WO 94/25144 PCT/AU94/00233 -1- ELECTROPHORETIC RESOLUTION OF CHARGED MOLECULES The present invention relates generally to the electrophoretic, and more particularly isotachophoretic resolution of charged molecules in a sample to thereby facilitate the isolation and purification of same. The present invention also relates to an isotachophoretic apparatus for the resolution of charged molecules. The method and apparatus of the present invention are particularly useful for laboratory aid large scale preparative electrophoresis of charged molecules such as proteinaceous molecules.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be muderstood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Electrophoretic separation of molecules such as proteins and nucleic acids has been widely used in experimental biology. However, notwithstanding that electrophoresis is capable of achieving high resolution of molecules, preparative electrophoresis has not achieved wide commercial usage. One problem has been the great difficulty in scaling up the electrophoretic process to deal with commercially significant quantities. A further difficulty is the tendency for particular recombinant molecules to form insoluble complexes during their production (often referred to as "inclusion bodies"), making purification of these molecules difficult, unpredictable, uneconomic and, due to the reagents involved in solubilisation, unacceptable for regulatory manufacturing requirements, especially for the production of clinical grade material, In fact, the production and purification of sufficient amounts of clinical grade material even for trial purposes frequently poses a major and often seemingly insurmountable hurdle to pharmaceutical and vaccine development. There is a need, therefore, to develop a high resolution preparative electrophoretic technique which WO 94/25144 PCT/AU94/00233 -2is capable of operating on a commercial scale and which is capable of purifying proteins and other molecules from biological or synthetic material and especially from insoluble complexes. The development of such an electrophoretic technique would also have significant advantages for routine laboratory practices as well as during pilot or scaling up purification runs prior to full scale commercial operation.
In work leading up to the present invention, the inventors sought to develop such a preparative electrophoretic technique based on isu achophoresis. In isotachophoresis, separation of charged molecules is carried out in a discontinuous buffer in which the molecules to be separated migrate between two electrolytes referred to herein as the "leading ions" and the "trailing ions", respectively, where the leading ions comprise ions of net mobility higher than those of the molecules while the trailing ions comprise ions of net mobility lower than the molecules. The molecules to be separated are resolved according to decreasing mobility from the leading to the trailing electrolyte.
Isotachophoresis has been principally used as the stacking process in Laemmli sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) (Laemmli, UK, Nature, 227: 680, 1970). By this process, the molecules, generally proteins, are concentrated and deposited as a "stack" on the surface of a resolving or separating gel. Furthermore, sample molecules are generally separated by insertion of "spacers" with mobilities intermediary of those of the molecules to be separated. Such "spacers" are also known as "carrier ampholytes", for example, "Ampholine" (Trademark, LKB Produkter AB). In accordance with the present invention, it has been surprisingly discovered that by inter alia continuing the isotachophoresis, the molecules are resolved and separated to an extent that they may be eluted off with a high degree of purity from other components in the mixture. In particular, the isotachophoresis system is modified, in accordance with the present invention, to enable its incorporation into a column chromatographic system. Furthermore, the technique obviates the need for ampholytic spacers which are expensive, complex and generally unacceptable for use in purifying clinical grade molecules. The technique of the present invention is suitable for both small scale and large scale preparation I WO 94/25144 WO 942S144FCTIAtJ94/00233 -3of molecules in highly purified form and is particularly beneficial for the purification of proteins, polypeptides and peptides of natural or synthetic recombinant) origin.
Accordingly, one aspect of the present invention contemplates a method for isolating, separating, purifying or otherwise resolving charged molecules in a sample, said method comprising introducing said sample to a matrix hydrated with a first buffer, applying a DC current to said hydrated matrix between a cathode and an anode to cause said charged molecules in said sample to migrate to said anode in the presence of a second buffer, wherein said first buffer comprises an ion of greater ionic mobility than the charged molecules to be isolated and said second buffer comprises an ion with lower ionic mobility than the charged molecules to be isolated, said migration being for a time and under conditions sufficient to isolate said charged molecules in the matrix and then subjecting said isolated charged molecules to eluting means.
The present invention also extends to an apparatus for isolating, separating, purifying or otherwise resolving charged molecules in accordance with the method of the first aspect of the invention.
H-ereinafter, reference is made to "isolating' charged molecules or to "isolated" charged molecules. The term "isolated" or similar terms such as "isolating" are used in their broadest sense and include separating, purifying or otherwise resolving charged molecules or separated, purified or otherwise resolved charged molecules.
The molecules to be isolated may be naturally charged or may be rendered charged in a particular buffer or aqueous environment. Alternatively, the molecules are rendered charged by the binding of small charged ligands including dodecyl sulphate.
The present invention applies to all suitably charged molecules such as but not limited to proteins (including peptides and polypeptides and their parts, fragments, derivatives and/or analogues) in native, recombinant and/or synthetic form including in the folded or unfolded state, nucleic acid molecules, charged oligosaccharides and WO 94/25144 PCT/AU94/00233 -4glycoproteins (including glycopeptides and glycopolypeptides and which may be freely soluble or membrane bound molecules). The molecules contemplated herein, therefore, are generally macromolecules.
The sample may be a heterogenous or homogenous, simple or complex composition of matter and may contain a single species of charged molecule, or molecule to be charged, or may contain more than one multiple) species of which one or more of said single species may be required to be isolated or resolved. The species to be isolated may be the major or dominant species in a sample as determined by percentage relative mass or activity) or may be a minor or even trace species. The present invention is particularly applicable to isolating recombinant products and, hence, may contain prokaryotic or eukaryotic cell components in addition to the recombinant product(s) to be resolved. Alternatively, the sample maybe supernatant fluid or fermentation fluid. The recombinant products referred to herein include recombinant proteins, polypeptides of prokaryotic, eukaryotic or viral origin. The charged molecules may also be isolated from insoluble complexes.
The first and second buffers generally constitute a discontinuous buffer system in which preferably the first buffer comprises Tris/Tris HC1 or Tris/Tris thioglycolate and the second buffer comprises glycine or Tris/Tris glycinate. Both buffers may also contain sodium dodecylsulphate (SDS). One skilled in the art will immediately appreciate that different types of buffers can be used provided that a discontinuous buffer system is achieved. Additionally, a multiple discontinuous buffer system may be employed. For example, a three buffer system with two discontinuities may be used comprising a first buffer of Tris HC a second buffer of thioglycolate and a third buffer of glycine. In this system, the first buffer is used for casting the gel and the second buffer is used in the upper electrode compartment. The gel is subjected to electrophoresis prior to loading the sample (pre-electrophoresis) which does not occur imtil the discontinuity reaches the bottom of the gel. The power is then turned off and the gel stored overnight to allow for the thioglycolate to react with residual acrylamride Such a method of pre-treatment improves the suitability of the gel for preparation of therapeutically important molecules.
WO 94/25144 PCT/AU94/06oJ The matrix preferably comprises polyacrylamide but may also comprise other gels or granular supports Sephadex). Where a polyacrylamide gel support is used, it is preferably prepared at a concentration of 4-10% w/v of polyacrylamide in a suitable first buffer. The amount of polyacrylamide used will depend on the molecules to be isolated. Generally, the amount of polyacrylamide must be sufficient to be self supporting although the acrylamide column may be supported by hydrostatic pressure at all times, allowing a lower acrylamide concentration to be used than would otherwise be possible,, The advantage of employing denaturing electrophoretic techniques where charged molecules such as proteins are denatured by binding to SDS in a purification protocol is the universality of the results, unlike chromatographic systems where the individual chemistry of each charged molecule makes applicability haphazard.
Isotachophoretic separation under these conditions occurs in the order of the molecular weights of the components and is predictable on that basis. Other denaturing conditions such as urea may be employed, however, the ordinary skilled artisan will be avxre that certain modifications to the isotachophoretic separation parameters may be required. All such modifications to the isotachophoretic procedures and apparatus are encompassed by the present invention, With isotachophoresis the charged molecules are trapped between leading and trailing buffer ion discontinuities, With glycine as the trailing ion, increasing the buffer pH accelerates the ionic mobility of glycine, allowing it to exceed that of high molecular weight charged molecules, excluding them from the stack. Additionally, increasing the matrix acrylamide) concentration gives the gel molecular sieving properties, retarding the mobility of high molecular weight charged molecules and causing them to be similaly excluded from the stack. Choosing a leading ion of slower mobility will cause lower molecular weight charged molecules to be precluded from the stack, whereas choosing a trailing ion of faster mobility will have the same effect as increasing buffer pH.
WO 9412514-4 PCT/AU94/00233 -6- All these physical properties allow further optimisation of the isotachophoresis process beyond the standard buffers exemplified herein. Given a charged molecule's molecular weight, a computer program based on Table 1 and published ionic mobilities of buffer components can predict optimal matrix acrylamide) concentrations, buffer pHs, and leading and trailing buffer ion species to exclude undesired higher or lower molecular weight proteins from the stack.
