Patent Application
20070111944
The present invention relates generally to a method of producing recombinant peptides, polypeptides and proteins. More particularly, the present invention provides a method of purifying recombinant peptides, polypeptides or proteins away from truncated or other non-full length forms of these molecules. Even more particularly, the present invention contemplates a method of purifying a vascular endothelial growth factor (VEGF) molecule or a derivative or homologue thereof including amino acid tagged forms or other peptide, polypeptide or protein by subjecting a preparation containing the molecule to be purified to affinity chromatography under chromatographic conditions sufficient for full length molecules but not truncated or non-full length molecules corresponding to said full length molecules to bind or otherwise associate by the affinity process. In a preferred embodiment, the purification involves optionally subjecting a preparation containing the molecule to be purified to an affinity column based on the properties of an exogenous amino acid sequence followed by a second affinity column based on properties inherent with the peptide, polypeptide or protein. The present invention is further directed to a peptide, polypeptide or protein such as a VEGF molecule or a derivative or homologue thereof purified by the methods of the present invention. Particularly preferred VEGF molecules are VEGF-B molecules including untagged VEGF-B167, hexa-His-tagged VEGF-B167, hexa-His-tagged VEGF-B186 and hexa-His-tagged VEGF-B10-108.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Recombinant DNA technology provides the means for the production of peptides, polypeptides and proteins in large quantity. This is especially required for molecules required for therapeutic interventionist purposes where vast quantities are required. However, the molecules also need to be highly purified.
Cytokines and growth factors are important molecules for which many are available in recombinant form. However, despite the available knowledge as to their structure and function, the therapeutic use of such molecules will depend upon the level of purity which can be obtained.
One particularly important growth factor is vascular endothelial growth factor (hereinafter referred to as “VEGF”). This molecule is also known as vasoactive permeability factor. VEGF is a secreted, covalently linked homodimeric glycoprotein that specifically activates endothelial tissues (Senger et al., 1993). A range of functions have been attributed to VEGF such as its involvement in normal angiogenesis including formation of the corpus luteum (Yan et al., 1993) and placental development (Sharkey et al., 1993), regulation of vascular permeability (Senger et al., 1993), inflammatory angiogenesis (Sunderkotter et al., 1994) and autotransplantation (Dissen et al., 1994) and human diseases such as tumour promoting angiogenesis (Folkman & Shing, 1992), rheumatoid arthritis (Koch et al., 1994) and diabetes related retinopathy (Folkman & Shing, 1992).
Based on a high level of sequence homology within a region incorporating 8 equally spaced cysteine residues (cystine knot motif/VEGF homology domain), four further proteins can be included within the VEGF family: placenta growth factor (PLGF), VEGF-B, VEGF-C and VEGF-D. Compared to VEGF-A relatively little is known about methods of production for these other members of the VEGF family. The five members of the family are now known to interact differentially with 3 distinct receptor tyrosine kinases. While VEGF-A binds VEGFR1 and R2, PLGF and VEGF-B bind only to VEGFR1. In contrast VEGF-C and D bind VEGFR2 and, in addition, a third receptor (VEGFR3 or Flt4) restricted to lymphatic endothelium. The functional significance of the distinct receptor binding characteristics of the additional family members remains unclear. The issue of functional activity of distinct family members is further complicated by their ability to form heterodimers when co-expressed in mammalian cells.
Like VEGF-A, VEGF-B is, therefore, an important molecule and may have utility as a therapeutic agent if it can be produced and purified to a sufficiently high level. VEGF-B comprises a series of isoforms and truncated isoforms, some of which retain the receptor binding domain. Examples of VEGF-B isoforms include VEGF-B167, VEGF-B186 and VEGF-B10-108. Due to a number of technical obstacles, VEGF-B isoforms have not previously been purified to near homogeneity as a homodimer and shown to be active.
VEGF-B is a member of the cystine knot family of cytokines that exhibit complex secondary structure elements, which include inter- and intra-molecular disulfide bonds. An ideal method of producing such complex eukaryotic proteins involves expression in a mammalian system, where it is likely that the protein will adopt its native conformation. However, mammalian systems produce endogenous VEGF family members, in particular VEGF-A, which form heterodimers with the expressed VEGF-B. Such heterodimers are difficult to separate from the desired homodimers and any such step would add substantially to the cost of production. An alternative method of producing pure homodimeric VEGF-B involves expression in non-mammalian systems such as Escherichia coli, where the protein is expressed most commonly as inclusion bodies. Inclusion bodies can in general only be solubilized under harsh denaturing conditions and proteins produced in such a way must be refolded into the correct conformation. For proteins with complex secondary structure, such as VEGF-B, this can create problems during refolding such that incorrectly folded and inactive proteins can result. Consequently, specific refolding conditions are required for VEGF-B. In addition to complex secondary structure, the hydrophobic nature of VEGF-B, and VEGF-B167 in particular, leads to aggregation during refolding and purification and this can result in complete loss of protein. This issue requires particular attention during purification. One further complication with some conventional purification techniques applied to VEGF-B is the inability to discriminate between full length VEGF-B molecules and truncated or “clipped” variants. Consequently, during refolding, hybrids can form between a full length molecule and a truncated variant leading to an inactive molecule or a molecule exhibiting undesirable properties.
The present invention describes a strategy that overcomes these technical obstacles to yield highly purified homodimeric VEGF-B isoforms that have demonstrated receptor binding characteristics. The molecules purified by the present invention are particularly useful in therapeutic protocols and in diagnostic assays.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood 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.
Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. <400>1, <400>2, etc. A sequence listing is provided after the claims.
One aspect of the present invention provides a method of purifying a peptide, polypeptide or protein from a biological sample said method comprising subjecting the biological sample to affinity chromatography comprising an affinity matrix under chromatographic conditions sufficient for the full length but not a truncated or non-full length peptide, polypeptide or protein corresponding to said full length peptide, polypeptide or protein to be bound to or otherwise associate with the affinity matrix and then eluting said bound or associated peptide, polypeptide or protein from the affinity matrix and collecting same.
Another aspect of the present invention is directed to a method of purifying a recombinant peptide, polypeptide or protein from a biological sample said method comprising subjecting said biological sample to affinity chromatography comprising an affinity matrix which has affinity for an N-terminal or C-terminal region of said peptide, polypeptide or protein but substantially not for the N-terminal or C-terminal region of a truncated or clipped form of said peptide, polypeptide or protein, said affinity chromatography being under chromatographic conditions sufficient to permit binding or association of full length but not truncated or non-full length peptide, polypeptide or protein, and then eluting the bound or associate peptide, polypeptide or protein from the affinity matrix and collecting same.
Yet another aspect of the present invention provides a method of purifying a peptide, polypeptide or protein from a biological sample comprising subjecting said biological sample to an optional first affinity chromatography comprising an affinity matrix which binds or associates said peptide, polypeptide or protein based on affinity to an N-terminal or C-terminal portion of said molecule, eluting off said bound or otherwise associated peptide, polypeptide or protein and subjecting same to a second affinity chromatography based on affinity or association with the other of an N-terminal or C-terminal portion of said molecule and eluting the peptide, polypeptide or protein bound or associated following said second affinity chromatography and collecting same.
Still yet another aspect of the present invention contemplates a method of purifying a full length VEGF-B isoform or a related polypeptide from a biological sample, said method comprising subjecting said biological sample to a first optional affinity chromatography comprising an affinity matrix based on affinity binding to multiple contiguous exogenous histidine (His) residues in the N-terminal portion of said VEGF-B isoform, eluting said VEGF-B isoform bound or otherwise associated with said first affinity chromatography and subjecting said eluted VEGF-B isoform to a second affinity chromatography based on affinity of the C-terminal portion of said VEGF-B isoform to heparin or like molecule, and then eluting and collecting said VEGF-B isoform bound or otherwise associated by said second affinity chromatography based on affinity of the C-terminal portion of said VEGF-B isoform to heparin or like molecule.
