Patent Application
20130012687
1. Field of the Invention
The present invention provides an inducible, self cleaving protease tag, and a method of purifying recombinant proteins using the same.
2. Description of the Background
The availability of simple, reliable, and cost-effective methods for recombinant protein purification is critical for the work of high throughput structural and proteomic centers and many individual researchers alike. While the addition of affinity tags such as poly-His and glutathione transferase (GST) to target proteins has greatly simplified purification strategies, it is often difficult to obtain soluble recombinant protein. As a result, affinit-tagged target proteins are often additionally fused to small proteins such as NusA and SUMO to improve their solubility, expression, and stability.
However, these tags can alter the biological activity of target proteins and interfere with protein crystallization studies. Therefore many biological and biomedical applications require tag removal from the target protein. Most commonly used methods require the addition of exogenous site-specific proteases to cleave the affinity tag off the target protein at engineered sites. Unfortunately, the need for high levels of endoprotease for extended periods of time can result in unwanted cleavage events within the target protein. Furthermore, these endoproteases are costly, often exhibit poor solubility, an require the inclusion of additional chromatography steps to remove the exogenous protease.
Accordingly, the present invention provides an inducible, sell-cleaving or autoprocessing protease tag, which may be used advantageously in the purification of recombinant proteins.
It is also an object of the present invention to provide a method of purifying recombinant proteins using tho inducible, self-cleaving protease tag.
It is, moreover an object of the present invention to provide a method of purifying recombinant proteins, wherein the purification, cleavage and separation of untagged protein from an endoprotease in condensed into a single step.
More particularly, it is an object of the present invention to provide a method of purifying a protein, which entails: a) fusing a site-specific affinity-tagged cysteine protease domain (CPD) protease to a target protein b) activating the site-specific CPD by subjecting the site-specific affinity-tagged CPD to an inducer, which induces autoprocessing at a cleavage site; thereby releasing untagged target protein; and c) isolating the untagged target protein.
To circumvent the above disadvantages, the present inventors have developed an on-bead cleavage purification system in which a site-specific affinity-tagged protease is fused directly to the target protein (
An important element of this purification method is the use of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD). The CPD exhibits several properties that make it amenable to its development into an inducible autocleaving protease tag First, the CPD is a highly specific protease that cleaves exclusively after Leu residues4. In the native toxin, the CPD processes the MARTX toxin within interdomain junctions to release discrete effector domains. Secondly, the CPD is selectively activated by the cukaryotic-specific small molecule inositol hexakisphosphate (InsP6). Since InsP6 is absent from bacterial cells6, when the CPD-His6 tag is fused to the C-terminus of target proteins and expressed in E. coli, a CPD-His6 fusion protein can be purified from bacterial lysates in a protease-inactive form using imidzaole affinity chromatography (IMAC,
To demonstrate the feasibility of this system, the present inventors constructed pET expression vectors in which DNA encoding the CPD was cloned in to the SalI restriction site to generate pET-CPD vectors (
As a proof-of-principle, the present inventors expressed and purified green fluorescent protein (GFP) as a fusion to CPD-His6 using IMAC; addition of increasing amounts of InsP6s stimulated the release of GFP from the Ni2+-NTA agarose beads (bead eluate,
We noticed that the expression of the ICD-CPD-His6 fusion protein was approximately two-fold higher than the ICD-His6 protein in E. coli lysates (
The CPD purification system also enhanced the expression, as well as purity, of a previously uncharacterized SUMO/Sentrin-specific peptidase 1 (SENP1) from the parasitic pathogen Plasmodium falciparum, the causative agent of malaria (
In addition to augmenting the expression of target proteins, the CPD-His6 fusions can protect target proteins from proteolytic degradation. This can be demonstrated by fusing the CRAC-activation domain (CAD) of the ER calcium sensor STIMI to the CPD (
Moreover, the CPD purification system also increased the solubility of difficult-to express proteins. Fusion of the mouse macrophage metalloelastase (MMP12) to CPDHis6 facilitated its purification from the soluble fraction of E. coli lysates, whereas His6-tagged MMP12 remained largely insoluble (FIG. 2c). The currently used method for purification of His6-tagged MMP12 is a laborious procedure that requires solubilization of MMP12 inclusion bodies, refolding over multiple days, followed by anion and cation exchange chromatography. The CPD purification system dramatically simplifies this purification procedure, allowing soluble, active MMP-12 to be isolated in approximately 7 hours (
Collectively, these results imply that the one-step purification systems such as the intein-chitinbinding-domain (CBD) and sortase-His6. While these systems simplify the purification of well-expressed proteins, the large size of the intein-CBD fusion tag can decrease target protein solubility, and sortase-His6 fusion tags do not increase target protein solubility. Furthermore, unlike self-cleaving elastin-like polypeptide (ELP) tags, fusion proteins do not need to be subjected to the temperature cycles, pH shifts, or high salt concentrations, a feature that is critical for the purification of the intractable proteins. Based on the properties reported here, the CPD could replace the intein-tag in the self-cleaving-ELP system and potentially improve the solubility of ELP-tagged proteins while retaining their self-cleavability. Indeed, a considerable strength of this method is that the CPD remains active over a wide range of conditions. CPD-mediated cleavage is complete within 1-2 hrs at temperatures between 4° C. and 37° C., requires only micromolar of the small molecule InsP6 (an abundant and inexpensive reagent), and occurs efficiently both in the presence of standard protease inhibitor cocktails and in the absence of salt. This latter property carries the additional advantage of allowing the user to determine the buffer system in which to elute the target protein, which eliminates the need for desalting or buffer exchange steps that can reduce protein yields. Thus, the CPD system allows for considerable flexibility in optimizing purification procedures, as is often necessary for uncharacterized target proteins.