Buffer ion concentration can also be used in a number of ways. The height of any charged molecule band within the stack can be proportionally increased or decreased by altering the buffer ion concentrations; halving the buffer concentrations will double the charged molecule band height by halving its concentration. In this way, for example, protein solubility problems may be overcome by reducing buffer concentrations, and the region of band interface to total band height minimised for maximum purification.
Isotachophoresis allows proteins to be loaded in the presence of SDS and pmercaptoethanol, overcoming solubility problems. Excess reagents do not contaminate eluted product as they in turn are electrophoretically separated from the protein. Endotoxins from the host bacteria are also electrophoretically separated and do not contaminate the product. For RAP2 (Example no E. colimaterial is detectable in the product by immunoblot with polyclonal rabbit antisera against E.
coli In accordance with the present invention, efficient scavenging of acrylamide monomers is also possible by pre-electrophoresing the gel with thioglycolate buffer, preventing product modification. The half-lives involved in thioglycolate scavenging illustrates that the usual methods for scavenging SDS-PAGE gels are inadequate.
Pre-electrophoresis is often done for the separating gel of Laemmli SDS-PAGE, for periods varying from 0.5 to 14 hours, using 0.1 mM thioglycolic acid in the separating gel buffer (Moos, M. et al. J. Bio. Chem. 263. 6005-6008, 1988). However, at 30 0
C,
the half-life of acrylamide for a far more concentrated 92 mM thioglycolate buffer WO 94/25144 PCT/AU94/00233 -7at pH 7.5 is 45 minutes. The inventors have determined that as much as 1% of acrylamide remains unpolym ',ed in a Laemmli gel, which is uncharged and will remain in the gel despite .eiectrophoresing unless scavenged. Further, loaded proteins in Laemmli geli J encounter this monomer acrylamide at pH 8.8 where the reaction rate is likely to be much faster as a number of reporters have demonstrated that the reactivity of primary amines with acrylamide monomers increases with pH. Thus, pre-electrophoresis for sufficient time such as for 14 hours or more, and preferably in a high pH buffer, will achieve particularly superior results for the isolation of certain charged molecules.
The application of the DC current is by any standard means and is most preferably supplied by way of constant current.
As the charged molecules migrate along the solid matrix, they are resolved and concentrated. Conveniently, the molecules of interest are eluted by the bolus of molecules electrophoreting out the end of the matrix and being collected by any suitable means. Generally, a fraction collector is used to collect the eluted molecules via a capillary tube. In an alternative embodiment, the matrix may be divided during the migration to elute the molecules at a given time or to retrieve a suitable portion of the matrix containing the charged molecules for purification and/or further analysis. In another embodiment, following elution of the bolus, air or other suitable gas is used to create a void separating the isolated molecules.
According to this latter embodiment there is provided an apparatus for introducing a gaseous space into a column of liquid, said apparatus comprising means for introducing air or other gaseous substance into said column of liquid. More particularly, the apparatus comprises a first passage connecting an entry port and an exit port for the passage of liquid and a second passage interconnecting said first passage wherein air or other gaseous material is capable of flowing from said second passage to said first passage such that in use, voids of air or other gaseous material are introduced into a substantially continuous column of liquid. In a particularly preferred embodiment, the first and second passages define a junction wherein WO 94/25144 PCT/AU94/00233 -8the angle at the junction between the exit port and the entry port of the first passage is from about 46 0 to about 120 0 C and more preferably from about 70 0 to 100 0
C.
In a most preferred embodiment, the exit port of the first passage is vertical, substantially vertical or approximately 0 0 to 30 0 5 0 or 10 0 or 15 0) from the vertical and the entry port of the first passage is horizontal, substantially horizontal or approximately 0 to 30 below the horizontal 5 10 15 or 20 The apparatus of this aspect of the present invention is referred to herein as a "bubbler".
This terminology is not intended to imply any limitation as to the mode of action of the apparatus.
In a preferred aspect of the invention, the greater density of the eluting bolus relative the surrounding electrode buffer is used to direct the emerging charged molecules into a collecting tube, the position of which may be altered during the run to allow for shrinkage and expansion of the matrix associated with the passage of large concentrations of the molecules of interest. This obviates the need for membranes, frits or other such devices for separating the eluting bolus from the electrode chamber. Alternatively, other fractionation techniques can be used, such as an electrically conductive membrane, the rapid flow of buffer across the matrix surface, a termination of the electric current prior to the bolus or partitioning of the matrix and elution of the molecule of interest from an isolated section of matrix.
The present method is applicable for small and large scale preparative electrophoresis. Amounts of from at least about 0.1 mg to about 1000 mg or from at least about 0.5 mg to about 1000 mg or from at least about 10 mg to about 800 mg or from at least about 100 mg to about 500 mg of, for example, protein, can be subject to the isolation procedure. However, greater or lesser amounts can be used depending on the type of molecules to be resolved.
The hydrated matrix may occupy any suitable apparatus although generally major stresses occur during the electrophoretic process which can result in shattering of the matrix as the resolving molecule migrates.
WO 94125144 PiCT/AU94/00233 -9- Accordingly, the matrix generally needs to be suitably supported to reduce the risk of shattering. Another consideration is the type of matrix. For example, acrylamide has difficulty polymerising when in contact with certain material, such as oxygen permeable material. However, the best support up to the present time is glass although other supports can be used. Electrophoresis may occur in any direction but conveniently it is in a substantially vertical direction and in particular in a downward substantially vertical direction during loading and in a direction approximately 0-30 but preferably about 0-15 5 or 10 to the horizontal during elution of the bolus. By "substantially vertical" is meant to include at least about up to 30 from vertical 5 or 10 or 15 Furthermore, the support is conveniently a glass tube of varying size and diameter. The glass tube may be continuous throughout its length or may be a series of multiple glass tubes wherein each tube is releasably connected for rapid retrieval of the matrix and the resolving molecules. In an alternative embodiment, the electrophoretic tube is substantially horizontal with the respective electrode housing devices mounted separately on either end of the tube at an appropriate angle.
Where desired, the progress of isolation, and purification during electrophoresis may be monitored by any convenient means such as using instrumentation to, for example, measure the voltage at points down the matrix via suitably embedded electrodes in the walls of the gel tube or by the refractive index changes which separate the various zones formed in the isotachophoresis, or by monitoring eluate by UV adsorption or any other method of determining the presence of protein.
In a particularly preferred embodiment, there is contemplated a method for isolating, separating, purifying or otherwise resolving charged molecules in a sample, said method comprising introducing said sample to a matrix hydrated with a first buffer, applying a DC current to said hydrated matrix between a cathode and an anode to cause said charged molecules in said sample to migrate to said anode in the presence of a second buffer, wherein said first buffer comprises an ion of greater mobility than the charged molecules to be isolated and said second buffer comprises an ion with lower ionic mobility than the charged molecules to be isolated, said migration being
L_
WO 94/25144 PCT/AU94/00233 for a time and under conditions sufficient to generate a greater density of eluting bolus relative the surrounding buffer to thereby direct the emerging charged molecules in the eluate into a collecting tube.
Preferably, the collecting tube is capable of being altered during the migration of the charged molecules through the matrix to allow for shrinkage and expansion of the matrix associated with the passage of large concentrations of molecules.
Preferably, the collecting tube is a capillary tube.
Another aspect of the present invention is directed to an apparatus for isolating charged molecules in a sample said apparatus comprising a matrix hydrated with a first buffer in a container such as to provide a sample loading end and an isolated molecule eluting end, said matrix having end surfaces in constant contact with a second buffer, wherein said first buffer comprises an ion of greater ionic mobility than the charged molecules to be isolated and said second buffer comprises an ion with lesser ionic mobility than the charged molecules to be isolated, wherein said container comprises electrode containing chambers at the sample loading end and isolated molecule end such that on application of a DC current, negatively charged molecules migrate to said isolated molecule eluting end.
The first and second buffers are as hereinbefore defined and preferably comprise a discontinuous buffer system. The apparatus may also comprise a multi-discontinuous buffer system. The container support is as hereinbefore described and is preferably a glass tube. For the production of clinical grade material, the apparatus is preferably constructed primarily of glass, with integral electrode compartments and with all buffer connections and vents via autoclavable tubing and filters to enable the electrophoresis to be performed under sterile conditions.
In a preferred embodiment, the apparatus also comprises a second apparatus as hereinbefore described for introducing air or other gaseous material into a column of eluted or eluting liquid to thereby facilitate the isolation of the charged molecules.