Still another aspect of the present invention contemplates a method of purifying a homomultimeric polypeptide such as a homodimeric VEGF-B isoform or similar molecule from a biological sample, said method comprising subjecting said biological sample to an optional first affinity chromatography based on affinity for exogenous basic amino acids such as polyHis or hexa-His in the N-terminal portion of said polypeptide; eluting and collecting fractions containing said polypeptide, subjecting said polypeptide to a second affinity chromatography based on affinity to heparin of the C-terminal portion of said polypeptide; eluting and collecting said polypeptide; subjecting said polypeptide to refolding conditions in the presence of Guanidine HCl (GdCl) or arginine and dialyzing refolded polypeptide against acetic acid and/or other acid with similar properties; and purifying said refolded polypeptide by reversed phase chromatography.
Yet still another aspect of the present invention contemplates a method of purifying a full length VEGF-B isoform or a related polypeptide from a biological sample, said method comprising subjecting said biological sample to a first optional affinity chromatography comprising an affinity matrix based on affinity binding to multiple contiguous exogenous histidine (His) residues in the N-terminal portion of said VEGF-B isoform, eluting said VEGF-B isoform bound or otherwise associated with said first affinity chromatography and subjecting said eluted VEGF-B isoform to a cation exchange chromatography, and then eluting and collecting said VEGF-B isoform bound or otherwise associated by said cation exchange chromatography.
Another aspect of the present invention contemplates a method of purifying a homomultimeric polypeptide such as a homodimeric VEGF-B isoform or similar molecule from a biological sample, said method comprising subjecting said biological sample to an optional first affinity chromatography based on affinity for exogenous basic amino acids such as polyHis or hexa-His in the N-terminal portion of said polypeptide; eluting and collecting fractions containing said polypeptide, subjecting said polypeptide to cation exchange chromatography, eluting and collecting said polypeptide; subjecting said polypeptide to refolding conditions in the presence of Guanidine HCl (GdCl) or arginine and dialysing refolded polypeptide against acetic acid and/or other acid with similar properties; and purifying said refolded polypeptide by reversed phase chromatography.
A further aspect of the present invention provides a method for the preparation and purification of a recombinant peptide, polypeptide or protein in homomultimeric form, said method comprising culturing a microorganism or animal cell line comprising a genetic sequence encoding a monomeric form of said peptide, polypeptide or protein under conditions sufficient for expression of said genetic sequence; obtaining cell lysate, culture supernatant fluid, fermentation fluid or conditioned medium from said microorganism or animal cell line and subjecting same to a first optional affinity chromatography step based on affinity to exogenous amino acids present in the N- or C-terminal region of said peptide, polypeptide or protein, collecting fractions containing said peptide, polypeptide or protein and subjecting said fractions to a second affinity chromatography step based on affinity to an inherent property of the amino acid sequence or structure in the C-terminal portion of said polypeptide such as binding to heparin or difference in charge; said affinity chromatography being under chromatographic conditions sufficient for full length but not truncated or non-full length peptide, polypeptide or protein to be bound or otherwise associated by said affinity chromatography; eluting and collecting said full length peptide, polypeptide or protein and subjecting same to refolding conditions in the presence of GdCl and dialyzing against acetic acid or other similar acid and then purifying the refolded polypeptide by reversed phase chromatography.
Yet another aspect of the present invention is directed to the use of a recombinant peptide, polypeptide or protein purified according to the methods herein described in the manufacture of a medicament for the treatment of a disease condition or the manufacture of an agent for use in diagnosis.
The present invention is predicated in part on the ability to discriminate between full length molecules and truncated or clipped variants during purification. This is particularly important for refolding of homomultimers such as homodimers. If truncated or non-full length molecules are co-purified with full length molecules, refolding can result in heteromultimers which may be inactive or exhibit undesirable properties.
Accordingly, one aspect of the present invention provides a method of purifying a peptide, polypeptide or protein from a biological sample said method comprising subjecting the biological sample to affinity chromatography comprising an affinity matrix under chromatographic conditions sufficient for the full length but not a truncated or non-full length peptide, polypeptide or protein corresponding to said full length peptide, polypeptide or protein to be bound to or otherwise associate with the affinity matrix and then eluting said bound or associated peptide, polypeptide or protein from the affinity matrix and collecting same.
Generally, the peptide, polypeptide or protein is in recombinant form. Furthermore, the biological sample is generally a cell lysate, membrane preparation, cytoplasmic extract or other form containing inclusion bodies. The present invention extends, however, to biological samples in the form of culture supernatant fluid, fermentation fluid and conditioned medium.
Preferably, the affinity chromatography is conducted in a column in which case the chromatography is said to be conducted in an affinity chromatography column. The present invention extends to all other forms of chromatography. Reference herein to an affinity matrix includes reference to the solid support within the column or other apparatus to which the peptide, polypeptide or protein binds or otherwise associates. For example, if the affinity chromatography involves a metal chelate affinity chromatography column, a metal cation such as Ni++ or Zn++ is attached to or forms part of the affinity matrix.
The preferred chromatographic conditions are generally described as being “harsh” or “highly stringent” and these conditions enable full length peptide, polypeptide or protein to be bound or otherwise associated during affinity chromatography whereas truncated or “clipped” forms of the molecule are not retained and tend to wash through ahead of the full length molecule. The harsh chromatographic conditions include reducing conditions of from about 5-100 mM DTT for from about 10 minutes to about 4 hours. More preferred reducing conditions are from about 10-60 mM DTT for from about 20 minutes to about 3 hours.
The chromatographic conditions selected assist in reducing non-specific affinity binding to the chromatographic column. For example, in one preferred embodiment, the affinity chromatography is based on a binding or interacting property of an N-terminal or C-terminal region of the peptide, polypeptide or protein being purified.
Truncated or clipped forms of the peptide, polypeptide or protein are generally those molecules which substantially lack that region of the polypeptide which binds to or otherwise associates with the affinity column.
Accordingly, another aspect of the present invention is directed to a method of purifying a recombinant peptide, polypeptide or protein from a biological sample said method comprising subjecting said biological sample to affinity chromatography comprising an affinity matrix which has affinity for an N-terminal or C-terminal region of said peptide, polypeptide or protein but substantially not for the N-terminal or C-terminal region of a truncated or clipped form of said peptide, polypeptide or protein, said affinity chromatography being under chromatographic conditions sufficient to permit binding or association of full length but not truncated or non-full length peptide, polypeptide or protein, and then eluting the bound or associated peptide, polypeptide or protein from the affinity matrix and collecting same. Substantial affinity is not intended to include non-specific affinity.
In order to facilitate the purification process, an optional two-step affinity chromatography protocol is also contemplated by the present invention. For example, a first optional affinity chromatography may target an affinity region in one of the N-terminal or C-terminal portions of the peptide, polypeptide or protein. A second affinity chromatography step would then target the other of the N-terminal or C-terminal portions of the same molecule.
According to this embodiment, there is provided a method of purifying a peptide, polypeptide or protein from a biological sample comprising subjecting said biological sample to an optional first affinity chromatography comprising an affinity matrix which binds or associates said peptide, polypeptide or protein based on affinity to an N-terminal or C-terminal portion of said molecule, eluting off said bound or otherwise associated peptide, polypeptide or protein and subjecting same to a second affinity chromatography based on affinity to the other of an N-terminal or C-terminal portion of said molecule and eluting the peptide, polypeptide or protein bound or associated following said second affinity chromatography and collecting same.
Alternatively, cation exchange chromatography is used in place of a second affinity chromatography.
Accordingly, another aspect of the present invention provides a method of purifying a peptide, polypeptide or protein from a biological sample comprising subjecting said biological sample to an optional first affinity chromatography comprising an affinity matrix which binds or associates said peptide, polypeptide or protein based on affinity to an N-terminal or C-terminal portion of said molecule, eluting off said bound or otherwise associated peptide, polypeptide or protein and subjecting same to cation exchange chromatography and eluting the peptide, polypeptide or protein bound or associated following said cation exchange chromatography and collecting same.
In one embodiment, the first optional affinity chromatography step is based on an exogenous amino acid sequence fused to or otherwise associated with the N-terminal or C-terminal of said peptide, polypeptide or protein and the second affinity chromatographic step is based on an inherent feature of an amino acid sequence or structure of the N-terminus or C-terminus of the molecule.