This versatility, combined with our observation that it can advantageously improve the solubility and integrity of difficult-to-express proteins (
“CPD” means cysteine protease domain.
“MARTX” means multifunctional, autoprocessing RTX toxins produced by certain bacteria. “Inducer” means a small molecule that induces autoprocessing at a cleavage site. In the present invention, that induced autocleavage releases an untagged target protein.
In more detail,
The results obtained and observed are summarized in the tables below.
GSHMPfSENP1
aProtein yield per litre of culture
bYield difficult to assess since GST-fusion protein degrades and falls out of solution over time
aRestriction enzyme sequences are underlines, and HA tag is shown in italics. bRestriction site
Bacterial growth conditions Overnight bacterial strains were grown at 37° C. in Luria-Bertrani (LB) broth. Antibiotics were used at 100 μg/mL carbenicillin for pET22b vectors expressed in E. coli.
Strain construction Primers used ate listed in Table 4; strains constructed are listed in Table 5. For construction of pET-CPDSalI vectors, DNA encoding Vibrio cholera MARTX toxin amino acids 3440-3650 from Vibrio cholerae N16961 was PCR amplified from genomic DNA using primers #1 and #2. The resulting PCR fragment was cloned into the SalI and XhoI sites of the pET22b and pET28a expression vectors, respectively (Novagen). For construction of the pET-CPDSacI vector, DNA encoding Vibrio cholerae MARTX toxin amino acids 3442-3650 from Vibrio cholerae N16961 was PCR amplified from genomic DNA using primers #3 and #2, and the resulting PCR fragment was cloned into the SacI and XhoI sites of pET22b. To construct the pET-HA-CPDSalI vectors, DNA encoding the HA epitope tag was added to the 5′ end of primer #4, and PCR amplification using primers #4 and #2 was used to fuse the HA tag directly to amino acid 3440 of V. cholera MARTX CPD. The resulting PCR fragment was cloned into the SalI and XhoI sites of the pET22b and pET28a expression vectors, respectively. For construction of the pET-CPDBamHI-Leu vector, DNA encoding Vibrio cholerae MARTX toxin amino acids 3440-3650 from Vibrio cholerae N16961 was PCR amplified from genomic DNA using primers #5 and #2, and the resulting PCR fragment was cloned into BamHI and XhoI sites of pET22b. For construction of the pETCPDBamHI vector, DNA Vibrio cholerae MARTX toxin amino acids 3440-3650 from Vibrio cholerae N 16961 was PCR amplified from genomic DNA using primers #6 and #2 were used, and the resulting PCR fragment was cloned into the BamHI and XhoI sites of pET22b. The pET22b-GFP-CPD construct was cloned by PCR amplifying GFP from pEGFPN3(Clontech) using primers #7 and #8. To construct the pET22b-gp130(ICD)-CPD vector, amino acid 642-918 of gp130 corresponding to the intracellular domain were PCR amplified using primers #9 and #10 and pET21a-gp130(ICD) as a template. The pET22b-BirA-CPD vector was constructed by PCR amplifying the birA gene from the pGEX4T1-BirA template using primers #9 and #10. The pET22b-STIMI(CAD)-CPD plasmid was constructed by PCR amplifying DNA encoding amino acids 342-369 of STIMI using pGEX6-CAD128 as a template and primers #13 and 14. The pET22b-nMMP12-CPD construct was constructed by PCR amplifying the catalytic domain of mouse MMP12 (aa 29-267) using pET41a-mMMP12 as a template using primers #15 and #16. In all cases, the resulting PCR products were cloned into the NdeI and SalI sites of pET22b-CPDSalI.