WO 94/25144 PCT/AU94/00233 11 The apparatus of the present invention may also further comprise means of elution based on the density of the eluting compound or movability of a collection tube or both. The apparatus may also be modified such that it can run under sterile conditions.
The present invention further extends to the apparatus with or without air introducing apparatus and/or with or without electrode containing chambers in kit form or in partially assembled form or in a form packaged for sale. Generally, the kit form of the apparatus or apparatuses will include instructions for use. The apparatus may not, therefore, contain the second buffer and/or the hydrated matrix.
The apparatus may further comprise a power supply to permit the application of a DC current along the hydrated matrix.
Yet another aspect of the present invention is directed to molecules and in particular charged molecules purified, separated, isolated or otherwise resolved by the method and apparatus of the present invention.
The present invention further extends to native isotachophoresis. For proteins with pi below 7.5 this is a relatively simple process since these proteins will be negatively charged under standard buffer systems (pH see Figure 14. For proteins with higher pIs, cationic isotachophoresis is required. The acidic nature of the buffers requires a photopolymerised gel, but beyond that little modification to the standard method is necessary (Figure However, the choice between cationic and anionic systems for native isotachophoresis purifications will for nirst proteins be more problematic, requiring optimisation for each protein of interest. For instance, with human serum albumin, cationic native isotachophoresis give the highest degree of purification. However, at the pHs utilised by the anionic system, the human serum albumin possesses more charges per protein than at the cationic pHs (anionic m/e 4004.3 to cationic m/e 4677.7). To carry the fixed current of 0.25 mAmps a lower density of protein molecules is therefore required under anionic conditions. Thus, despite a lower WO 94/25144 PCT/AU94/00233 -12protein load, under anionic conditions the protein stack volume was greater than twice that of the cationic system. This phenomena may be of use in cases where for a particular protein species neither native ITP system produces a markedly cleaner purification. Broadening the stack height would serve to minimise the region of protein hand interface within the stack compared to protein band volume.
Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: Figure 1 is a front elevation of one form of the isotachophoretic apparatus of the present invention.
Figure 2 is a front elevation of the elution chamber at the resolved molecule elution en'd of the apparatus in a position used during the elution phase.
Figure 3 is a photographic representation of isotachophoretic fractions following SD$PAGE obtained during the purification of topoisomerase. Lane A, molecular weight markers; Lane B, topoisomerase-GST prior o purification; Lane C, topoisomerase-GST after isotachophoretic purification.
Figure 4 is a photographic representation following SDS-PAGE of isotachophoretic fractions of U1RNP collected at one minute intervals.
Figure 5 is a photographic representation following SDS-PAGE of isotachophoretic fractions of topoisomerase-GST fusion protein collected at one minute intervals.
Figure 6 is a photographic representation following SDS-PAGE of isotachophoretic fractions of nuclear antigen La collected at one minute intervals.
Figure 7 is a photographic representation following SDS-PAGE of isotachophoretic fractions of R060 collected at one minute intervals.
WO 94/25144 PCT/AU94/00233 -13- Figure 8 is a diagrammatic representation of an isotachophoretic apparatus incorporating a bent column. The apparatus incorporates a central glass column flanked by two buffer reservoirs containing electrodes. The central column is interchangeable with tubing having a range of ID values such as but not limited to 3 to 19 mm ID. To avoid the necessity to rotate the column during the run, the central column is bent into an obtuse J-shape, the protein stack regaining focus past the curve. In this particular embodiment, the obtuse J-shape is 80 but the present invention is clearly not so limited.
Figure 9 is a photographic representation of SDS-PAGE analysis of fractions obtained from the removal of contaminants from a commercial batch of bovine serum albumin. An amount of 500 mg BSA Fraction V (50 mg/ml) was loaded onto a 5% w/v isotachophoresis column (19 mm ID x 500 mm). Samples of the starting material (1 pl of BSA Fraction V) are compared with 1 pl samples from 1 ml fractions eluted from the isotachophoresis run (fractions 46 Lo 136). The size of the standard protein markers is indicated.
Figure 10 is a photographic representation of SDS-PAGE analysis of the purification of minor contaminants in a commercial batch of bovine serum albumin. Commercial bovine serum albumin (BSA Fraction V) was first separated into fractions containing predominantly low molecular weight contaminants and pure bovine serum albumin in a 5% w/v isotachophoresis run. Fraction 51 from this first run was then reelectrophoresed on a 3 mm ID x 500 mm isotachophoresis column and 64 pl fractions collected. An aliquot of 1 pl of both the original Fraction V BSA and isotachophoresis run 1 fraction 51 are compared with 4 ul of each fraction eluted from the second isotachophoresis run. The size of the standard protein markers is indicated.
Figure 11 is a photographic representation of SDS-PAGE analysis of fractions obtained from the removal of contaminants from recombinant RAP2. An amount of 50 mg of RAP2 starting material (10 mg/ml) was loaded onto a 7.5% w/v isotachophoresis column (10 mm ID x 30 mm). Aliquots of 0.1 p samples of the WO 94/25144 PCT/AU94/00233 -14starting material are compared with 1 pl samples from 300 pl fractions eluted from the isotachophoresis run and a pool of 21 fractions (63-83). The size of the standard protein markers is indicated.
Figure 12 is a photographic representation of SDS-PAGE analysis of fractions obtained from the removal of contaminants from recombinant RAP1. Amounts of 2.8 mg of RAP1 starting material '9.3 mg/ml) was loaded onto a 5% w/v isotachophoresis coluimn 3 mm ID x 500 mm. Aliquots of 1 pl samples of the starting material are compared with 6 pl samples from 115 pI fractions eluted from the isotachopboresis run. The size of the standard protein markers is indicated.
Figure 13 is a photographic representation of SDS-PAGE analysis of fractions obtained from the removal of contaminants from recombinant RAP1. Incremental stepping of protein bands up the stack is demonstrated by loading 0.6 pl of the central fractions from Figure 12. Starting material load is 1 pl, and size of the standard protein markers is indicated.
Figure 14 is a photographic representation of SDS-PAGE analysis of fractions obtained from an anionic native isotachophoresis purification of Human Sera combined with IEF markers, performed in a 7% wfv 3 mm ID x 500 mm column.
Starting material consisted of 1.3 mg of whole human serum mixed with 1.32 mg of IEF markers at pH 7.5. Trailing buffer pH was 8.8. Aliquots of 0.25 pl samples of the starting material are compared with 6 pl samples from 115 pi fractions eluted from the native isotachophoresis run. The size of the standard protein markers is indicated.
Figure 15 is a photographic representation of SDS-PAGE analysis of fractions obtained from a cationic native isotachophoresis purification of human sera combined with IEF markers, performed in a 5% 3 w/v mm ID x 500 mm column.
Starting material consisted of 2.0 mg of whole human serum mixed with 1.32 mg of IEF markers at pH 5.0. Trailing buffer pH was 4.5. Aliquots of 0.25 p1 samples of the starting material are compared with 6 p1 samples from 115 p1 fractions eluted WO 94/25144 PCT/AU94/00233 15 from the native isotachophoresis run. The size of the standard protein markers is indicated.
Figure 16 is a diagrammatic representation of a form of the isotachophoresis apparatus located in a housing and incorporating a shaped tube.
Figure 17 is a diagrammatic representation of an emitter [54 of Figure 16] and a collector [53 of Figure 16].
Figure 18 is a photographic representation following SDS-PAGE of isotachophoretic fractions of nuclear antigen La collected at one minute intervals using the J-curve apparatus substantially as described in Figure 8.
Figure 19 is a diagrammatic representation of a form of the isotachophoresis apparatus located in a housing, incorporating a horizontal tube emitter module and collector module (71).
Figure 20 is a diagrammatic representation of an emitter collector probe housing for a near horizontal straight column.
Figure 21 is a diagrammatic representation of an emitter module for a near horizontal straight column.
Figure 22 is a diagrammatic representation of a collector module for a near horizontal straight column or a bent column.
The apparatus shown in the accompanying drawings (see in particular Figures 1 and 2) comprises a support container 1 in the form of a glass tube. Typically, a 600 mm x 20 mm diameter glass tube is used fitted with female B24/29 ground glass joints 2a, 2b which enable connection to anode 3 and cathode 4 chambers, generally also made of glass. The support container 1 is maintained in a vertical position by a series of clamps.