In a particularly preferred example, the optional first affinity chromatographic step is based on a polymer of basic amino acids such as polyHis or hexa-His residues. Such residues have an affinity for metal cations such as a Ni++ or Zn++. The second affinity chromatographic step is, in a particularly useful example, based on an inherent heparin binding property of the peptide, polypeptide or protein.
On the basis of the highly charged putative heparin binding domain which exists in the COOH-terminus of VEGF-B167, the charge of the truncated VEGF-B167 species is expected to substantially different from the full length form. A more preferred method would include the optional first affinity chromatographic step based on a polymer of basic amino acids such as polyHis or hexa-His residues, which have an affinity for metal cations such as a Ni++ or Zn++, followed by a second affinity chromatographic step based on the inherent charge difference in the C-terminal region of the full length protein as compared to the truncated form. As stated above, cation exchange chromatography may be used to substitute for the second affinity chromatographic step.
The preferred peptide, polypeptide or protein of the present invention is a growth factor, cytokine or haemopoietic regulator of mammalian and preferably human origin. Reference to “mammalian” includes primates, humans, livestock animals, laboratory test animals and companion animals. A more preferred polypeptide or protein is a growth factor such as VEGF and in particular human-derived VEGF. A particularly preferred polypeptide or protein is VEGF-B or more particularly an isoform thereof such as VEGF-B167, VEGF-B186 or VEGF-B10-108 (tagged or untagged with an amino acid sequence such as His6). The amino acid sequence of VEGF-B167 is shown in
In a preferred embodiment, the VEGF-B isoform comprises a hexa-His at its N-terminal amino acid end portion and exhibits inherent heparin binding properties at its C-terminal amino acid end portion. This is referred to herein as a “tagged” VEGF-B isoform.
Accordingly, another aspect of the present invention contemplates a method of a purifying full length VEGF-B isoform or a related polypeptide from a biological sample, said method comprising subjecting said biological sample to a first optional affinity chromatography comprising an affinity matrix based on affinity binding to multiple contiguous exogenous His residues in the N-terminal portion of said VEGF-B isoform, eluting said VEGF-B isoform bound or otherwise associated with said first affinity chromatography and subjecting said eluted VEGF-B isoform to a second affinity chromatography based on affinity of the C-terminal portion of said VEGF-B isoform to heparin or like molecule, and then eluting and collecting said VEGF-B isoform bound or otherwise associated by said second affinity chromatography.
Generally, the second and optional first affinity chromatography are conducted under chromatographic conditions sufficient for the full length but not truncated or non-full length VEGF-B isoform to be bound to or associated with the affinity chromatography.
In an alternative embodiment, cation exchange chromatography is used in place of the second affinity chromatographic step.
Accordingly, the present invention contemplates a method of purifying a full length VEGF-B isoform or a related polypeptide from a biological sample, said method comprising subjecting said biological sample to a first optional affinity chromatography comprising an affinity matrix based on affinity binding to multiple contiguous exogenous histidine (His) residues in the N-terminal portion of said VEGF-B isoform, eluting said VEGF-B isoform bound or otherwise associated with said first affinity chromatography and subjecting said eluted VEGF-B isoform to a cation exchange chromatography, and then eluting and collecting said VEGF-B isoform bound or otherwise associated by said cation exchange chromatography.
The collected, purified VEGF-B isoform or other polypeptide is generally subjected to refolding. The essence of this aspect of the present invention is that only full length monomers be available for refolding otherwise heteromultimers will result which may be inactive or exhibit undesirable properties. In a preferred embodiment, the peptide, polypeptide or protein and in particular the VEGF-B isoform is subjected to a cleavage reaction to remove any exogenous basic amino acids such as those introduced or otherwise associated with the N-terminal region.
Preferably, the purified monomeric forms of a VEGF-B isoform or other polypeptide are subjected to refolding conditions in 0.1-10 M GdCl, and more preferably 0.3-5 M GdCl followed by dialyzing against acetic acid or other suitable acid. Alternatively, arginine may be employed in the refolding conditions. The refolded multimeric polypeptides, and more preferably homomultimeric polypeptides are then subjected to purification by reversed phase chromatography or other convenient means.
Accordingly, in a particularly preferred embodiment, the present invention contemplates a method of purifying a homomultimeric polypeptide such as homodimeric VEGF-B167 or similar molecule from a biological sample, said method comprising subjecting said biological sample to an optional first affinity chromatography based on affinity for exogenous basic amino acids such as polyHis or hexa-His in the N-terminal portion of said polypeptide; eluting and collecting fractions containing said polypeptide, subjecting said polypeptide to a second affinity chromatography based on affinity to heparin of the C-terminal portion of said polypeptide; eluting and collecting said polypeptide; subjecting said polypeptide to refolding conditions in the presence of GdCl or arginine and dialyzing the refolded polypeptide against acetic acid and/or other acid with similar properties; and purifying said refolded polypeptide by reversed phase chromatography.
In an alternative embodiment, the present invention provides a method of purifying a homomultimeric polypeptide such as a homodimeric VEGF-B isoform or similar molecule from a biological sample, said method comprising subjecting said biological sample to an optional first affinity chromatography based on affinity for exogenous basic amino acids such as polyHis or hexa-His in the N-terminal portion of said polypeptide; eluting and collecting fractions containing said polypeptide, subjecting said polypeptide to cation exchange chromatography, eluting and collecting said polypeptide; subjecting said polypeptide to refolding conditions in the presence of GdCl or arginine and dialyzing the refolded polypeptide against acetic acid and/or other acid with similar properties; and purifying said refolded polypeptide by reversed phase chromatography.
In a preferred aspect of the abovementioned embodiments, the refolded polypeptide is subjected to cleavage conditions to remove some or all of the exogenous basic amino acids such as polyHis or hexa-His prior to purification.
In a particular embodiment, the purification protocol enables purification of all isoforms of VEGF-B including isoforms -167, -186 and 10-108. A particularly useful purification protocol is as follows:
Purified refolded His-tagged VEGF-B may be suitable in its own right. However, for lyophilization of the refolded His-tagged VEGF-B the following steps are employed:
In the above steps, Steps 6 and 10 can be replaced by purification by polyhydroxyethyl A hydrophilic chromatography.
The present invention further contemplates compositions comprising purified peptide, polypeptide or protein prepared by the method of the present invention such a composition comprising purified homomultimeric forms of said peptide, polypeptide or protein. Preferred compositions comprise purified homodimeric forms of VEGF-B isoform or related molecule. The composition may also contain one or more pharmaceutically acceptable carriers and/or diluents.
Still another aspect of the present invention provides a method for the preparation and purification of a recombinant peptide, polypeptide or protein in homomultimeric form, said method comprising culturing a microorganism or animal cell line comprising a genetic sequence encoding a monomeric form of said peptide, polypeptide or protein under conditions sufficient for expression of said genetic sequence; obtaining cell lysate, culture supernatant fluid, fermentation fluid or conditioned medium from said microorganism or animal cell line and subjecting same to a first optional affinity chromatography step based on affinity to exogenous amino acids present in the N- or C-terminal region of said peptide, polypeptide or protein, collecting fractions containing said peptide, polypeptide or protein and subjecting said fractions to a second affinity chromatography step based on affinity to an inherent property of the amino acid sequence or structure in the C-terminal portion of said polypeptide such as binding to heparin or difference in charge; said affinity chromatography being under chromatographic conditions sufficient for full length but not truncated or non-full length peptide, polypeptide or protein to be bound or otherwise associated by said affinity chromatography; eluting and collecting said full length peptide, polypeptide or protein and subjecting same to refolding conditions in the presence of GdCl or arginine and dialysing against acetic acid or other similar acid and then purifying the refolded polypeptide by reversed phase chromatography.
