述诱导培养的培养基的溶剂是水，溶质是10g/L NaCl，5g/L酵母提取物，10g/L蛋白胨和100μg/L氨苄青霉素。According to a preferred embodiment herein, the solvent of the medium for the above induction culture is water, the solute is 10g/L NaCl, 5g/L yeast extract, 10g/L peptone and 100μg/L ampicillin.
对于制备含硫酸软骨素ABC酶突变体融合蛋白的粗产物的方法，没有特别地限制，可以使用各种的方法。通常，可以如下制备：利用上述的适当的培养基培养的转化体，通过使用超声波、压榨、渗透压冲击等物理方法，或者利用了表面活性剂等化学方法，或者酶处理等破碎或可溶化，以及离心分离或过滤等操作，从而得到含硫酸软骨素ABC酶突变体融合蛋白的粗产物。The method for preparing the crude product of the chondroitin sulfate-containing ABC enzyme mutant fusion protein is not particularly limited, and various methods can be used. Usually, it can be prepared by crushing or dissolving the transformant cultured in the above-mentioned appropriate medium by physical methods such as ultrasonication, pressing, osmotic shock, or chemical methods such as surfactants, or enzyme treatment, etc., And operations such as centrifugation or filtration, so as to obtain the crude product of the fusion protein containing chondroitin sulfate ABC enzyme mutant.
根据本文一个优选的实施方案，将上述培养的转化体培养物菌体采用超声波、压榨、渗透压冲击等物理方法进行破碎而得到含硫酸软骨素ABC酶突变体融合蛋白的粗产物。According to a preferred embodiment herein, the above-mentioned cultivated transformant culture cells are crushed by physical methods such as ultrasonic waves, pressing, osmotic pressure shock, etc., to obtain a crude product containing chondroitin sulfate ABC enzyme mutant fusion protein.
根据本文一个优选的实施方案，采用超声破碎获得含突变的硫酸软骨素ABC酶融合蛋白的粗产物。According to a preferred embodiment herein, the crude product containing the mutated chondroitin sulfate ABC enzyme fusion protein is obtained by ultrasonication.
根据本文一个优选的实施方案，将是上述含硫酸软骨素ABC酶突变体融合蛋白的粗产物通过直链淀粉树脂(amylose树脂)实现一步亲和分离。直链淀粉树脂是任何本领域普通技术人员能够可以根据常规选择而确定的，可以使用可以商购的直链淀粉树脂，例如MBP Trap HP(商品号28-9187-78，GE Healthcare。在一个优选的实施方案中，通过直链淀粉树脂的上清液的流速为1mL/min。在一个优选的实施方案中，用于洗脱的麦芽糖的浓度为10mM。在一个优选的实施方案中，用于洗脱的麦芽糖的流速为1mL/min。在一个优选的实施方案中，该直链淀粉树脂是经过预平衡的。According to a preferred embodiment herein, the crude product of the chondroitin sulfate-containing ABC enzyme mutant fusion protein is subjected to one-step affinity separation through amylose resin. Amylose resin is any person of ordinary skill in the art can be determined according to routine selection, can use commercially available amylose resin, such as MBP Trap HP (product number 28-9187-78, GE Healthcare. In a preferred In a preferred embodiment, the flow rate of the supernatant by the amylose resin is 1mL/min.In a preferred embodiment, the concentration of maltose used for elution is 10mM.In a preferred embodiment, for The flow rate of eluted maltose is 1 mL/min. In a preferred embodiment, the amylose resin is pre-equilibrated.
实施例Example
下面结合具体实施例对本文作进一步说明，但本文并不限于以下实施例。This paper will be further described below in conjunction with specific examples, but this paper is not limited to the following examples.
下述实施例中，如无特殊说明，均为常规方法。所述引物合成及测序工作均由华大基因完成，Q5TM High-Fidelity 2×Master Mix和所有的Dpn I购自New England Biolabs公司；所有感受态细胞(如：DH5α和BL21(DE3))购自北京博迈德生物技术有限公司)；硫酸软骨素ABC酶酶活测定底物硫酸软骨素A和B购自南京奥多福尼生物科技有限公司，其它的化学药品均为一般分析纯试剂，购自北京化工厂。In the following examples, unless otherwise specified, all are conventional methods. The primer synthesis and sequencing work were completed by BGI, Q5TM High-Fidelity 2×Master Mix and all Dpn I were purchased from New England Biolabs; all competent cells (such as: DH5α and BL21(DE3)) were purchased from From Beijing Bomaide Biotechnology Co., Ltd.); Chondroitin Sulfate ABC Enzyme Activity Assay Substrates Chondroitin Sulfate A and B were purchased from Nanjing Audofoni Biotechnology Co., Ltd. Other chemicals were general analytical reagents. purchased from Beijing Chemical Plant.