IWO 94/25144 PCT/AU94/00233 -16- During loading of the sample and initial electrophoresis, the apparatus is held in a vertical position with the sample loading port 12 at the top. The support container 1 carries the first buffer hydrated matrix 5 which is preferably a 500 mm polyacrylamide gel cast which forms the electrophoretic support medium. A removable platinum anode 6 and cathode 7 are inserted into the respective chambers 3, 4 and connected to in appropriate source of direct current. The electrode chambers 3, 4 are filled with and constantly flushed with the second buffer through tubes connecting to an external pump through ports 8,9. Spent electrode buffer flows to waste via tubes connected at ports 10,11. Identical sample loading 12 and sample elution 13 port assemblies are inserted into the electrode compartments. Details of the port assembly are shown in Figure 2. For elution, the apparatus is rotated so that the support container 1 is approximately 10" above horizontal (see Figure 2) with the elution port 13 higher than the sample loading port 12. During this reorientation, the electrode assemblies are maintained in a substantially vertical position by rotating the chambers 3, 4 with respect to the assembly around ground glass joints 14, In Figure 2, the support container 1 with first buffer hydrated matrix 5 in the form of polyacrylamide gel is orientated at approximately 10 to the horizontal with the elution port assembly 13 upper most and chamber 3 with the anode electrode 6 in a vertical orientation. Preferably, the elution port 13 is inserted into the electrode chamber via a B24/29 ground glass joint 14. It consists of a glass tube 16 sealed at the lower end with silicon rubber 17 passing through a silicon rubber compression ring 18 held in place by a threaded cap 19. This assembly allows the position of tube 16 to be adjusted during electrophoresis to accommodate shrinkage or expansion of buffer hydrated matrix 5. Two glass capillary tubes pass through the silicon seal 17 at the lower end of tube 16. The sample elution capillary 20 touches the wall of the support container 1 and the position of the elution assembly is adjusted so that the end of capillary 20 is approximately 1 mm from the surface of the buffer hydrated matrix 5. The other end of capillary 20 is connected via tubing to a peristaltic pump and a fraction collector. A second glass capillary 21 is used as an inlet for electrode buffer. Buffer pumped into the apparatus via this capillary, flushes the space above WO 94/25144 PCT/AU94/00233 -17the buffer hydrated matrix removing traces of sample not collected and provides a barrier preventing ions generated at the electrode assembly 6 from contaminating the eluted sample. A third capillary tube 22 is joined to the sample elution capillary within tube 16. Sterile air pumped into this tube forms spacer bubbles in the sample elution line 20 which minimise loss of resolution of the eluted sample components during their passage through this line and the connected peristaltic pump and fraction collector.
Another embodiment of the present invention is shown in Figure 8 in which the central glass column 30 is interchangeable from about 3 to about 19 mm ID and flanked by two buffer reservoirs 31, 32 containing electrodes. The column is in the shape of an obtuse J-shaped column. The entry portion of the tube at reservoir 31 is substantially vertical whereas the eluting end portion of the column at reservoir 32 is generally but not essentially about 10 0 from the horizontal.
A particularly preferred form of the apparatus is shown in Figure 16 having a collector and an emitter as shown in Figure 17. The apparatus conveniently comprises a housing 50 such as but not exclusively defined by upstanding walls, a bottom 51 and a top 52 and is divided into two sections by a petition and a collector 53 and an emitter 54 mounted on the petition spaced from one another. The collector 53 and emitter 54 are shown in more detail in Figure 17 and are substantially identical and each comprises an electrode chamber 55 which is fixed to the petition 56 by securing means in the form of, for example, screws 57 and a connector block 58. Each electrode chamber has a spigot 59 which passes through the partition 56 and which is adapted to sealingly receive the respective connector block 58 thereon. The connector blocks of the emitter and collector are connected one to the other by the column or tube comprising the matrix which in a most preferred embodiment is "J"-shape (see Figures 10 and 17). However, as described hereinbefore, other shapes may be readily adapted for use in accordance with the method of the present invention.
The electrode containing chambers shown in Figure 17 are each provided with a IWO 94/25144 PCT/AU94/00233 -18probe acting as either a cathode or anode, an inlet 62 for the infeed of buffer solution and/or sample into the chamber and an outlet 61 in fluid communication with the inlet.
In a more preferred embodiment of the present invention, air or other gaseous substance is introduced into the connection block of the collector by a "bubbler" (as hereinbefore described) which causes air or gas bubbles to be entrained in the fluid flow which minimises loss of resolution of the eluted sample. Preferably, the electrode containing chambers and connector blocks are formed of respective blocks of polycarbonate although clearly other suitable material may be used.
In yet a further embodiment, the electrophoretic tube is located in a horizontal, substantially horizontal or near horizontal position and is substantially straight rather than bent with the electrode housing devices placed at appropriate angles at the molecule entry end portion and the molecule elution end portion. One such nonlimiting embodiment is exemplified in Figure 19 which shows a form of the isotachophoresis apparatus located in a housing, incorporating a horizontal tube 72, emitter module 70 and collector module 71. The emitter module 70 is further shown in Figure 21 and the collector module 71 is shown in more detail in Figure 22. The latter module is suitable for use in a near horizontal straight column or a bent column such as a J-curved column. The near horizontal substantially straight tubed isotachophoretic apparatus also employs an emitter collector probe housing (Figure This housing incorporates a buffer in portion 74 which reduces any eddy currents. At the waste end portion 75 and specifically shown as insert A there is a modification which increases buffer flow past the electrode causing products of the electrophoresis to be flushed away from the elutirg charged molecule.
The results of isotachophoretic trials using a form of the apparatuses hereinbefore described are presented in the following non-limiting examples: WO 94/25144 PCT/AU94/00233 -19- EXAMPLE 1 PURIFICATION OF BOVINE SERUM ALBUMIN 1. Materials and Methods Sample Preparation An aliquot of 10 ml of 50 mg/ml Bovine Serum Albumin (BSA; Sigma, USA, Fraction 10% w/v SDS (BioRad, USA), 124 mM Trizma Base (Sigma, USA), 92 mM thioglycolic acid (Sigma, USA) pH 7.5 was placed into a dialysis membrane (Spectrum, USA) and dialysed at 22 *C against 500 ml of thioglycolic buffer (124 mM Trizma Base, 92 mM thioglycolic acid) for 90 hours.
An aliquot of 10 pl of retentate was diluted 1/50 in dialysate and a uv/vis spectrum was taken from 320 nm to 220 nm (Cary, model 4E) against dialysate as background.
An aliquot of 50 pl of prestained molecular weight markers (BioRad Broadrange) was added to the dialysate.
Polyacrylamide Gel Casting An aliquot of 30 ml of 30% w/v 29:1 Acrylamide (BioRad, USA): Bisacrylamide (BioRad, USA); 18 ml of 10 X Gel Buffer (1.5 M Tris-Cl, pH 7.5 when 1 132 ml of MQ-H 2 0 were mixed, filtered in a 0.22 pm disposable filter (Millipore, USA) then degassed by sonication under 900 mbar vacuum for 10 minutes.
Aliquots of 270 p1 10% w/v ammonium persulphate (BioRad, USA) and 90 pi TEMED (LKB Pharmacia, Sweden) were added, and the solution laid over 10% w/v sucrose (BDH, Australia) in the bottom electrode chamber to give a sharp gel bottom. To give a sharp upper end, 5 ml of 1 X Gel buffer was laid over the gel, resulting in a 500 mm x 19 mm ID 5% w/v polyacrylamide gel.
Column Preunning The sucrose solution in the bottom electrode chamber was replaced with 1 X gel buffer. The upper gel face was washed several times with thioglycolic buffer to 1 WO 94/25144 PCT/AUJ94/00233 remove any unpolymerised acrylamide solution, before the upper electrode chamber was filled with the thioglycolic buffer. A 200 pl aliquot of marking dye v/v glycerol BDH, Australia), 5% v/v methanol (BDH, Australia), 0.005% w/v Bromophenol Blue (BioRad, USA) in thioglycolic buffer was loaded, and a constant current of 20 mAmps was applied between the electrodes and the column preelectrophoresed for 36 hours until the dye had run off.
During electrophoresis, the electrode chamber buffers were flushed with respective buffers at 65 ml/hr (cathode) and 120 ml/hr (anode). Twelve hours prior to the completion of the pre-running the buffer used to flush the bottom (anode) electrode chamber was changed from the gel casting buffer to thioglycolic buffer.
A final 2 hours of pre-electrophoresis at 60 mAmps constant current was done to heat the column to ensure that remaining traces of acrylamide reacted with the thioglycolate. The column was allowed to cool for one hour with no current prior to sample loading.
Electrophoresis The top electrode (cathode) chamber buffer was changed to 25 mM Trizma base, 200 mM glycine (BDH, Australia), 0.01% w/v SDS. The dialysed sample was loaded onto the top of the gel and electrophoresed for 21 hours at 20 mAmps constant current with buffer flushing through the electrode chambers as before. This was followed by 6.5 hours at 30 mAmps. The electrode assembly was rotated and electrophoresis continued for a further 2.5 hours at 30 mAmps while the product eluted from the assembly. Eluate was collected at 1 ml/min in 160 x 1 ml fractions, using a "bubbler" to prevent sample mixing in the elution tube.