The present invention is further described by the following non-limiting Examples.
pET15b-VEGF-B167
The coding region of the mature human VEGF-B167 protein was amplified using PCR (94° C./2 min—1 cycle; 94° C./15 sec, 60° C./15 sec, 72° C./2 min—35 cycles; 72° C./5 min B 1 cycle; Stratagene pfu turbo; Corbett Research PC-960-G thermal cycler) to introduce in frame Nde I and BamH1 restriction enzyme sites at the 5′ and 3′ ends, respectively, using the following oligonucleotides:
The resulting PCR derived DNA fragment was gel purified, digested with Nde I and BamH1, gel purified again, and then cloned into NdeI/BamH1 digested pET15b (Novagen, Madison Wis., USA). When expressed in E. coli the VEGF-B167 protein has an additional 21 amino acids at the N-terminus that incorporates a hexa-His tag and a thrombin cleavage site (
pET15b-VEGF-B167 was transformed into BL21(DE3) GOLD E. coli (Stratagene, Catalogue #230132) using an Electroporator (BioRad, USA) according to the manufacturer's instructions. The transformation reaction was plated onto LB ampicillin plates and incubated overnight at 37° C. Four ampicillin resistant colonies were picked, grown overnight and DNA extracted using a standard miniprep protocol (Bio101). Miniprep DNA was analyzed using the restriction enzymes BamH1 and Nde1. A colony giving the appropriate fragment was used for preparation of a glycerol stock for subsequent studies.
For preparation of a seed culture a 50 ml LB broth (10 g tryptone, 5 g yeast extract, 5 g NaCl, pH 7.0) was inoculated with pET15b-VEGF-B167 transformed BL21(DE3) GOLD from the glycerol stock. The culture was allowed to grow at 37° C. (with continuous shaking) to OD600 0.7 and stored at 4° C. until required (usually no more than 4 days).
For protein production one litre of LB medium was inoculated with 5 ml of seed culture and incubated at 37° C. Cells were grown to OD600 0.7 (typically 5 hrs) and induced with 1 mM IPTG (Amersham Pharmacia, Sweden) for two hrs. Yields were typically 3-4 g wet cells per litre of culture (
Cell Lysis
Frozen cell pellets were thawed and 3 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl) was added per gram of cells. Once thoroughly mixed, 40 μl PMSF (10 mM) (phenylmethylsulfonyl fluoride: Sigma-Aldrich, USA) and 40 μl lysozyme (20 mg/ml) were added per gram of cells. The solution was mixed thoroughly and allowed to stand for 30 min at 37° C. Deoxycholic acid (4 mg/gram cells) was added and the solution mixed until viscous. DNase I (1 mg/ml: 20 μl/g of cells) was mixed with the cell lysate and allowed to stand for 30 min at 37° C., or until no longer viscous. Insoluble material (including inclusion bodies) was pelleted by centrifugation at 13,500 rpm for 30 min at 4° C. (
Washing of Inclusion Bodies
Pelleted insoluble material was resuspended in 35 ml of 100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT, 2 M urea, 2% v/v Triton-X100 (Buffer 1) per litre of starting fermentation product. The suspension was placed on ice and subjected to sonication (6×1 min on high power with 2 min intervals) using a Braun sonicator, followed by centrifugation (13,500 rpm, 4° C.) for 30 min. This wash method was repeated two additional times. After the third wash, the pelleted material was resuspended in 25 ml of 100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT (Buffer 2) per litre of starting fermentation product, sonicated for one min at 4° C. and centrifuged (13,500 rpm, 4° C.) for 30 min. This second wash step was also repeated twice (
Solubilization
The washed inclusion bodies were solubilized by the addition of 10 ml 6M GdCl, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.5 (Buffer 3). In order to fully solubilize inclusion bodies, the suspension was placed on ice and subjected to sonication for one minute at high power. The solution was centrifuged at 18,000 rpm for 15 min in order to separate undissolved material. The solution was reduced by the addition of 20 mM DTT and allowed to stand at 37° C. for 30 min.
Ni2+ Affinity Chromatography
10 ml metal chelating resin was packed in a BioRad EconoPak column using Chelating Sepharose Fast Flow resin (Amersham Pharmacia, Sweden). The column was washed with three column volumes milliQ H2O, followed by five column volumes of 0.1 M NiSO4. A further three column volumes of milliQ H2O followed by three column volumes of 6 M GdCl, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.5 (Buffer 3) were used to equilibrate the column. The reduced protein solution was loaded onto the column at 3 ml/min using a Pharmacia P1 peristaltic pump. To enhance recovery, the flow through was reapplied to the column five times prior to washing the column with three column volumes of the same buffer. The column was then washed with 5 column volumes of 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.5 (Buffer 4), followed by 5 column volumes of 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 6.3 (Buffer 5). The bound fraction was eluted with 6-10×5 ml volumes of 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, 0.5 M Imidazole, pH 5.9 (Buffer 6). Fractions containing protein were identified by Bradford assay and an aliquot of each fraction was subjected to ethanol precipitation to remove the high salt content for subsequent analysis by SDS-PAGE electrophoresis. Samples were electrophoresed on an SDS-PAGE gel under reducing conditions. Coomassie staining revealed the major band to be running with an apparent molecular weight of 22 kDa (
Subsequent autoradiography indicated that this band was indeed VEGF-B167 with additional bands corresponding to clipped forms of VEGF-B167 also being observed (
A second major band runs with an apparent molecular weight of approximately 18 kDa on SDS-PAGE under reducing conditions. Failure to remove this clipped variant would result in heterogenous forms of VEGF-B after refolding. Consequently, it was essential to develop a technique to remove the clipped form from the full-length VEGF-B167 altogether. The use of heparin-sepharose under both reducing and denaturing conditions was successful in achieving this objective. It is likely that the clipped form does not possess the same charge profile as the putative C-terminal heparin-binding domain present on full-length VEGF-B167.
Heparin Sepharose affinity: Removal of C-terminally clipped VEGF-B The pooled fractions from Ni2+ purification were reduced with 40 mM DTT for 1-2 hrs. A 10 ml heparin-sepharose CL6B column was prepared by first washing with 5 column volumes of milliQ H2O and equilibrating with 4 column volumes of 6 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, 1 mM EDTA, 20 mM DTT, pH 8.5 (Buffer 7). The urea concentration of the protein solution was reduced from 8 M to 6 M with 0.1 M NaH2PO4, 10 mM Tris-HCl, 1 mM EDTA, 20 mM DTT, pH 8.5. The protein solution was loaded onto the column at 3 ml/min. The C-terminally clipped VEGF-B eluted in the flow through and wash (
An Alternative Approach for the Removal of C-Terminally Clipped VEGF-B: Cation Exchange Chromatography
Pooled fractions from Ni2+ purification were reduced with 40 mM DTT for 1-2 hours. A 50 mL SP-Sepharose fast flow column (Amersham Pharmacia, Sweden) was prepared by equilibrating with five column volumes of 6 M urea, 10 mM NaH2PO4, 10 mM Tris-HCl, pH 5.8 (Buffer 9). The protein solution was diluted three-fold with Buffer 9, and loaded onto the column at 10 mL/min. Full length monomeric VEGF-B167 was separated from the truncated form using a linear gradient formed between buffer A and 6 M urea, 10 mM NaH2PO4, 10 mM Tris-HCl, 1M NaCl, pH 5.8 (Buffer 10) (see
1. Incorporation of GdCl in Refolding Buffer
Purified monomeric His6-VEGF-B167 from the heparin-sepharose purification was reduced with 20 mM DTT for 45 minutes at 37° C., followed by dilution to 60-200 μg/ml with Buffer 7 (6 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, 1 mM EDTA, 20 mM DTT, pH 8.5). The protein solution was dialyzed at room temperature against Buffer 11 (100 mM Tris-HCl, 5 mM cysteine, 1 mM cystine, 0.5 M GdCl, pH 8.5) for one to three days.
2. Incorporation of Arginine in Refolding Buffer
Purified monomeric His6-VEGF-B167 from the heparin-sepharose purification was reduced with 20 mM DTT for 45 minutes at 37° C., followed by dilution to 60-200 μg/ml with Buffer 7 (6 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, 1 mM EDTA, 20 mM DTT, pH 8.5). The protein solution was dialyzed at room temperature against Buffer 27 (100 mM Tris-HCl, 5 mM cysteine, 1 mM cystine, 0.4 M arginine, pH 8.5) for one to three days.