Protein expression and purification. For purification of His6-tagged CPU fusion proteins, overnight cultures of the appropriate strain were diluted 1:500 into 1 L 2YT media and grown shaking at 37° C. When an OD600 of 0.6 was reached. IPTG was added to 250 μM, and cultures were grown for 3-4 hrs at 30° C. Cultures were pellatized, resuspended in 25 mL lysis buffer (500 nM NaCl, 50 mM Tris-HCL, pH 7.5, 15 nM imidazole, 10% glycerol) and flash frozen in liquid nitrogen. Lysates were thawed, then lysed by sonication and cleared by centrifugation at 15,000×g for 30 minutes. His6-tagged CPD fusion proteins were affinity purified by incubating the lysates in batch with 0.5-1.0 mL Ni-NTA Agarose beads (Qiagen) with shaking for 2-3 hrs at 4° C. the binding reaction was pelleted at 1,500×g, the supernatant was aside, and the pelleted Ni2+-NTA agarose beads were washed three times with lysis buffer. In some cases, 10% of the Ni2+-NTA beads containing immobilized CPD-His6 fusion proteins were removed, pelleted and then His6-tagged eluted using high imidazole buffer (500 nM NaCl, 50 mM Tris-HCL, pH 7.5, 175 nM imidazole, 10% glycerol).
To liberate untagged target proteins into the supernatant fractions, 300-500 μL lysis buffer was added to the Ni2+-NTA beads containing CPD-His6 fusion proteins and the indicated amount of inositol hexakisphosphate (InsP6, Calbiochem) was added. In general, on-bead cleavage was allowed to proceed by nutating the beads in the presence of 50-100 μM InsP6 for 1-2 hr at room temperature or 4° C. The beads were pelleted at 1,500×g, and the supernatant fraction was removed. The beads were then washed 3-4 times with 300-500 μL lysis buffer, and supernatant fractions retained. His6-tagged proteins remaining on the beads (i.e. cleaved CPD-His6) were eluted using high imidazole buffer (500 nM NaCl, 50 mM Tris-HCL, pH 7.5, 175 nM imidazole, 10% glycerol) in 300-500 μL volumes. The elute was repeated 3-4 times, and elute fractions were collected. Purification of His6-tagged proteins lacking the CPD was performed in parallel. This general procedure was followed with the following exceptions: for purification of MMP12 constructs, the cultures were grown at 16° C. overnight after IPTG induction, and 1 mM tris(2-carboxyethyl)phosphine (TCEP) was added to the lysis buffer to prevent misfolding of the protein. PfSENP1 and BirA protein purifications were performed exclusively at room temperature, since at 4° C., protein aggregation was observed. For removal of the His6-tag from His6-PfSENPI, thrombin beads (Calbiochem) that had been washed in PBS were added to the elute His6-PfSENP1, which had been buffer exchanged into PBS. Thrombin cleavage was allowed to proceed with shaking overnight for 12 hr at room temperature. Aliquots were taken before and after thrombin addition to monitor cleavage efficiency. Thrombin cleaved, untagged PfSENP1 was enriched by performing a subtractive Ni2+-NTA pull-down. Untagged PfSENP1 from both methods was then buffer-exchanged into gel filtration buffer (50 mM NaCl, 20 mM Iris pH 8.0). Protein purifications were analyzed by SDS-PAGE and Coomassie staining using GelCode Blue (Pierce). Purified protein concentrations of purified were determined by Bradford assay (Pierce).
Purification of MMP12-His6. MMP12-His6 was purified as previously described with the following modifications. The cell pellet was resuspended in 100 mM NaCl, 100 mM Tris pH 8.0, 5.0 mM EDTA, 0.5 mM DTT, 100 μg/mL lysozyme and stirred for 2 hr. The cells were sonicated then centrifuged at 10,000 rpm for 10 min. The resulting inclusion bodies were washed two times and then resuspended in 50 mL 6M guanidine hydrochloride, 10 mM Iris pH 8.0 by stirring at 4° C. overnight. The mixture was centrifuged at 15,000 rpm for 30 min, and 2 mL aliquots of supernatant were prepared. The supernatant was diluted 1:100 into denaturing buffer (6M Urea, 50 mM Tris pH 8.0, 10 mM CaCl2, 30 mM NaCl, 5 mM DTT) to a final concentration of 0.1-0.2 mg/mL. The protein was then dialyzed for 24 hr in 2 L refolding buffer 1 (3 M Urea, 50 mM Tris pH 8.0, 10 mM CaCl2, 30 mM NaCl, 5 mM DTT). The partially refolded protein was then dialyzed in 4 L of refolding buffer 2 (1 M Urea, 50 mM HEPES pH 7.4, 10 mM CaCl2, 5 mM DTT). The buffer exchanged protein was then purified using tandem 5 mL MonoQ and SP Sepharose (GE Healthcare) at 4° C. After loading the protein on the column, the column was washed with 50 mL of refolding buffer 2 without DTT at 1 M, 0.5, and 0 Murea, respectively. The protein was eluted from the SP column in 500 mM NaCl, 50 mM HEPES ph 7.4, 10 mM CaCl2.