Sample Analysh AM quots of 1 pl samples from each of the fractions were added to 2% w/v SDS, 63 rmM Tris-Cl pH 6.8, 1% v/v glycerol, 5% v/v p-mercoptoethanol (Sigma, USA :r'ri boiled for 5 minutes. These, together with molecular size standards and 1 pi of the starting material were then electrophoresed on 10% w/v SDS-PAGE WO 94/25144 PCT/AU94/00233 21 after the method Laemmli (1970) Supra These gels were then Coomassie Blue (BioRad, USA) stained and quantitative densitometry performed to determine purity and yields of BSA containing fractions (Molecular Dynamics Computing Densitometer and ImageQuant software).
ii) Fractions 64 to 100 were pooled to total 64.6 ml and dialysed 1 in 500 000 against 150 mM Tris-C1 pH 7.5, 0.05% w/v SDS at 4"C. A uv spectrum was measured from 340 to 220 nm (Cary, model 4E).
iii) Acrylamide assays were performed on the pooled dialysate as follows.
A 25 pl sample was loaded onto a Whatman Partisil 10 ODS-2 HPLC analytical column and eluted with water at a flow rate of 2 ml/min with a detector sensitivity of 0.02 Absorbance at 280 nm. The sample peak was compared with acrylamide standards and the concentration of acrylamide present in the sample determined.
iv) Determination of the SDS concentration associated with the protein in the dialysed pool was done according to the procedure of Waite and Wang Anal.
Biochem. 701: 270-280, 1976. Essentially, triplicate 20 pl SDS standards and protein samples were made up to 200 pl. To each tube, 200 pl of 0.03 M HC1 (BDH, Australia), 48 mg/ml Basic fuschin (Sigma, USA) and 400 pl chloroform (BDH, Australia) was added. The tubes were thoroughly vortexed and incubated at 60 *C for 15 minutes, remixed and allowed to cool, After a 10 minute centrifuge at 15 000 rpm the aqueous phase was removed. Approximately 300 pi of the lower organic phase containing SDS-basic fuschin complex was transferred to new tubes containing 700 pl chloroform, The sample absorbances at 553 nm were then read in a spectrophotometer (Pye Unican, model PU 8600).
Results SDF \.GE of eluted fractions demonstrated that the BSA containing fractions largix. ,eparated from low molecular weight contaminants to be those between fractions 60 and 130 (Figure Quantitative densitometry of these fractions demonstrated a BSA purity of 95% in peak fractions, compared with 72% in the 4 .WO 94/25144 PCT/AU94/00233 -22starting material. There was a total exclusion of contaminants <50 kDa and kDa from the pooled peak material. Protein recovery as a percentage of total protein loaded was The pooled and dialysed fractions subjected to SDS and acrylamide content assays returned values of 0.10% w/v SDS content (1.0 mg/ml compared with 2.02 mg/ml total protein by uv spectroscopy) and an acrylamide content of below the limiting vAue of the assay sensitivity of 0.000005% w/v The rate of acrylamide scavenging by thioglycolic acid was determined by incubating 100 pg/ml acrylamide in thioglycolic buffer at 50 40 and 30 *C and taking various time points.
To prevent reaction of acrylamide monomers with proteins during electrophoresis, the inventors developed an assay for measuring monomer acrylamide via HPLC detection at 208 nm as described above. Assay sensitivity was determined to be 17.6 pmoles. This assay was then used to measure the rate of free monomer acrylamide scavenging by thioglycolic buffer at 3 temperatures by incubating 100 pg/ml acrylamide in thioglycolate buffer at 50 40 and 30 and taking various time points. Scavenging half-lives of 20, 30 and 45 ininutes were found for 50, 40 and C, respectively. Pre-running the columns overnight in thioglycolic buffer therefore removes a theoretically calculated 99.999976% of the unpolymerised acrylamide.
EXAMPLE 2 PURIFICATION OF A MINOR CONTAMINANT IN COMMERCIAL BOVINE SERUM ALBUMIN Materials and Methods Sample Fraction 51 from Example 1.
WO 94/25144 PCT/AU94/00233 -23- Electrophoresis Following the method detailed in Example 1, a 5% w/v polyacrylamide gel was cast 500 mm x 3 mm ID. The gel was pre-electrophoresed as described in Example 1 but only at a 2 mAmps constant current with re-circulating buffers at 6.5 ml/hr for the top chamber and 12 ml/hr for the bottom chamber. After pre-electrophoresis, the top buffer was changed to 25 mM Trizma base, 200 mM glycine, 0.01% w/v SDS as in Example 1 and the sample loaded. The sample was fraction 51 from the electrophoresis run in Example 1 plus 2 pl pre-stained molecular weight markers.
The column was electrophoresed for 4 hours at 2 mAmps, then 26 hours at 1 mAmps. For the final 80 minutes, 80 x 1 min 64 pl fractions were collected again using a bubbler.
Sample Analysis and Sequencing Aliquots of 4 pl of each fraction were run on a 12.5% w/v SDS-PAGE after the method of Laemmli (1970) Supra as described for Example 1.
A 10 pl aliquot from Fraction 41 of this second run was sequenced directly A Prospin (Applied Biosystems, USA) cartridge was pre-wet with 25 pl of methanol, the methanol removed and replaced with 10 pl of fraction 41 80 pi of MQ-H 2
O.
The cartridge was then spun to dryness at 4"C at 5,000 g. The membrane was removed from the cartridge and loaded directly into a model 473A Applied Biosystems Protein Sequencer.
Results Fraction 51 from the first isotachophoresis run (Example 1) was combined again with pre-stained molecular weight markers and loaded directly onto a 3 mm ID isotachophoresis column. SDS-PAGE of the product from the second run clearly showed separation of four major brands (Figure 10), none of which was clearly visible. The middle of these bands was contained in fraction 41, had an apparent molecular weight of 30 kDa and, when sequenced,, the N terminus proved to be amino acid 25 of BSA (GenBank Locus ABBOS). These results indicate that the isotachophoretic process of the present invention is capable of substantially purifying WO 94/25144 PCT/AU94/00233 -24a minor component in a complex mixture and furthermore that, as judged by the ability to obtain an N terminal sequence, the procedure as described results in minimal modification of the protein.
EXAMPLE 3 PURIFICATION OF TOPOISOMERASE 1 Materials and Methods GST Topoisomerase 1 sample An IPTG induced culture of E. colitrans med with the pGEX (Smith and Johnson Gene 67: 31-40, 1988) topoisomerase (Shero et al. Science 231: 737-740, 1986) recombinant plasmid was pelleted and resuspended in Phosphate Buffered Saline (PBS). Cells were lysed by treatment with 1% v/v Triton X-100 and sonicated, inclusion bodies were removed from the cell lysate by centrifugation. The pellet was washed with PBS twice and solubilised in 8 M Urea in PBS and non solubilised proteins were removed by centrifugation. An 8.5 ml extract containing 140 mg protein (approximately 75 mg full length fusion protein GST-Topo) was used in the purification procedure. Prior to loading on the acrylamide gel, 150 mg SDS, 425 pl p mercaptoethat. 1 and 0.1% w/v Bromophenol were added to the sample.
Electrophoresis Gel The gel was cast in the support container illustrated in Figure 1. The gel consisted of 8% w/v acrylamide/bisacrylamide (29:1 150 mM Tris/Tris HC1 pH 7.5 and polymerised by the addition of 14 mg/100 ml of ammonium persulphate and 45 pl per 100 ml of N, N, N'N'-tetramethylethylene diamine.
Electrophoresis The electrophoresis gel was mounted in the apparatus illustrated in Figure 1. The elution chamber was fitted to the lower end of the gel and the lower container filled with gel buffer. The upper container was filled with electrode buffer (25 mM Tris, 200 mM glycine, 0.01% w/v SDS). The sample was layered on the top of the gel beneath the electrode buffer. Electrophoresis was performed at 10 mAmps. The WO 94/25144 PCT/AU94/00233 position of the protein band was noted at various times and was moving through 3 pl of gel per mAmps per minute, i.e. the protein band was moving at 30 pl per minute at 10 mAmps. Under these conditions, the protein band is visible in the gel tube since this region of the gel has a markedly different refractive index to the remainder of the gel. From the width of the band, the volume occupied by the protein was 5.0 ml at an average protein concentration of 20 mg/ml towards the end of the run. A series of protein bands could be discerned within the region occupied by protein as a series of refractive index changes in the gel. Running in front of the protein band is a faster moving discontinuity indicating a region of buffers, salts and detergent.
Collection of Protein Collection of eluate commenced as the buffer/detergent front commenced to slute.