Major bands positioned at approximately 48 kDa and 22 kDa in Western blot analysis correspond to dimeric and monomeric forms of His6-VEGF-B167, respectively, under non-reducing conditions. In addition, higher oligomeric forms of His6-VEGF-B167 are present (
The acidified protein solution was loaded onto a Brownlee C8 reversed-phase column pre-equilibrated at 45° C. in Buffer 12 (0.15% v/v Trifluoroacetic acid, TFA) using a Beckman GOLD liquid chromatographic system. Fractions were collected at one min intervals and monitored by SDS PAGE (
The acidified protein solution was loaded onto a Vydac 300 C8 reversed-phase column (2.2×10 cm; Higgins Analytical, USA) pre-equilibrated in Buffer 12 (0.15% v/v TFA) using a Beckman GOLD liquid chromatographic system. The column was washed with two column volumes of Buffer 12 followed by two column volumes of 35% Buffer 14 (60% v/v acetonitrile, 0.13% TFA). A linear gradient was formed with 35-60% Buffer 14 over 50 mins at a flow rate of 20 ml/min. Fractions containing dimeric His6-VEG-B167 were pooled (as in Example 6), diluted ten-fold with Buffer 15 (80% v/v n-propanol, 10 mM NaCl, pH 2) and loaded on a Polyhydroxyethyl A hydrophilic column (2.1×25 cm; PolyLC, USA) pre-equilibrated with 25% Buffer 15. Dimeric protein was eluted using a linear gradient formed with 25-45% Buffer 16 (10 mM NaCl, pH 2.0). The purified dimeric His6-VEGF-B167 was diluted 10-fold with Buffer 12, reapplied to the C8 column and eluted with 100% v/v Buffer 14 to minimize sample dilution. Purified material was analysed by SDS PAGE and Western blot analysis (
To separate dimeric His6-VEGF-B167 from mono- and multimeric species the acidified protein solution was diluted five-fold with Buffer 15 (80% v/v n-propanol, 10 mM NaCl, pH 2.0) and loaded onto a Polyhydroxyethyl A hydrophilic column (2.1×25 cm; PolyLC, USA) pre-equilibrated with three column volumes of Buffer 15 at 20 ml/min. The column was washed with two column volumes of 25% Buffer 16 (10 mM NaCl, pH 2.0). A linear gradient was formed with 25-45% Buffer 16 (10 mM NaCl, pH 2.0) over 40 minutes using a flow rate of 10 ml/min. Fractions containing dimeric His6-VEGF-B167 were combined, diluted four-fold with Buffer 12 (0.15% TFA), and loaded onto a Vydac 300 C8 reversed-phase column (2.2×10 cm; Higgins Analytical, USA) pre-equilibrated with Buffer 12. The column was equilibrated with two column volumes of Buffer 12 followed by two column volumes of 35% Buffer 14 (60% v/v acetonitrile, 0.13% TFA). A linear gradient was formed with 35-60% Buffer 14 over 50 mins at 20 ml/min. Fractions containing dimeric His6-VEGF-B167 were pooled, diluted with Buffer 12, and reapplied to the C8 column. The protein was eluted with 100% Buffer 14 to minimize sample dilution.
Modified pET15b-VEGF-B167
The coding region of the mature human VEGF-B167 protein was amplified using PCR (96° C./2 min—1 cycle; 96° C./10 sec, 55° C./10 sec, 72° C./1 min—35 cycles; 72° C./2 min—1 cycle; Stratagene pfu turbo; Corbett Research PC-960-G thermal cycler) to introduce in frame Nco I and BamH1 restriction enzyme sites at the 5′ and 3′ ends, respectively, using the following oligonucleotides:
The resulting PCR derived DNA fragment was gel purified, digested with NcoI and BamH1, gel purified again, and then cloned into NcoI/BamH1 digested pET15b (Novagen, USA), resulting in the removal of the His6-tag and thrombin cleavage site. When expressed in E. coli the untagged VEGF-B167 protein has an additional glycine residue at the N-terminus.
The modified pET15b-VEGF-B167 was transformed into BL21(DE3) GOLD E. coli using an electroporator (BioRad, USA) according to the manufacturer's instructions. The transformation reaction was plated onto LB ampicillin plates and incubated overnight at 37° C. Sixteen ampicillin resistant colonies were picked, grown overnight and DNA extracted using a standard miniprep protocol (Bio101). Miniprep DNA was analyzed using the restriction enzymes BamH1 and Nco1. A colony giving the appropriate fragment was used for preparation of a glycerol stock for subsequent studies.
For preparation of a seed culture a 50 ml LB broth (10 g tryptone, 5 g yeast extract, 10 g NaCl, pH 7.5) was inoculated with pET15b-VEGF-B167 transformed BL21(DE3) GOLD from the glycerol stock. The culture was allowed to grow at 37° C. (with continuous shaking) to OD600 0.7 and stored at 4° C. until required (usually no more than 4 days).
For protein production one litre of LB medium was inoculated with 20 ml of seed culture and incubated at 37° C. Cells were grown to OD600 0.7 (typically 3-4 hrs) and induced with 1 mM IPTG (Amersham Pharmacia Biotech, Sweden) for two hours. Yields were typically 3-4 g wet cells per litre of culture. Cells were pelleted by centrifugation and pellets stored frozen at −80° C. until required.
Cell Lysis
Frozen cell pellets were thawed and 3 ml lysis buffer (50 mM Tris-HCl, pH8.0, 1 mM EDTA, 100 mM NaCl) was added per gram of cells. Once thoroughly mixed, 40 μl PMSF (10 mM) and 40 μl lysozyme (20 mg/ml) were added per gram of cells. The solution was mixed thoroughly and allowed to stand for 1 hour at 37° C. Deoxycholic acid (4 mg/gram cells) was added and the solution mixed until viscous. DNase I (1 mg/ml: 20 μl/g of cells) was mixed with the cell lysate and allowed to stand for 30 min at 37° C., or until no longer viscous. Insoluble material (including inclusion bodies) was pelleted by centrifugation at 13,500 rpm for 45 min at 4° C.
Washing of Inclusion Bodies
Pelleted insoluble material was resuspended in 35 ml of Buffer 1 (100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT, 2 M urea, 2% v/v Triton X-100) per litre of starting fermentation product. The suspension was placed on ice and subjected to sonication (6×1 min on high power with 2 min intervals), followed by centrifugation (13,500 rpm, 4° C.) for 30 min. This wash method was repeated two additional times. After the third wash, the pelleted material was resuspended in 25 ml of Buffer 2 (100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT) per litre of starting fermentation product, sonicated for one min at 4° C. and centrifuged (13,500 rpm, 4° C.) for 30 min. This second wash step was also repeated twice. The washed inclusion bodies were pelleted as above and stored at −70° C. until required.
Solubilization
The washed inclusion bodies were solubilized by the addition of 20 ml Buffer 3 (6 M GdCl, 10 mM NaH2PO4, 10 mM Tris-HCl, pH 8.5). In order to fully solubilize inclusion bodies, the suspension was placed on ice and subjected to sonication for one minute at high power. The solution was centrifuged at 18,000 rpm for 15 min in order to separate undissolved material. The solution was reduced by the addition of 20 mM DTT, 1 mM EDTA and allowed to stand at 37° C. for 2 hours.
Cation Exchange Chromatography
A 50 ml SP-Sepharose column (Amersham Pharmacia Biotech, Sweden) was prepared by equilibrating the column with five column volumes of Buffer 9 (6 M urea, 10 mM NaH2PO4, 10 mM Tris-HCl, pH 5.8). The protein solution was adjusted to pH 5.8, and loaded onto the column at 5 ml/min. Full length monomeric VEGF-B167 was separated from the truncated form and other contaminating host cell proteins using a linear gradient formed between Buffer 9 and Buffer 10 (6 M urea, 10 mM NaH2PO4, 10 mM Tris-HCl, 1M NaCl, pH 5.8).