Gel filtration chromatography. Untagged PfSENP1 obtained from either thrombin or InsP6-mediated cleavage was concentrated using 10 kDa Centricon concentrator (Millipore) and buffer exchanged into 50 mM NaCl. 20 mM Tris pH 8.0 and purified on a Superdex 200 10/30 column (GE Healthcare) equilibriated in the same buffer. For MMP12, the gel filtration buffer contained 150 mM NaCl, 50 Mm Tris pH 7.4, 10 Mm TCEP. Gel filtrations were performed at 4° C.
Activity assays. Flourescence of purified GNP at 511 nm was verified using a Molecular Devices fmax plate reader in black 96-well plates and 488 nm excitation. The activity of MMP12 was determined using the flourogenic substrate Mea-PLGLDL(Dpa)AR (Mea, (7-methoxycoumarin-4-yl)acetyl, Dpa, N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl, Anaspec). Reactions were preformed in the assay buffer (50 Mm Tris pH 7, 150 mM NaCl, 10 Mm CaCl2, 0.02% NaN3, 5 mM TCEP) at 37° C. The substrate was used at 10 μM and the protein at 0.2 μM. The substrate hydrolysis was monitored continuously in a fluorescent plate reader (Molecular Devices) using an excitation wavelength of 325 nm and an emission wavelength of 395 mn.
In addition to the embodiments described above, other variations thereof may also be used on accordance with the present invention without departing from the spirit and scope thereof.
For example, aside from the Vibrio cholera MARTX cysteine protease domain (CPD), related MARTX CPDs from Pholorhabdus luminescens and Vibrio vulnificus also autoprocess in the presence of InsP6. Further, Clostridium toxin CPDs also work in the system.
Generally, MARTX toxins produced by Vibrio sp. and Clostridium sp. may be used. Exemplary species of Vibrio are V. anguillarum, V. splendidus and V. vulnificus in addition to V. cholera. Further, these toxins arc specifically described as CPDs. For example, the Clostridium toxins are CPDs derived from the large glucosylating toxins produced by lostridium sp. All of these bacterial species are commercially or readily available to the artisan.
Further, in addition to InsP6 (inositol hexakisphosphate), inositol pentakisphosphate (InsP5) may also be used but generally higher concentrations of the later are required to induce autocleavage.
Moreover, while E. coli is used as a well-known expression system the present invention, other bacterial hosts may also be used to produce the target recombinant protein. For example, Bacillus systems and Lactobacillus lactis systems may be used. Generally, any bacterial host system may be used provided that the expressed target protein is secreted into the media, and the media does not contain either InsP6 or InsP5, for example. Further, it is also possible to use the system in eukaryotic cells when the CPD is mutated to be less responsive to InsP6 and InsP5, for example. Basic cage mutations described in recent literature require higher than physiological concentrations of InsP6 (cystolic concentrations have been reported to be between 5-100 micromolar InsP6) to become activated.
Generally, the present method is conducted at a pH range of about 6.5 to 9.5. However, it is preferred that a pH range of about 7.5 to 8.5 be used. Further, the MARTX CPDs used in the present invention are preferably insensitive, i.e., no loss of protease activity, to salt concentration. For example, the MARTX CPDs used in the present invention generally exhibit little or no loss of activity in the presence of NaCl in a concentration of from 0 to 500 mM.
As noted above, the present method generally affords an increased expression of target proteins. In general, although the extent of increased expression varies from protein to protein, at least a two-fold increase in expression is commonly observed. Yet, greater increases are also observed. See the discussion above regarding a comparison of the expression level for (BirA-CPD-His6) in E. coli and (GST-BirA).
Additionally, various affinity tags may be used in accordance with the present invention being fused to the CPD. For example, steptavidin binding tags (SBP or Nanotag), CBP (Calmodulin binding tag) and Protein C-epitope tag may be mentioned. Further, it is also acceptable to combine the use of affinity-tagged CPD with other fusion or affinity tags. For example, the present inventors have constructed vectors in which the protein of interest can itself carry an affinity tag in addition to the affinity-tagged CPD (like the HA tag depicted in
Finally, the CPD used may be modified to function as an N-terminal fusion. For example, the present inventors have observed that the CPD can cleave itself off a streptavidin support when a C-terminal biotinylated peptide sequence is fused to the CPD.
Having described the above embodiments, it will remain clear that various other changes and modifications may be made without departing from the spirit and scope of the present invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/02571 | 9/21/2010 | WO | 00 | 9/17/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61272401 | Sep 2009 | US |