Gel buffer was sucked from the electrode compartment through the elution chamber at 0.15 ml/min at 5 times the elution rate of protein) and collected in 2 min (0.3 ml) fractions.
Results Analysis of Fractions Aliquots of 1 pl of every second fraction were analysed by SDS PAGE and the results are shown in Figure 3. Included on the gel were standard proteins (Lane A); 500 ng Pharmacia Low Molecular Weight Standards, the starting material (Lane B) and the purified sample (Lane Samples were diluted 1:5 in sample buffer mM Tris-HCl, 1 mM EDTA, 2.4% w/v SDS, 5% Bromphenol blue at pH Aliquots of 1 p1 of samples were loaded onto an 8-25% gradient polyacrylamide gel.
The extent of purification of GST-Topo is clearly shown in Figure 3 (compare I ane B and Lane Following electrophoresis, the gel was stained with Coomassi blue.
WO8 94125144 PCT/AU94/00233 -26- EXAMPLE 4 PURIFICATION OF U1 RIBONUCLEAR PROTEIN (U1RNP) An 0.5 ml aliquot of a culture of pGEX2T-UIRNP (Smith and Johnson Supra; Query et al. Cell 57: 89-101, 1989) in E. coli strain JPA101 grown from a single colony for 5 hours in Super Broth containing 50 pg/ml ampicillin was used to inoculate a 500 ml flask of Super Broth containing 50 pg/ml ampicillin grown overnight at 32 An amount of 50 ml of the overnight culture was added to each of ten 500 ml flasks of Super Broth (1:10 dilution) containing 50 pg!ml ampicillin and grown until the OD6 was approximately 1.2. Expression was induced with IPTG for 3 hours at 32 C and then the bacteria harvested by centrifugation and frozen at -70 *C, A 64.22 g cell pellet was resuspended in PBS at a concentration of 0.2 g/ml and adjusted to 1% v/v Triton prior to sonication for 3 minutes. The solution was cleared by centrifugation. Glutathione Sepharose affinity resin was added to the supernatant (1:50 dilution) and incubated for 1 hour, the resin was retrieved and a new aliquot added for a further 30 minutes. The resin was washed extensively with PBS and 150 mM NaCI, 50 mM Tris-HC1, pH 8.0 before being resuspended in mM CaCI 2 150 mM NaCI, 50 mM Tris-HCI, pH8.0 and incubated with thrombin for 1 hour at 37 *C and subsequent elution with 150 mM NaC1, 50 mM Tris-HC1, pH Fractions were pooled and concentrated by ethanol precipitation. The precipitate was collected by centrifugation and resuspended in 2 ml of 10% w/v SDS. Excess SDS was removed by extensive dialysis against 25 mM Tris, 200 mM glycine, 0.1% w/v SDS resulting in 7.5 ml at 32 mg/ml. An aliquot of 3 ml of this solution was adjusted to 0.1% w/v Bromophenol Blue and loaded onto a 1.2 x 40 cm 8% w/v acrylamide, 150 mM Tris-HCI column. Tris(25 mM), 200 mM glycine and 0.01% w/v SDS was circulated at 0.5 ml/min at the cathode. Isotachophoresis was performed for 14 hours at 8 mAmps and a further 4 hours at 16 mAmps prior to elution at 5 mAmps. One minute fractions were collected at 0.5 ml/min at an angle of 15 0 to the horizontal and subjected to SDS-PAGE (Figure All fractions which appeared greater than 95% pure by Pharmacia Phast Gel (gradient 8-25%) stained WO 94/25144 PCT/AU94/00233 -27with Coomassie Blue were pooled. An amount of 25.5 mg U1RNP at 3 mg/ml concentration was recovered.
EXAMPLE PURIFICATION OF TOPOISOMERASE-GST An 0.5 ml aliquot of a culture of pGEX2T-Topo (Smith and Johnson Supra) in E.
coli strain JPA101 grown from a single colony for 5 hours in Super Broth containing pg/ml ampicillin was used to inoculate a 500 ml flask of Super Broth containing 50 pg/ml ampicillin grown overnight at 37 An amount of 50 ml of the overnight -alture was added to each of ten 500 ml flasks of Super Broth (1:10 dilution) containing 50 pg/ml ampicillin and grown until the OD, was approximately 1.2.
Expression was induced with IPTG for 3 hours at 37 C and then the bacteria harvested by centrifugation and frozen at -70 "C, A 6 g cell pellet was washed 3 times with 45 ml IPBS and resuspended in 35 ml PBS and adjusted to 1% v/v Triton prior to sonication for 3 minutes. The insoluble material was collected by centrifugation and resuspended in 12 nrl 8 M Urea in PBS before repeating the sonication procedure. The suspension was cleared by centrifugation. An amount of 5 ml of this solution was adjusted to 5% v/v p- Mercaptoethanol, 0.1% w/v Bromophenol Blue, 150 mM Tris-HCI, 20 mg/ml SDS and loaded onto a 1.2 x 40 cm 5% w/v acrylamide, 150 mM Tris-HCl column. Tris mM), 200 mM glycine, 0.01% w/v SDS was circulated at 0.5 ml/min at the cathode and 150 mM Tris-HC1, pH 7.5 was circulated at 0.5 ml/min at the anode.
Isotachophoresis was performed for 15 hours at 10 mAmps and a further 2 hours at mAmps prior to elution at 5 mAmps. One minute fractions were collected at ml/min at an angle of 15 to the horizontal and subjected to SDS-PAGE (Figure All fractions which appeared greater than 95% pure by Pharmacia Phast Gel (gradient 8-25%) stained with Coomassie Blue were pooled. An amount of 18.2 mg of TOPO-GST at 2.8 mg/ml concentration was recovered.
WO 94/25144 PCT/AU94/00233 -28- EXAMPLE 6 PURIFICATION OF NUCLEAR ANTIGEN LA (STRAIGHT COLUMN) An 0.5 ml aliquot of a culture of pQEll-La (Chambers et al. J. Biol. Chem. 263: 18043-18051, 1988) in E. coli SG13009 grown from a single colony for 5 hours in liquid broth containing 50 pg/ml ampicillin and kanamycin was used to inoculate an 800 ml flask of liquid broth containing 50 pg/ml ampicillin and kanamycin grown overnight at 37 An amount of 80 ml of the overnight culture was added to each of ten 800 ml flasks of liquid broth (1:10 dilution) containing 50 pg/ml ampicillin and kanamycin and grown until the ODa 6 was approximately 1.2. Expression was induced with IPTG for 3 hours at 32 °C and then the bacteria harvested by centrifugation and frozen at -70 0
C.
A 6 g cell pellet was resuspended in 6 M guanidine-HC1, 100 mM KH 2
PO
4 pH at a concentration of 0.2 g/ml and sonicated for 3 minutes. The solution was cleared by centrifugation. The supernatant was bound to Ni-agarose affinity resin and washed with a pH step gradient at pH 6.3 and 5.9 prior to elution at pH Guanidine was removed by extensive dialysis against PBS before concentration by ethanol precipitation. The precipitate was collected by centrifugation and resuspended in 4 ml 10% w/v SDS. Excess SDS was removed by extensive dialysis against 25 mM Tris, 200 mM glycine, 0.1% w/v SDS resulting in 24 ml at 8.2 mg/ml.
An aliquot of 4 ml of this solution was adjusted to 5% v/v p-mercaptoethanol, 0.1% w/v Bromophenol Blue and loaded onto a 1.2 x 40 cm 5% w/v acrylamide, 150 mM Tris-HCI column. Tris (25 mM), 200 mM glycine, 0.01% w/v SDS was circulated at ml/min at the cathode and 150 mM Tris-HC1, pH 7.5 was circulated at ml/min at the anode. Isotachophoresis was performed for 11 hours at 8 mAmps and a further 5 hours at 16 mAmps prior to elution at 5 mAmps. One minute fractions were collected at 0.5 ml/min at an angle of 15 to the horizontal and subjected to SDS-PAGE (Figure All fractions which appeared greater than 95% pure by 8- Pharmacia gradient Phast Gel stained with Coomassie Blue were pooled. An amount of 2.35 mg of La at 4.7 mg/ml concentration was recovered.
WO 94/25144 PCT/AU94/00233 -29- EXAMPLE 7 PURIFICATION OF R060 An 0.5 ml aliquot of a culture of pQE9-R060 (Deutscher et al. Proc. Nat. Acad. Sci.