Purified monomeric untagged VEGF-B167 from the cation exchange purification was reduced with 20 mM DTT for 45 minutes at 37° C., followed by dilution to 60-100 μg/ml with Buffer 7 (6 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, 1 mM EDTA, 20 mM DTT, pH 8.5). The protein solution was dialyzed at room temperature against Buffer 11 (100 mM Tris-HCl, 5 mM cysteine, 1 mM cystine, 2 mM EDTA, 0.5 M GdCl, pH 8.5) for one to three days. Major bands positioned at approximately 48 kDa and 22 kDa in Western blot analysis correspond to dimeric and monomeric forms of untagged VEGF-B167, respectively, under non-reducing conditions. In addition, higher oligomeric forms of untagged VEGF-B167 are present.
The protein solution was dialyzed against 0.1 M acetic acid overnight and filtered through a 0.22 μM cellulose acetate filter (Corning, USA) to remove particulate matter.
To separate dimeric untagged VEGF-B167 from mono- and multimeric species the acidified protein solution was diluted five-fold with Buffer 15 (80% v/v n-propanol, 10 mM NaCl, pH 2.0) and loaded onto a Polyhydroxyethyl A hydrophilic column (2.1×25 cm; PolyLC, USA) pre-equilibrated with three column volumes of Buffer 15 at 20 ml/min. The column was washed with two column volumes of 25% Buffer 16 (10 mM NaCl, pH 2.0). A linear gradient was formed with 25-45% Buffer 16 over 40 minutes using a flow rate of 10 ml/min. Fractions containing dimeric VEGF-B167 were combined, diluted four-fold with Buffer 12 (0.15% TFA), and loaded onto a Vydac 300 C8 reversed-phase column (2.2×10 cm; Higgins Analytical, USA) pre-equilibrated with Buffer 12. The column was washed with two column volumes of Buffer 12 followed by two column volumes of 35% Buffer 14 (60% v/v acetonitrile, 0.13% TFA). A linear gradient was formed with 35-60% Buffer 14 over 50 mins at 20 ml/min. Fractions containing dimeric VEGF-B167 were pooled, diluted with Buffer 12, and reapplied to the C8 column. The protein was eluted with 100% Buffer 14 to minimize sample dilution.
pET15b-VEGF-B186
The coding region of the mature human VEGF-B186 protein was amplified using PCR (94° C./2 min—1 cycle; 94° C./15 sec, 60° C./15 sec, 72° C./2 min—35 cycles; 72° C./5 min—1 cycle; Stratagene pfu turbo; Corbett Research PC-960-G thermal cycler) to introduce in frame Nde I and BamH1 restriction enzyme sites at the 5′ and 3′ ends, respectively, using the following oligonucleotides:
The resulting PCR derived DNA fragment was gel purified, digested with NdeI and BamH1, gel purified again, and then cloned into NdeI/BamH1 digested pET15b (Novagen, USA). When expressed in E. coli the VEGF-B186 protein has an additional 21 amino acids at the N-terminus that incorporates a hexa-His tag and a thrombin cleavage site.
The pET15b-VEGF-B186 was transformed into BL21(DE3) GOLD E. coli using an electroporator (BioRad, USA) according to the manufacturer's instructions. The transformation reaction was plated onto LB ampicillin plates and incubated overnight at 37° C. Four ampicillin resistant colonies were picked, grown overnight and DNA extracted using a standard miniprep protocol (Bio101). Miniprep DNA was analyzed using the restriction enzymes BamH1 and Nde1. A colony giving the appropriate fragment was used for preparation of a glycerol stock for subsequent studies.
For preparation of a seed culture a 50 ml LB broth (10 g tryptone, 5 g yeast extract, 5 g NaCl, pH 7.0) was inoculated with pET15b-VEGF-B186 transformed BL21(DE3) GOLD from the glycerol stock. The culture was allowed to grow at 37° C. (with continuous shaking) to OD600 0.7 and stored at 4° C. until required (usually no more than 4 days).
For protein production one litre of LB medium was inoculated with 5 ml of seed culture and incubated at 37° C. Cells were grown to OD600 0.7 (typically 5 hrs) and induced with 1 mM IPTG for two hrs. Yields were typically 3-4 g wet cells per litre of culture. Cells were pelleted by centrifugation and pellets stored frozen at −80° C. until required.
Cell Lysis
Frozen cell pellets were thawed and 20 ml lysis buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 100 mM NaCl) was added per gram of cells. Once thoroughly mixed, 40 μl PMSF (10 mM) and 40 μl lysozyme (20 mg/ml) were added per gram of cells. The solution was mixed thoroughly and allowed to stand for 30 min at 37° C. Deoxycholic acid (4 mg/gram cells) was added and the solution mixed until viscous. DNase I (1 mg/ml: 20 μl/g of cells) was mixed with the cell lysate and allowed to stand for 30 min at 37° C., or until no longer viscous. Insoluble material (including inclusion bodies) was pelleted by centrifugation at 13,500 rpm for 30 min at 4° C.
Washing of Inclusion Bodies
Pelleted insoluble material was resuspended in 100 ml of Buffer 22 (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl) per litre of starting fermentation product. The suspension was placed on ice and subjected to sonication (6×1 min on high power with 2 min intervals), followed by centrifugation (13,500 rpm, 4° C.) for 30 min. The pelleted material was resuspended in 50 ml of Buffer 23 (2 M urea, 100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA) per litre of starting fermentation material, sonicated for one min at 4° C. and centrifuged (13,500 rpm, 4° C.) for 30 min. This second wash step was repeated twice. The washed inclusion bodies were pelleted as above and stored at −70° C. until required.
Solubilization
The washed inclusion bodies (2.5 g) were solubilized by the addition of 1 L of Buffer 24 (8 M urea, 100 mM Tris-HCl, 50 mM NH4SO4, 5% (v/v) Triton X-100, 100 mM DTT, pH 9.0). In order to fully solubilize inclusion bodies, the suspension was homogenized with an Ultra-turrax T8 homogenizer (Janke & Kunkel GmbH, Germany) for 3 min at full power and then incubated at 45° C. for 1 hour.
Cation Exchange Chromatography
This method describes a means by which a truncated component of His6-VEGF-B186 may be selectively separated from full length His6-VEGF-B186. This shortened His6-VEGF-B186 component appears to non-covalently associate with the full-length material. This interaction can be disrupted by the presence of the non-ionic detergent Triton X-100.
The solubilized inclusion bodies suspension was adjusted to pH 5.8 prior to loading on a 100 ml SP-sepharose cation exchange column (Amersham Pharmacia Biotech, Sweden) pre-equilibrated with three column volumes of Buffer 25 (4 M urea, 100 mM Tris-HCl, 50 mM NH4SO4, 1% Triton X-100, 2.5 mM β-mercaptoethanol, pH 5.8). The sample was loaded through the system pump of an ÅKTA Explorer 100 (Amersham Pharmacia Biotech, Sweden) at a flow rate of 10 ml/min. Bound material was washed with 10 column volumes of Buffer 25. The bound material was eluted with a gradient generated over 5 column volumes from 0-100% Buffer 26 (4 M urea, 0.1 M Tris-HCl, 50 mM NH4SO4, 1% Triton X-100, 2.5 mM β-mercaptoethanol, 1 M NaCl, pH 5.8). Eluant was fractionated into 1 minute/10 ml fractions. Those fractions within the conductivity range of 15-75 mS/cm were pooled and diluted 10-fold with Buffer 24 (8M urea, 100 mM Tris-HCl, 50 mM NH4SO4, 5% v/v Triton X-100, 100 mM DTT pH 9.0). The solution was adjusted to pH 9.0 and incubated at 45° C. for 1 hr. The solution was readjusted to pH 5.8 and the previous chromatography step repeated. Collected fractions were analyzed by SDS-PAGE Coomassie and Western blot analysis using VEGF-B-specific monoclonal antibodies.
The purified monomeric His6-VEGF-B186 was reduced with 20 mM DTT for 45 minutes at 37° C., followed by dilution to 60-200 μg/mL with Buffer 7 (6 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, 1 mM EDTA, 20 mM DTT, pH 8.5). The protein solution was dialyzed at room temperature against Buffer 11 (100 mM Tris-HCl, 5 mM cysteine, 1 mM cystine, 0.5 M GdCl, pH8.5) for one to three days. Major bands corresponding to dimeric and monomeric forms of His6-VEGF-B186 were identified in addition to higher oligomeric forms of His6-VEGF-B186. Coomassie staining suggested >20% conversion to dimer. The protein solution was dialyzed against 0.1 M acetic acid overnight and filtered through a 0.22 μM cellulose acetate filter (Corning, USA) to remove particulate matter.
