Exploring the activity of tobacco etch virus protease in detergent solutions

Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.     

An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins

In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin–agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.     

High level expression and single-step purification of hexahistidine-tagged L-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set

Affinity tags as fusions to the N- or C-terminal part of proteins are valuable tools to facilitate the production and purification of proteins. In many cases, there may be the necessity to remove the tag after protein preparation to regain activity. Removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease. Here, we report the construction of an expression vector set that combines N- or C-terminal fusion to either a hexahistidine tag or Streptag with the possibility of tag removal by factor Xa or recombinant tobacco etch virus protease (rTEV), respectively. The vector set offers the option to produce different variants of the protein of interest by cloning the corresponding gene into four different Escherichia coli expression vectors. Either immobilized metal affinity chromatography or streptactin affinity chromatography can be used for the one-step purification. Furthermore, we show the successful application of the expression vector for C-terminal hexahistidine tagging. The expression and purification of His-tagged L-2-hydroxyisocaproate dehydrogenase yields fully active enzyme. The tag removal is here accomplished by a derivative of rTEV.     

High level expression and single-step purification of hexahistidine-tagged L-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set

Affinity tags as fusions to the N- or C-terminal part of proteins are valuable tools to facilitate the production and purification of proteins. In many cases, there may be the necessity to remove the tag after protein preparation to regain activity. Removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease. Here, we report the construction of an expression vector set that combines N- or C-terminal fusion to either a hexahistidine tag or Streptag with the possibility of tag removal by factor Xa or recombinant tobacco etch virus protease (rTEV), respectively. The vector set offers the option to produce different variants of the protein of interest by cloning the corresponding gene into four different Escherichia coli expression vectors. Either immobilized metal affinity chromatography or streptactin affinity chromatography can be used for the one-step purification. Furthermore, we show the successful application of the expression vector for C-terminal hexahistidine tagging. The expression and purification of His-tagged L-2-hydroxyisocaproate dehydrogenase yields fully active enzyme. The tag removal is here accomplished by a derivative of rTEV.     

Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives

Tobacco etch virus (TEV) protease is widely used to remove tags from recombinant fusion proteins because of its stringent sequence specificity. It is generally accepted that the high concentrations of salts or other special agents in most protein affinity chromatography buffers can affect enzyme activity, including that of TEV protease. Consequently, tedious desalination or the substitution of standard TEV reaction buffer for elution buffer are often needed to ensure TEV protease activity when removing fusion tags after purifying target proteins using affinity chromatography. To address this issue, we used SOE PCR technology to synthesize a TEV protease gene with a codon pattern adapted to the codon usage bias of Escherichia coli, recovered the purified recombinant TEV protease, and examined its activity in various elution buffers commonly used in affinity chromatography as well as the effects of selected additives on its activity. Our results showed that the rTEV protease maintained high activity in all affinity chromatography elution buffers tested and tolerated high concentrations of additives commonly used in protein purification procedures, such as ethylene glycol, EGTA, Triton X-100, Tween-20, NP-40, CHAPS, urea, SDS, guanidine hydrochloride and β-mercaptoethanol. These results will facilitate the use of rTEV protease in removing tags from fusion proteins.     

Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium

A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His(6)-tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry.     

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (enterokinase, factor Xa, human rhinovirus 3C protease, SUMOstar, tobacco etch virus protease, and thrombin) by use of a panel of 94 individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and factor Xa were only affected by a small number of detergents, making them good choices as well.     

Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.     

Expression of a kallikrein-like protease from the snake venom: engineering of autocatalytic site in the fusion protein to facilitate protein refolding

In order to circumvent the difficulty encountered in the expression and purification of the recombinant products in E. coli system, we have developed a novel and facile method of removing the polyhistidine tag from target proteins after heterologous gene expression. The expression of a serine protease (Tm-5) from Taiwan habu (Trimeresurus mucrosquamatus) is taken as an exemplar to illustrate the basic rationales and protocols involved. In place of an enterokinase recognition site, a polyhistidine tag linked to an autocatalyzed site based on cleavage specificity of the serine protease flanking on the 5'-end of Tm-5 clone sequence was engineered before protein expression in E. coli system. Renaturation of the fusion protein after expression revealed that the recombinant protease had refolded successfully from the inclusion bodies. Upon autocleavage of the expressed protease, the polyhistidine tag with additional amino acid residues appended to the N-terminus of the coding sequence is found to be removed accordingly. The protein expressed and purified by this new strategy possesses a molecular weight of approximately 28,000 in accord with the expected value for this venom protease. Further characterization of the recombinant protein employing a variety of techniques which include immunoblot analysis, RP-HPLC, ESI-MS, and N-terminal amino acid sequencing all shows indistinguishable properties to those of the isolated native protease. Most noteworthy is that the recombinant Tm-5 protease also exhibits amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, a unique and strict substrate for native Tm proteases reported previously.     