USA 85:9479-9483, 1988) in M15 E. coli grown from a single colony for 5 hours in Liquid Broth containing 50 pg/ml ampicillin and kanamycin was used to inoculate an 800 ml flask of liquid broth containing 50 pg/ml ampicillin and kanamycin grown overnight at 37 C. An amount of 80 ml of the overnight culture was added to each of ten 800 ml flasks of Liquid Broth (1:10 dilution) containing 50 pg/ml ampicillin and kanamycin and grown until the OD600 was approximately 1,2. Expression was induced with IPTG for 3 hours at 37 *C then harvested by centrifugation and frozen at A 17.4 g cell pellet was resuspended in 50 mM phosphate, 300 mM NaC, pH8.0 at a concentration of 0.2 g/ml and sonicated for 3 ix nutes. The solution was cleared by centrifugation. The pellet was resuspended in 8 M urea, 100 mM NaH-P0 4 mM Trs-HC1, pH 8.0 at 200 pg/ml and mixed for 2 hours at 4 The suspension was cleared by centrifugation and 80 ml supernatant retained. An aliquot of 5 ml of this solution was adjusted to 5% v/v p-mercaptoethanol, 0.1% w/v Bromophenol Blue and 60 mg/ml SDS and loaded onto a 1.2 x 40 cm 5% w/v acrylamide, 150 mM Tris-HCl column. Tris (25 mM), 200 mM glycine, 0.01% w/v SDS was circulated at ml/mmi at the cathode and 150 mM Tris-HC1, pH 7.5 was circulated at ml/min at the anode. Isotachophoresis was performed for 1 hour at 18 mAmps, 16 hours at 10 mAmps and a further 3 hours at 16 mAmps prior to elution at 5 mAmps.
One minute fractions were collected at 0.5 ml/min at an angle of 15" to the horizontal and subjected to SDS-PAGE (Figure All fractions which appeared greater than 95% pure by 8-25% Pharmacia gradient Phast Gel stained with Coomassie Blue were pooled. An amount of 7.65 mg of R060 at 1.9 mg/ml concentration was recovered.
WO 94/25144 PCT/AU94/00233 EXAMPLE 8 PURIFICATION OF RAP2 Recombinant RAP2 was expressed from Escherichia coli SG13009 containing the hexaHis expression vector pDS56/RBSII ligated with DNA coding for the mature protein sequence from the D10 Plasmodiumfalcipanun isolate (Saul, A. et al. Mol.
Biochem. Parasit. 50: 139-150, 1992). Inclusion bodies in transformed cells are solubilised with two 60 minute extractions at room temperature in 6 M guanidine- HC1, 0.01 M p-mercaptoethanol (BioRad, USA), 20% v/v ethanol (BDH Chemicals, Australia), pH 7.9. An initial purification over a Ni +-chelating resin (Qiagen, USA) was performed using the above buffer and an imidazole (Kodak, USA) gradient.
This product was precipitated in 20 x volume ethanol to remove the guanidine, redissolved in 10% w/v SDS and dialysed into 0.1% w/v SDS, thioglycolate buffer.
Denaturing isotachophoresis was performed in cylindrical columns, varying in ID from 19 mm to 3 mm depending on the protein load and 500 mm in length.
Denaturing polyacrylamide gels of varying percentages of 29:1 acrylamide (BioRad, USA):bisacrylamide (BioRad, USA) were prepared in 150 mM Tris-HCI pH filtered in a 0.22 jpm disposable filter (Millipore, USA) then degassed by sonication under 900 mr vacuum for 10 minutes. Polymerisation was initiated with 0.015% w/v ammonium persulphate (BioRad, USA) and 0.05% v/v TEMED (LKB Pharmacia, Sweden).
Fractions were analysed by SDS-PAGE (Laemmli, 1970 Supra), and purity and yields quantitated by densitometry (Molecular Dynamics Computer Densitometer and ImageQuant software). Fractions of highest puritywere pooled, dialysed 1 in 500,000 against 150 mM Tris-Cl pH 7.5, 0.05% w/v SDS at 4 0 C. Spectral and amino acid analysis was performed to determine concentrations and product modifications.
Columns were pre-electrophoresed with thioglycolic buffer for 36 hours at 20 rJunps constant current for the 19 mm ID gels, with a final 2 hours at 60 mAmps to heat the column to ensure that any remaining traces of monomer acrylamide reacted with WO 94/25144 PCT/AU94/00233 -31 the thioglycolate prior to loading; alternately 3 mm ID gels were pre-run 16 hours at 2.0 mAmps constant current. During both pre-electrophoresis and the actual run the electrode chambers were continuously flushed with fresh buffer at 65 ml/hr or ml/hr (cathode) and 120 ml/hr or 12 ml/hr (anode), depending on the column size. Columns were allowed to cool for one hour with no current prior to sample loading, and the trailing electrode (cathode) chamber buffer was changed to 127 mM Tris, 90 mM glycine (BDH Chemicals, Australia) and 0.01% w/v SDS.
Protein samples were sequenced using a model 473A Applied Biosystems protein sequencer, either directly by loading a sample aliquot into a Propsin cartridge (Applied Biosystems, USA), or sequenced after SDS-PAGE and electro-blotting to polyvinyl diflouride membranes (Applied Biosystems, USA) and the band of interest being excised.
With denaturing isotachophoresis under the conditions used and described herein, an optimal load for a 19 mm ID column was 500 mg total protein, with power scaled to protein size and minimum gel volume required for stacking being equal to the sample load volume. These limits are then scaled down proportionally for columns with smaller IDs.
The isotachophoresis apparatus comprised a central glass column interchangeable from 3 to 19 mm ID flanked by two buffer reservoirs containing electrodes. When a straight column is used Figure 1) the column is pre-run, and the samples loaded in the vertical position. The column is rotated approximately 100 to allow sample elution from the upper end with the column just off the horizontal.
Alternatively, where a bent column is used, the column incorporates an obtuse Jshaped column, obviating the necessity to rotate the column during the run, while the protein stack still re-focuses past the curve.
Real-time elution occurs through a small capillary tube running to the bottom of the gel face, The proteins eluting from the gel are highly concentrated, and the nearly vertical, gel face allows density driven elution. For convenience, in this particular WO 94/25144 PCT/AU94/00233 -32example, the eluted sample is divided into discrete blocks in the capillary tube by introducing air bubbles into the line at 50% of the elution rate. These bubbles reduce sample mixing in the elution line by over 50%. The introduction of dye into the buffers flushing the electrode chambers allowed a number of measures to be taken to minimise heating in the elution chamber and subsequent elimination of convection currents which interfere with sample elution and greatly reduce yields.
These include locating the terminal electrode so that the path of the by-products of electrolysis at the terminating electrode (concentrated HC1 amongst others) is directly into the waste line.
Similarly, a wide bore path between the gel face and the electrode provides a sufficiently large volume of buffer to allow dissipation of the heat resulting from the power loss, in the voltage drop between the gel face and the terminating electrode.
And finally, empirically determining a sufficient buffer flow rate to reinforce the above two effects and maintain buffering pH at the gel face. Substantial yield losses may result if these measures are not taken.
The form of RAP2 used here was expressed as a recombinant protein in E. coliwith a hexaHis N-terminal, and passed over a Ni" chelating resin as an initial purification step. Figure 11 shows the purification profile of this material after denaturing isotachophoresis with 50 mg total protein loaded. Fractions 63 to 83 were pooled, giving 6.3 ml at 1.82 mg/ml.
Repeating the procedure using a re-run column allowed the sequencing of the minor bands purified in the early fractions. These were found to be N-terminal breakdown products of RAP2, i'dth no blocking of the N-terminal to prevent the sequencing.
In a similar experimtent where 280 mg total protein from non-Ni" purified solubilised inclusion bodies was loaded directly onto a pre-run 19 mm ID x 500 mm column, amino acid analysis revealed no modified residues, and the entire procedure was performed to meet GMP standards for the production of clinical grade material.
WO 94/25144 PCT/AU94/00233 -33- EXAMPLE 9 PUPIFICATION OF RAP1 Recombinant RAP1 was expressed as for RAP2 and was obtained from Roche Pharmaceuticals, Switzerland and dialysed into thioglycolic buffer, 0.01% w/v SDS.
All other conditions used were as substantially described in Example; 8.
RAP1 is an 80 kDa Plasmodium falciparwn protein (Ridley et al. Mol. Biochem.
Parasit. 41: 125-134, 1990). The form used here was expressed as a recombinant protein in E. coliwith a hexaHis N-terminal, and passed over a Ni chelating resin in an initial purification step.
A 3 mm ID x 500 mm column f 5%'w/v T acrylamide was pre-run with thioglycolate and 2.8 mg of recombinant protein was loaded in 2% w/v sucrose and 5% v/v p-mercaptoethanol. Fractions of 80 x 115 pi were collected and displayed in Figures 14 and 15. Interestingly, increasing the RAP1 protein load caused precipitation of proteins within the stack and subsequent loss of resolution. Chasing the protein stack with SDS or lower pH buffers, or adding dithiothreitol to the trailing buffer failed to redissolve protein that had precipitated from the stack, indicating that precipitation was not due to oxidisation. SDS removal or the higher pH of the trailing buffer allowed protein-protein interactions. However, lowering the current flux to 40 pAmps per mm 2 (3 mAmps per a 3 mm gel) allowed separation without precipitation.