To separate dimeric His6-VEGF-B186 from mono- and multimeric species the acidified protein solution was diluted five-fold with Buffer 15 (80% v/v n-propanol, 10 mM NaCl, pH 2.0) and loaded onto a Polyhydroxyethyl A hydrophilic column (2.1×25 cm; PolyLC, USA) pre-equilibrated with three column volumes of Buffer 15 at 20 ml/min. The column was washed with two column volumes of 25% Buffer 16 (10 mM NaCl, pH 2.0). A linear gradient was formed with 25-45% Buffer 16 (10 mM NaCl, pH 2.0) over 40 minutes at a flow rate of 10 ml/min. Fractions containing dimeric His6-VEGF-B186 were combined, diluted four-fold with Buffer 12 (0.15% TFA), and loaded onto a Vydac 300 C8 reversed-phase column (2.2×10 cm; Higgins Analytical, USA) pre-equilibrated with Buffer 12. The column was washed with two column volumes of Buffer 12 followed by two column volumes of 35% Buffer 14 (60% v/v acetonitrile, 0.13% TFA). A linear gradient was formed with 35-60% Buffer 14 over 50 mins at 20 ml/min. Fractions containing dimeric His6-VEGF-B186 were pooled, diluted with Buffer 12, and reapplied to the C8 column. The purified dimeric protein was eluted with 100% Buffer 14 to minimize sample dilution (
pQE30-VEGF-B10-108
The coding region of the mature human VEGF-B10-108 protein was amplified using PCR (95°2 min—1 cycle; 94° C./1 min, 60° C./1 min, 72° C./1 min—30 cycles; 72° C./15 min—1 cycle; 1.5 U Expand High Fidelity PCR System enzyme mix (Roche Diagnostics GmbH, Germany; Corbett Research PC-960-G thermal cycler) to introduce in frame BamHI and HindIII restriction enzyme sites at the 5′ and 3′ ends, respectively, using the following oligonucleotides:
5′Oligo 5′-CACGGATCCGCAGCACACTATCACCAGAGGAAAG-3′ SEQ ID NO: 9
3′Oligo 5′-GCATAAGCTTTCACTTTTTTTTAGGTCTGCATTC-3′ SEQ ID NO: 10
The resulting PCR derived DNA fragment was gel purified, digested with BamHI and HindIII, gel purified again, then cloned into BamHI and HindIII digested pQE30 (QIAGEN GmbH, Germany). The ligated DNA was transformed into DH5α E. coli using an electroporator (BioRad, USA) according to the manufacturer's instructions. The transformation reaction was plated onto LB ampicillin plates and incubated overnight at 37° C. Six ampicillin resistant colonies were picked for colony PCR analysis using pQE30 primers (QIAGEN GmbH, Germany) to identify fragment insertion. Colonies with the appropriate fragment were grown overnight and the plasmid DNA extracted using a standard midiprep protocol (QIAGEN GmbH, Germany). The DNA was sequenced using a BigDye Sequencing Kit (Applied Biosystems, USA). When expressed in E. coli the VEGF-B10-108 protein has an additional 16 amino acids at the N-terminus that incorporates a hexa-His tag and a Genenase I (New England Biolabs, USA) cleavage site.
The pQE30-VEGF-B10-108 was transformed into M15[pREP4] E. coli (QIAGEN GmbH, Germany) using an electroporator (BioRad, USA) according to the manufacturer's instructions. The transformation reaction was plated onto LB ampicillin and kanamycin plates and incubated overnight at 37° C. A single ampicillin and kanamycin resistant colony was picked, grown overnight and used for preparation of a glycerol stock for subsequent studies.
For preparation of a seed culture a 50 ml LB broth (10 g tryptone, 5 g yeast extract, 5 g NaCl, pH 7.0) with ampicillin and kanamycin was inoculated with pQE30-VEGF-B10-108 transformed M15[pREP4] from the glycerol stock. The culture was allowed to grow overnight at 37° C. with continuous shaking.
For protein production one litre of LB medium with ampicillin and kanamycin was inoculated with 20 ml of seed culture and incubated at 37° C. Cells were grown to OD600 0.7 (typically 4 hrs) and induced with 1 mM IPTG (Amersham Pharmacia Biotech, Sweden) for 4 hrs. Yields were typically 5-6 g wet cells per litre of culture. Cells were pelleted by centrifugation and pellets stored frozen at −80° C. until required.
Cell Lysis
Frozen cell pellets were thawed and 3 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl) was added per gram of cells. Once thoroughly mixed, PMSF (40 μl, 10 mM) and lysozyme (40 μl, 20 mg/ml) were added per gram of cells. The solution was mixed thoroughly and allowed to stand for 30 min at 37° C. Deoxycholic acid (4 mg/gram cells) was added and the solution mixed until viscous. DNase I (1 mg/ml: 20 μl/g of cells) was mixed with the cell lysate and allowed to stand for 30 min at 37° C., or until no longer viscous. Insoluble material (including inclusion bodies) was pelleted by centrifugation at 13,500 rpm for 30 min at 4° C.
Washing of Inclusion Bodies
Pelleted insoluble material was resuspended in 35 ml of Buffer 1 (100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT, 2 M urea, 2% v/v Triton X-100) per litre of starting fermentation product. The suspension was placed on ice and subjected to sonication (6×1 min on high power with 2 min intervals; Braun, Germany), followed by centrifugation (13,500 rpm, 4° C.) for 30 min. This wash method was repeated two additional times. After the third wash, the pelleted material was resuspended in 25 ml of Buffer 2 (100 mM Tris-HCl, pH 7.0, 5 mM EDTA, 10 mM DTT) per litre of starting fermentation product, sonicated for one min at 4° C. and centrifuged (13,500 rpm, 4° C.) for 30 min. This second wash step was also repeated twice. The washed inclusion bodies were pelleted as above and stored at −70° C. until required.
Solubilization
The washed inclusion bodies were solubilized by the addition of 20 ml Buffer 3 (6M GdCl, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.5). In order to fully solubilize inclusion bodies, the suspension was placed on ice and subjected to sonication for one minute at high power. The solution was reduced by the addition of 20 mM β-mercaptoethanol and incubated at 37° C. for 30 min. Insoluble material was removed by centrifugation at 18,000 rpm for 15 mm.
Ni2+ Affinity Chromatography
A column containing 20 ml Ni-NTA Superflow resin (QIAGEN GmbH, Germany) was washed with 10 column volumes of milliQ H2O followed by five column volumes of Buffer 3. The reduced protein solution was loaded onto the column at 4 ml/min and washed with five volumes of Buffer 3. The bound non-specific endogenous bacterial proteins were removed from the column by washing with five column volumes of Buffer 17 (6M GdCl, 0.1 M NaH2PO4, 10 mM Tris-HCl, 20 mM imidazole, pH 6.3) followed by five column volumes of Buffer 3. The bound protein was eluted with 10 column volumes of Buffer 18 (6M GdCl, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 4.5). The fractions containing His6-tagged VEGF-B10-108, as determined by Western blot analysis using a polyclonal N-terminal VEGF-B peptide specific antibody and corresponding to the single peak on the elution profile, were pooled and stored at 4° C.
The purified monomeric His6-VEGF-B10-108 was adjusted to pH 8.5 with 5 M NaOH and reduced with 20 mM DTT for 2 hrs at 37° C. The protein solution was diluted 10-fold by the slow drop-wise addition of Buffer 11 (100 mM Tris-HCl pH 8.5, 5 mM cysteine, 1 mM cystine, 0.5 M GdCl, 2 mM EDTA, pH 8.5) at 4° C., followed by overnight dialysis against 0.1 M acetic acid. Major bands positioned at approximately 13 kDa and 26 kDa in Western blot analysis correspond to monomeric and dimeric forms of His6-VEGF-B10-108, respectively, under non-reducing conditions. Coomassie staining suggested 30-40% conversion to dimer.