实施例1、硫酸软骨素ABC酶突变体融合蛋白(MBP-ChSase ABC)的表达Embodiment 1, expression of chondroitin sulfate ABC enzyme mutant fusion protein (MBP-ChSase ABC)
一、突变的pMAL-c2x-ChSase ABC I突变载体的获得1. Obtaining the mutant pMAL-c2x-ChSase ABC I mutant vector
突变载体pMAL-c2x-ChSase ABC I获得的具体过程如下：The specific process of obtaining the mutant vector pMAL-c2x-ChSase ABC I is as follows:
1、引物的设计及合成1. Design and synthesis of primers
经Genbank查询得到普通变形杆菌硫酸软骨素ABC酶的DNA序列(Tam K.W.Studyof chondroitin sulphate abc lyases and their use in combination for promotionof neurite growth.University of Hong Kong,2010.)，突变Asn771Ala所用的上下游引物分别为：The DNA sequence of Proteus vulgaris chondroitin sulphate abc lyases and their use in combination for promotion of neurite growth.University of Hong Kong,2010. was obtained through Genbank query. The upstream and downstream primers used for the mutation Asn771Ala for:
上游引物P1：5’-GCCATTACTCCAACATTAGCTACCCTTTGG-3’(SEQ ID NO:5)(带下划线的为突变碱基)，下游引物P2：5’-GCTAATGTTGGAGTAATGGCATGTTGGAATAAG-3’(SEQ ID NO:6)(带下划线的为突变碱基)。Upstream primer P1: 5'-GCCATTACTCCAACATTAGC TACCCTTTGG-3'(SEQ ID NO:5) (underlined bases are mutant bases), downstream primer P2: 5'-GC TAATGTTGGAGTAATGGCATGTTGGAATAAG-3'(SEQ ID NO:6)( Mutation bases are underlined).
2、PCR获得pMAL-c2x-ChSase ABC I突变体2. Obtain pMAL-c2x-ChSase ABC I mutant by PCR
PCR扩增的反应体系为：50ng普通变形杆菌基因组DNA模版，100pmol每种引物，响应体积的2×Master Mix；扩增程序为：98摄氏度预变性30秒，98摄氏度变性7秒，60摄氏度引物退火30秒，72摄氏度延伸5分钟，30个循环后，72摄氏度延伸10分钟结束反应。该PCR结果如图1所示，表明扩增得到了9.6kb的pMAL-c2x-ChSase ABC I，PCR得到质粒后用Dpn I，37摄氏度酶切消化为未突变的pMAL-c2x-ChSase ABC I模板，消化后转化到DH5α中，并进行测序,测序结果表明，已成功将Asn771突变成Ala。图1中，泳道M为分子量marker(条带大小依次为10kb、8kb、6kb、5kb、4kb、3kb、2kb、1kb)，泳道1为扩增得到的pMAL-c2x-ChSase ABCI突变体，箭头所指处为9.6kb目标片段，表明已成功PCR得到pMAL-c2x-ChSase ABC I突变体。The reaction system for PCR amplification is: 50ng Proteus vulgaris genomic DNA template, 100pmol of each primer, 2×Master Mix in the response volume; the amplification program is: pre-denaturation at 98°C for 30 seconds, denaturation at 98°C for 7 seconds, primer at 60°C Anneal for 30 seconds, extend at 72°C for 5 minutes, and after 30 cycles, extend for 10 minutes at 72°C to end the reaction. The PCR result is shown in Figure 1, indicating that the 9.6kb pMAL-c2x-ChSase ABC I was amplified, and after the plasmid was obtained by PCR, it was digested with Dpn I and digested at 37 degrees Celsius to an unmutated pMAL-c2x-ChSase ABC I template , transformed into DH5α after digestion, and sequenced, the sequencing results showed that Asn771 had been successfully mutated into Ala. In Figure 1, lane M is the molecular weight marker (band sizes are 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb), lane 1 is the amplified pMAL-c2x-ChSase ABCI mutant, indicated by the arrow Points to the 9.6kb target fragment, indicating that the pMAL-c2x-ChSase ABC I mutant has been successfully obtained by PCR.