EXAMPLE NATIVE ISOTACHOPHORESIS For anionic and cationic native isotachophoresis, 4 ml O' Human Serum supplied by the Australian Red Cross was dialysed into 150 mM Tris-HCI pH Anionic and cationic native isotachophoresis were carried out in 3 mm ID x 500 mm length columns, with 7% or 5% (total monomer, acrylamide bisacrylamide at WO 94/25144 PCT/AU94/00233 -34- 29:1), respectively. Neither system was pre-electrophoresed.
Anionic native isotachophoresis was performed with the same leading and trailing buffers as detailed above for denaturing systems, without the SDS (150 mM Tris-HCl pH 7.5 leading, 127 mM Tris, 90 mM glycine trailing).
Cationic native isotachophoresis gels were prepared in a leading buffer of 60 mM potassium hydroxide (BDH Chemicals, Australia) 121.8 mM acetic acid (BDH Chemicals, Australia) pH 5.0. These gels were photopolymerised, catalysed by 0.01% w/v ammonium persulphate, 0.0008% w/v riboflavin (Sigma, USA) under fluorescent lamps. Cationic terminating buffer was 202 mM glycine, 190 mM acetic acid pH For both systems, samples were loaded in each leading buffer. Samples consisted of 300 pl of 1 x leading buffer, 3% w/v sucrose containing an amount of 1.32 mg IEF markers (BioRad, USA) and either 1.3 or 2.0 mg of dialysed human serum (anionic and cationic, respectively). Electrophoresis was performed at 0.25 mAmps constant current for 12 or 30 hours (anionic or cationic), prior to the gravity fed elution of x 1 min 64 pl fractions.
Figures 16 and 17 demonstrate protein purification usinLg anionic and cationic native isotachophoresis systems. In both systems a similar protein load of whole human serum combined with IEF markers was used.
The order of elution for the anionic system is p-lactoglobulin B (18.4 kDa, pi 5.1); equine myoglobin (17.5 kDa, pi human serum albumin (66.47 kDa, pi 6.03), which co-eluted with initially some lower molecular weight human serum proteins, and then some higher molecular weight human serum proteins; and a second equine myoglobin protein (17.5 kDa, pi Human haemoglobin (64.5 kDa, pi 7.1 to and cytochrome C (12.2 kDa, pI 9.6) were excluded from the protein stack.
Conversely for the cationic system the order of elution was cytochrome C; bovine and human carbonic anhydrase (31 and 28 kDa, pi 6.0 and equine myoglobin WO 94/25144 PCT/AU94/00233 (both hands); p-lactoglobulin B; human serum albumin; some higher molecular weight human serum proteins; and then some lower molecular weight human serum proteins.
Despite a human serum load only 2/3 that of the cationic system, the anionic system protein stack occupied a volume in the column greater than twice that of the cationic stack, and travelled 3 times as fast.
Optimal purity depends on the protein of interest, but for human serum albumin clearly the cationic system is optimal, while for p-lactoglobulin B it is the anionic system.
EXAMPLE 11 PROTEIN MOBILITY Protein mobilities at different percentages of acrylamide and buffer pH were determined using 2-D capillary gels. A series of duplicate capillary gel sets were prepared ranging in concentration from 3 to 11% T. Each set of gels was prepared in a buffer ranging in pH from 6.8 to 8.8, but at the same ionic ratio as the other sets. Broad-range protein standards (BioRad, USA) were loaded onto each gel and electrophoresed using a Mini Tube Cell Module (BioRad, USA) until 3/4 down the length of the capillary. The protein stack form each capillary was excised and run in a second dimension in 12.5% w/v T SDS-PAGE gels and silver-stained by the method of Schoenle, EJ. et al. J. Biol. Chem. 259:12112, 1984.
Optimisation of stacking is possible for each individual protein of interest.
Increasing the gel pH at constant acrylamide concentration was found to cause higher molecular weight proteins to be excluded from the stack, likewise increasing the adrylamide concentration at a constant buffer pH (Table 1).
WO 94/25144 PCT/AU94/00233 36- CONSTANT ACRYLAMIDE PERCENTAGE CONSTANT I H 3% 5%1 7%I 9% 11%I pH 6.8 200.00 200.00 116.20 116.20 97.40 9740 9740 66.20 66.20 66.20 66.20 66.20 45.00 45.00 45.00 45.00 45.00 31.00 31.00 31.00 31.00 31.00 21.50 21.50 21.50 21.50 21.50 14.40 14.40 14.40 14.40 14.40 pH 7.5 200.00 116.20 116.20 97.40 97.40 97.40 66.20 66.20 66.20 66.20 66.20 45.00 45.00 *45.00 45,00 45.00 31.00 31.00 31.00 31.00 31.00 21540 21.50 21.50 21.50 4.014.40 14.40 1 14,40 pH 8.0 200.00 116.20 116.20 97.40 97.40 97.40 66.20 66.20 66.20 66.20 45.00 45.00 45.00 45.00 45.00 31.00 31.00 31.00 31.00 31.00 21.50 21.501 21.50 21.50 21.50 14.40 14.40 14A40 14.40 14.40 pH 84 200.00 116.20 97.40 97.40 66.20 66.20 66.20 45.00 45.00 45.00 45.00 45.00 3100 31.00 31.00 31.00 31.00 21.50 21.0 21.50 21.50 21.50 1440 14.40 14.40 1440 14.40 pH 8.8 200.00 116.20 91.40 97.40 6.20 66.20 66.20 4M00 .00 45. 00 45.00 31,00 3100 31.00 00 31.00 3 21.50 21.50 21.50 21.50 21.51 1440 144AD 14.40 140 14,4w' ,ABLE 1: Columns indicate the molecular weight of proteins which were trapped in the stack of capillary gels using a discontinuous buffer system. The effect of varyrir.E 1el percentage and buffer pH was examined by 2D gel elcctrophoresis as detail' in the text.
WO 94/25144 PCT/AU94/00233 -37- EXAMPLE 12 PURIFICATION OF NUCLEAR ANTIGEN LA (BENT COLUMN) An 0.5 ml aliquot of a culture of pQE11-La (Chambers et al. J. Biol. Chem. 263: 18043-18051, 1988) in E. coli SG13009 grown from a single colony for 5 hours in liquid broth containing 50 pg/ml ampicillin and kanamycin was used to inoculate an 800 ml flask of liquid broth containing 50 pg/ml ampicillin and kanamycin grown overnight at 37 An amount of 80 ml of the overnight culture was added to each of ten 800 ml flasks of liquid broth (1:10 dilution) containing 50 pg/ml ampicillin and kanamycin and grown until the OD6, was approximately 1.2. Expression was induced with IPTG for 3 hours at 320C and then the bacteria harvested by centrifugation and frozen at -70 0
C.
A 6 g cell pellet was resuspended in 6 M guanidine-HCl, 100 mM KH 2
PO
4 pH at a concentration of 0.2 g/ml and sonicated for 3 minutes. The solution was cleared by centrifugation. The supernatant was bound to Ni-agarose affinity resin and washed with a pH step gradient at pH 6.3 and 5.9 prior to elution at pH Guanidine was removed by extensive dialysis against PBS before concentration by ethanol precipitation. The precipitate was collected by centrifugation and resuspended in 4 ml 10% w/v SDS. Excess SDS was removed by extensive dialysis against 25 mM Tris, 200 mM glycine, 0.1% w/v SDS resulting in 24 ml at 8.2 mg/ml.
An aliquot of 4 ml of this solution was adjusted to 5% v/v p-mercaptoethanol, 0.1% w/v Bromophenol Blue and loaded onto a 1.2 x 35 cm 5% w/v acrylamide, 150 mM Tris-HCl J-curved column. Tris (25 mM), 200 mM glycine, 0.01% w/v SDS was circulated at 0.5 ml/min at the cathode and 150 mM Tris-HCI, pH 7.5 was circulated at 0.5 ml/min at the anode. Isotachophoresis was performed for 11 hours at 8 mAmps and a further 5 hours at 16 mAmps prior to elution at 5 mAmps. One minute fractions were collected at 0.5 ml/min at an angle of 15" to the horizontal and subjected to SD5-PAGE (Figure 18). All fractions which appeared greater than pure by 8-25% Pharmacia gradient Phast Gel stained with Coomassie Blue were pooled. An amount of 2.35 mg of La at 4.7 mg/ml concentration was recovered.
WO 94/25144 PCT/AU94/00233 -38- Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.
It is to be understood that the inention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.