The acidified protein solution was concentrated five-fold with a 10 kDa cut-off EasyFlow concentrator (Sartorius AG, Germany), and adjusted to contain 80% n-propanol, 10 mM NaCl, pH 2.0. The material was loaded onto a Polyhydroxyethyl A hydrophilic column (2.1×25 cm; PolyLC, USA) attached to an ÅKTA FPLC system (Amersham Pharmacia Biotech, Sweden) at 10 ml/min, and equilibrated with Buffer 15 (80% n-propanol, 10 mM NaCl, pH 2.0). The bound material was eluted with a 10-40% linear gradient over 60 min of Buffer 16 (10 mM NaCl, pH 2.0).
Fractions containing dimeric His6-VEGF-B10-108 were pooled and diluted five-fold with Buffer 12 (0.15% v/v TFA). The material was loaded onto a Vydac 300 C8 Reverse-phase column (2.2×10 cm; Higgins Analytical, USA) previously equilibrated with Buffer 12 (0.15% v/v TFA) at 10 ml/min. The bound material was eluted with a 50-65% linear gradient over 60 min of Buffer 14 (0.13% v/v TFA, 60% v/v acetonitrile). Fractions containing dimeric VEGF-B10-108 were pooled, diluted five-fold in Buffer 12 and re-loaded on to the C8 column equilibrated with Buffer 12. Purified dimeric His6-VEGF-B10-108 was eluted with 100% Buffer 14 and freeze dried (
The following method is applicable to all forms of VEGF-B isoforms including by not limited to VEGF-B-167, -186 and 10-108 isoforms. The following steps are employed:
Purified refolded His-tagged VEGF-B may be suitable in its own right. However, for lyophilization of the refolded His-tagged VEGF-B the following steps are employed:
In the above steps, Steps 6 and 10 can be replaced by purification by polyhydroxyethyl A hydrophilic chromatography.
Furthermore, methods are devised which specifically purify VEGF-B from bacterially produced recombinant VEGF-B. The method steps include running a sample on metal-infinity columns.
Members of the VEGF family of cytokines have been shown to bind differentially to a family of three receptor tyrosine kinases (RTKs) designated VEGF receptor 1 (VEGF-R1), 2 (VEGF-R2) and 3 (VEGF-R3). Demonstration of binding to one or more of these receptors is important to establish that the purified homodimer has refolded correctly. The inventors used two methods, biosensor analysis (surface plasmon resonance) and an ELISA based assay, to demonstrate that the refolded dimeric VEGF-B167 is able to bind to VEGF-R1
Biosensor Analysis of Receptor Binding
Analysis of binding of VEGF-B167 to VEGF-R1 and VEGF-R2 was monitored using surface plasmon resonance (Biacore 2000, Pharmacia-Biosensor, Sweden) and commercially available receptor proteins. For control purposes binding of the receptors to VEGF-A165 was also monitored. Both VEGF-B167 and VEGF-A165 were individually immobilised to a sensorchip using NHS/EDC chemistry according to the manufacturer's instructions. Briefly, 35 μl of NHS and EDC was injected onto the sensorchip at a flow rate of 5 μl/min to activate the sensor surface and enable covalent coupling of either VEGF-A165 or VEGF-B167. The VEGF-A165 (Peprotech, USA, 100 μg/ml) was diluted (1:10) in 20 mM sodium acetate, pH 4.2 and injected directly onto the sensor surface (35 μl). Post coupling, diaminoethane (50 mM, pH 9.0) was used to block any unbound activated sites on the sensor surface. Concentrated dimeric VEGF-B167 (200 μg/ml) was diluted (1:10) in 20 mM sodium acetate and immobilized onto a separate channel on the sensorchip. Post coupling, diaminoethane (50 mM, pH 9.0) was used to block any unbound activated sites on the sensor surface.
At the end of each run, the surface of the sensorchip was regenerated using 2 cycles of phosphoric acid (0.1 M, 30 μl) at a flow of 50 μl/min. Both VEGF-R1 (R&D systems, USA) and VEGF-R2 (R&D systems, USA) were obtained as chimeric proteins incorporating the human immunoglobulin Fc domain. Both were diluted into 0.1% w/v BSA in PBS as a stock solution (50 μg/ml, storage −20° C.).
Biosensor analysis of binding of VEGF-A165 or VEGF-B167 to VEGF-R2/Fc is shown in
ELISA Based Analysis of Receptor Binding
An ELISA based assay to facilitate competitive receptor binding studies was developed using the chimeric receptor proteins described above and, in addition, a biotinylated polyclonal antibody specific for VEGF-A165. In the first instance, surface plasmon resonance was used to verify the specificity of the antibody. Binding to sensorchip immobilised (see above) VEGF-A165 and VEGF-B167 is shown in
The potential of VEGF-B167 to compete with VEGF-A165 for binding to VEGF-R1 was examined in an ELISA based assay using the VEGF-R1/Fc chimeric receptor. Briefly, the assay utilised the following protocol:
Receptor binding data obtained using Biosensor and ELISA based analysis clearly indicate that the production, refolding and purification protocol gives rise to VEGF-B167 that is refolded into the conformation capable of binding to the receptor. In addition the competitive binding analysis suggests that the majority of purified dimer is active, consistent with appropriately folded conformation.
Naturally occurring VEGF-B isoforms (VEGF-B167 and 186) as well as artificial truncated versions of the protein (VEGF-B10-108) that retain the core structural domain bind to VEGF receptor-1 or Flt-1. While it has been possible to demonstrate binding of recombinant forms of VEGF-B to isolated recombinant receptor proteins using a variety of biochemical strategies, a cell based assay, where VEGF-B binds to and dimerizes cell associated receptors to trigger activation of downstream substrates and subsequently a biological response that can be quantitated, has not been available. To address this issue, the inventors used splice-overlap-PCR techniques to generate chimeric receptors consisting of the extracellular and transmembrane domain of VEGFR1 fused to the cytoplasmic domain of the shared receptor component gp130. Dimerization of gp130 cytoplasmic domains leads to activation of the Jak/STAT signal transduction pathway and subsequently transcription of genes that incorporate appropriate STAT binding elements within their promoter region.
The chimeric receptor was co-transfected along with a gene encoding hygromycin resistance into 293A12 cells. 293A12 are an engineered version of standard 293T cells that have been transfected with the luciferase reporter gene under the control of a STAT responsive promoter. Stimulation of these cells with cytokines that dimerize gp130, including LIF and IL-6, leads to activation of luciferase gene transcription and subsequently quantifiable luciferase reporter activity. Following selection in hygromycin resistant clones were isolated and selected for luciferase production in response to the control protein VEGF-A. VEGF-A is a commercially available cytokine related to VEGF-B that also binds to and dimerizes the VEGFR1 receptor. Resistant clones producing luciferase in response to VEGF-A were expanded, recloned and further characterized prior to analysis of VEGF-B isoforms. Analysis of the response to VEGF-A indicated an ED50 at between 10-50 ng/ml of the recombinant protein.
The cloned cell line with the highest signal to background ratio in response to VEGF-A (clone 2.19.25) was selected for analysis of refolded VEGF-B isoforms. Experiments demonstrated the both naturally occurring VEGF-B isoforms as well as the artificial truncated form, were able to stimulate luciferase activity. For VEGF-B186 and the artificial truncated form in particular the dose response was identical to that of the recombinant VEGF-A. Furthermore this activity could be blocked by incorporating soluble VEGFR1-Ig chimeric (commercially available, R&D Systems) protein into the assay. These results demonstrate that the recombinant VEGF-B proteins are correctly refolded and able to dimerize their cognate receptor in a biologically appropriate manner.
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 invention 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.
This application is a continuation in part of U.S. patent application Ser. No. 10/204,070, filed Aug. 16, 2002 as a 371 application based on PCT/AU01/00160 having an international filing date of Feb. 16, 2001.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10204070 | Aug 2002 | US |
| Child | 11541848 | Oct 2006 | US |