二、硫酸软骨素ABC酶突变的融合蛋白MBP-ChSase ABC的表达2. Expression of fusion protein MBP-ChSase ABC with chondroitin sulfate ABC enzyme mutation
提取步骤一中含有突变的pMAL-c2x-ChSase ABC的大肠杆菌DH5α中的质粒，按照常规方法转化大肠菌BL21(DE3)。经过氨苄青霉素筛选和利用步骤一中步骤1提供的引物进行菌落PCR鉴定，得到含有突变的pMAL-c2x-ChSase ABC的大肠杆菌BL21(DE3)，即BL21(DE3)/pMAL-ChSase ABC作为表达突变的MBP-ChSase ABC I的工程菌。Extract the plasmid in Escherichia coli DH5α containing the mutated pMAL-c2x-ChSase ABC in step 1, and transform Escherichia coli BL21(DE3) according to conventional methods. After ampicillin screening and colony PCR identification using the primers provided in step 1 in step 1, Escherichia coli BL21(DE3) containing mutated pMAL-c2x-ChSase ABC was obtained, that is, BL21(DE3)/pMAL-ChSase ABC was used as the expression mutant The engineering bacteria of MBP-ChSase ABC I.
以质粒pMAL-c2x转化大肠杆菌BL21(DE3)，得到空载体对照BL21(DE3)/pMAL-c2x。Escherichia coli BL21(DE3) was transformed with plasmid pMAL-c2x to obtain the empty vector control BL21(DE3)/pMAL-c2x.
将空载体对照和工程菌分别在含氨苄青霉素抗性的LB培养基(NaCl10g/L，酵母提取物为5g/L，蛋白胨10g/L，含100μg/L氨苄青霉素)37摄氏度培养2.5小时后，加入终浓度为0.5mM IPTG 16摄氏度诱导20小时。6000rpm，6分钟离心收集菌体并用20mM Tris-HCl(pH7.4)洗涤两次，然后重悬菌体。将重悬液进行超声破碎(输出功率为300W，每次超声5秒和间歇6秒，处理总时间为15分钟)，6000rpm，6分钟离心，超声破碎后离心所得的上清液即为含硫酸软骨素ABC酶突变体的粗产物。The empty vector control and engineered bacteria were cultured at 37 degrees Celsius for 2.5 hours in ampicillin-resistant LB medium (NaCl 10g/L, yeast extract 5g/L, peptone 10g/L, containing 100μg/L ampicillin), Add a final concentration of 0.5mM IPTG to induce at 16°C for 20 hours. The cells were collected by centrifugation at 6000 rpm for 6 minutes, washed twice with 20 mM Tris-HCl (pH 7.4), and then the cells were resuspended. The resuspension is subjected to ultrasonic crushing (output power is 300W, each ultrasonic 5 seconds and intermittent 6 seconds, the total processing time is 15 minutes), 6000rpm, centrifuged for 6 minutes, and the supernatant obtained after ultrasonic crushing is the sulfuric acid-containing solution. Crude product of chondroitin ABC enzyme mutant.
实施例2、通过直链淀粉柱纯化硫酸软骨素ABC酶突变体融合蛋白Embodiment 2, purifying chondroitin sulfate ABC enzyme mutant fusion protein by amylose column
本实施例利用融合伴侣(fusion partner)麦芽糖结合蛋白MBP能够与直链淀粉亲和吸附实现一步分离。具体的亲和分离步骤如下：将终浓度为0.5mM IPTG诱导表达20小时的菌体100mL，6000rpm离心6分钟；同时设未诱导表达的菌体对照。接着按以下两个方案分别操作：In this example, the fusion partner (fusion partner) maltose-binding protein MBP can be used to achieve one-step separation with amylose affinity adsorption. The specific steps of affinity separation are as follows: 100 mL of bacterium whose final concentration was 0.5 mM IPTG induced expression for 20 hours was centrifuged at 6000 rpm for 6 minutes; at the same time, a bacterium with no induced expression was set as a control. Then proceed with the following two options:
用柱平衡液Column buffer(20mM Tris-HCl，200mM NaCl，pH7.4)洗涤两次，重悬在30mL Column buffer中，进行超声破碎(输出功率为300W，每次超声5秒和间歇6秒，处理总时间为15分钟)。Wash twice with column equilibration solution Column buffer (20mM Tris-HCl, 200mM NaCl, pH7.4), resuspend in 30mL Column buffer, and perform sonication (output power is 300W, each sonication is 5 seconds and interval is 6 seconds, The total processing time was 15 minutes).
离心后上清液以1mL/min通过1mL预平衡的直链淀粉亲和分离柱，通过10mM 1mL/min麦芽糖洗脱并收集。After centrifugation, the supernatant was passed through a 1 mL pre-equilibrated amylose affinity separation column at 1 mL/min, eluted and collected by 10 mM 1 mL/min maltose.
目标蛋白经过直链淀粉(amylose)树脂吸附后，用10mM麦芽糖在2个柱体积下能够将目标蛋白洗脱。结果如图2所示，表明经过直链淀粉树脂一步纯化后目标蛋白可占90％以上。图2中，泳道M为marker(由上至下分子量依次均为250kDa、150kDa、100kDa、70kDa)，泳道1为空白对照、泳道2为E.coli BL21(DE3)/pMAL-ChSase ABC突变体的可溶蛋白，箭头所指处为MBP-ChSase ABC I突变体融合蛋白(140kDa)。After the target protein is adsorbed by amylose resin, the target protein can be eluted with 10 mM maltose in 2 column volumes. The results are shown in Figure 2, indicating that the target protein can account for more than 90% after one-step purification with amylose resin. In Figure 2, lane M is the marker (molecular weights are 250kDa, 150kDa, 100kDa, 70kDa from top to bottom), lane 1 is the blank control, lane 2 is the E.coli BL21(DE3)/pMAL-ChSase ABC mutant Soluble protein, the MBP-ChSase ABC I mutant fusion protein (140kDa) indicated by the arrow.
酶活力(单位为IU/mL)的检测采用232nm的光吸收法，1IU的酶活定义为37摄氏度每分钟产生1μmoL不饱和二糖的反应效力。将硫酸软骨素A或B配制成1mg/mL的底物溶液，取底物溶液2mL，加入直链淀粉柱纯化所得的50μL的纯化产物，最终的反应体积为2.05mL，测单位时间内在232nm的吸光度变化△A232。消光系数ε＝3800M-1。比酶活(单位为IU/mg蛋白)的定义为酶活力与蛋白浓度(单位为mg/L)的比值。蛋白浓度监测采用常规的Bradford法。Enzyme activity (unit: IU/mL) was detected by 232nm light absorption method, and 1 IU of enzyme activity was defined as the reaction efficiency of producing 1 μmoL of unsaturated disaccharide per minute at 37 degrees Celsius. Prepare chondroitin sulfate A or B into a substrate solution of 1 mg/mL, take 2 mL of the substrate solution, add 50 μL of the purified product obtained by amylose column purification, and the final reaction volume is 2.05 mL, measured at 232 nm per unit time Absorbance change ΔA232 . Extinction coefficient ε=3800M-1 . Specific enzyme activity (in IU/mg protein) is defined as the ratio of enzyme activity to protein concentration (in mg/L). Protein concentration was monitored using the conventional Bradford method.
综上，通过定点突变使MBP-ChSase ABC I的酶活提高了：以硫酸软骨素A为底物，Asn771Ala的酶活和比酶活分别为24.96IU/mL enzyme和31.32IU/mg protein，以硫酸软骨素B为底物，酶活和比酶活分别为21.58IU/mL enzyme和27.07IU/mg。未突变的MBP-ChSaseABC I，以硫酸软骨素A为底物，酶活和比酶活分别为20.18IU/mL enzyme和22.52IU/mgprotein，以硫酸软骨素B为底物，酶活和比酶活分别为13.08IU/mL enzyme和14.60IU/mgprotein。In summary, the enzyme activity of MBP-ChSase ABC I was improved by site-directed mutation: with chondroitin sulfate A as the substrate, the enzyme activity and specific activity of Asn771Ala were 24.96IU/mL enzyme and 31.32IU/mg protein, respectively. Chondroitin sulfate B was used as the substrate, and the enzyme activity and specific enzyme activity were 21.58IU/mL enzyme and 27.07IU/mg, respectively. Unmutated MBP-ChSaseABC I, with chondroitin sulfate A as the substrate, the enzyme activity and specific enzyme activity are 20.18IU/mL enzyme and 22.52IU/mgprotein, respectively, with chondroitin sulfate B as the substrate, the enzyme activity and specific enzyme activity The activities were 13.08IU/mL enzyme and 14.60IU/mgprotein respectively.
本发明还可通过亲和分离实现了该融合蛋白的一步纯化。与现有技术中使用的鱼精蛋白沉淀(参考ANA LYDIA TKALEC,DOMINIQUE FINK等人，Isolation and Expressionin Escherichia coli of cslA and cslB,Genes Coding for the ChondroitinSulfate-Degrading Enzymes Chondroitinase AC and Chondroitinase B,Respectively,from Flavobacterium heparinum.APPL.ENVIRON.MICROBIOL.2000,66(1):29-35)和一步柱纯化法相比(参见表1)，可以大大缩短纯化需要的时间，并且纯化所用的设备明显减少，降低了纯化硫酸软骨素酶的生产成本。利用直链淀粉树脂的纯化仅需要进行2个小时左右，即可获得能够用于工业生产的硫酸软骨素ABC酶突变体融合蛋白。纯度达到可以应用的95％以上。The present invention can also realize one-step purification of the fusion protein through affinity separation. Precipitate with protamine used in the prior art (with reference to ANA LYDIA TKALEC, DOMINIQUE FINK et al., Isolation and Expressionin Escherichia coli of cslA and cslB, Genes Coding for the ChondroitinSulfate-Degrading Enzymes Chondroitinase AC and Chondroitinase B, Respectively, from Flavobacterium heparinum.APPL.ENVIRON.MICROBIOL.2000,66(1):29-35) compared with one-step column purification (see Table 1), can greatly shorten the time required for purification, and the equipment used for purification is significantly reduced, reducing the purification time. Production cost of chondroitin sulfate enzyme. The purification by using the amylose resin only takes about 2 hours to obtain the chondroitin sulfate ABC enzyme mutant fusion protein that can be used in industrial production. The purity reaches more than 95% that can be used.
表1 鱼精蛋白沉淀和本发明一步柱纯化法相比Table 1 Protamine precipitation compared with the one-step column purification method of the present invention
经过菌株优化后，大肠杆菌BL21(DE3)(pMAL-c2x-ChSase ABC I)突变株，在37摄氏度培养2.5小时后，加入0.5mM IPTG，诱导温度16摄氏度，生产的硫酸软骨素ABC酶突变体酶活可达7488IU/L发酵液(以硫酸软骨素A为底物)，表达量可达620mg/L发酵液，比酶活可达31.32IU/mg(以硫酸软骨素A为底物)。After strain optimization, Escherichia coli BL21(DE3) (pMAL-c2x-ChSase ABC I) mutant strain was cultured at 37 degrees Celsius for 2.5 hours, then added 0.5mM IPTG, and the induction temperature was 16 degrees Celsius, and the chondroitin sulfate ABC enzyme mutant produced The enzyme activity can reach 7488IU/L fermentation broth (with chondroitin sulfate A as substrate), the expression level can reach 620mg/L fermentation broth, and the specific enzyme activity can reach 31.32IU/mg (with chondroitin sulfate A as substrate).
本发明将在硫酸软骨素ABC酶的生产中发挥重要作用，并且通过本发明生产的硫酸软骨素ABC酶突变体融合蛋白可以与硫酸软骨素ABC酶同样地应用于各领域，且酶活性更高。The present invention will play an important role in the production of chondroitin sulfate ABC enzyme, and the chondroitin sulfate ABC enzyme mutant fusion protein produced by the present invention can be applied to various fields in the same way as chondroitin sulfate ABC enzyme, and the enzyme activity is higher .
以上全面描述了本发明的优选实施例，但是可对它们进行各种替代和修改。因此，不应参照以上描述来决定本发明的范围，而是应参照所附权利要求书及其全部等同物来决定本发明的范围。任何特征，(不论是否为优选)均可与任何其他特征(不论是否为优选)相结合。本发明的权利要求书不应被理解为具有方法+功能的限制，除非在某一权利要求中通过术语“…的方法”明确地列举出此类限制。The preferred embodiments of the present invention have been fully described above, but various substitutions and modifications can be made thereto. The scope of the invention should, therefore, be determined not with reference to the foregoing description, but should be determined with reference to the appended claims and their full equivalents. Any feature, whether preferred or not, may be combined with any other feature, whether preferred or not. The claims of the present invention should not be read as having a means+function limitation unless such limitation is expressly recited in a certain claim by the term "means of...".
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| CN201510125696.5ACN104711245B (en) | 2015-03-20 | 2015-03-20 | A kind of chondroitin sulfate A (CSA) BC enzyme mutants fusion protein and application |
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| CN201510125696.5ACN104711245B (en) | 2015-03-20 | 2015-03-20 | A kind of chondroitin sulfate A (CSA) BC enzyme mutants fusion protein and application |
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