Purification and characterization of RGD tumor-homing peptide conjugated human tumor necrosis factor alpha over-expressed in Escherichia coli

A number of approaches have been investigated to enhance the selective toxicity of tumor necrosis factor alpha (TNFalpha) to permit its systemic use in cancer therapy. Because vascular targeting has been proven to be a valid strategy for improving the therapeutic index of TNFalpha, we prepared RGD-hTNF consisting of human TNF fused with the ACDCRGDCFCG peptide, a ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins. Recombinant RGD-hTNF was produced in Escherichia coli as a polyhistidine fusion protein. Between polyhistidine tag and RGD-hTNF, a tobacco etch virus (TEV) protease cleavage site (ENLYFQG) was introduced to ensure the release of intact RGD-hTNF. The purification strategy consisted of the target protein capture step by immobilized metal affinity chromatography (IMAC), TEV protease cleavage of fusion protein, the subtractive depletion of removed His-tag by IMAC and the final gel filtration step. As a result, about 18 mg of intact RGD-hTNF was obtained from 1l of bacteria culture. The purified RGD-hTNF was characterized by SDS-PAGE, Western blot, mass spectroscopy and gel filtration. Since the RGD-hTNF molecule retained the cytotoxic activity of the TNF moiety and the integrin binding ability of the RGD moiety, the purification method provided material for assessing its anti-tumor activity in animal model.     

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.     

A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6-maltose binding protein fusion tag

We describe a generic protocol for the overproduction and purification of recombinant proteins in Escherichia coli. The strategy utilizes a dual His6-maltose binding protein (HisMBP) affinity tag that can be removed from the target protein by digestion of the fusion protein at a designed site by tobacco etch virus protease. The MBP moiety serves to enhance the solubility and promote the proper folding of its fusion partners, and the polyhistidine tag facilitates its purification to homogeneity. This protocol is divided into three stages, each of which takes approximately 1 week to complete: (i) construction of a HisMBP fusion vector; (ii) a pilot experiment to assess the yield and solubility of the target protein; and (iii) the large-scale production and purification of the target protein.     

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.     

The Xenopus laevis Atg4B Protease: Insights into Substrate Recognition and Application for Tag Removal from Proteins Expressed in Pro- and Eukaryotic Hosts

During autophagy, members of the ubiquitin-like Atg8 protein family get conjugated to phosphatidylethanolamine and act as protein-recruiting scaffolds on the autophagosomal membrane. The Atg4 protease produces mature Atg8 from C-terminally extended precursors and deconjugates lipid-bound Atg8. We now found that Xenopus laevis Atg4B (xAtg4B) is ideally suited for proteolytic removal of N-terminal tags from recombinant proteins. To implement this strategy, an Atg8 cleavage module is inserted in between tag and target protein. An optimized xAtg4B protease fragment includes the so far uncharacterized C-terminus, which crucially contributes to recognition of the Xenopus Atg8 homologs xLC3B and xGATE16. xAtg4B-mediated tag cleavage is very robust in solution or on-column, efficient at 4°C and orthogonal to TEV protease and the recently introduced proteases bdSENP1, bdNEDP1 and xUsp2. Importantly, xLC3B fusions are stable in wheat germ extract or when expressed in Saccharomyces cerevisiae, but cleavable by xAtg4B during or following purification. We also found that fusions to the bdNEDP1 substrate bdNEDD8 are stable in S. cerevisiae. In combination, or findings now provide a system, where proteins and complexes fused to xLC3B or bdNEDD8 can be expressed in a eukaryotic host and purified by successive affinity capture and proteolytic release steps.     

Construction,non-denaturing affinity purification,and characterization of baculovirally expressed human secretory leukocyte protease inhibitor

Secretory leukocyte protease inhibitor (SLPI) is a 11.7 kDa mucosal protein with potent anti-microbial, anti-inflammatory, and wound healing activities. Previous efforts to express and purify the non-glycosylated cationic protein as a recombinant protein in bacteria required extensive denaturation and renaturation to refold the disulfide-rich protein into its biologically active form. To overcome this limitation, we have expressed human SLPI as a polyhistidine-tagged protein (bvHisSLPI) using a recombinant baculovirus expression system. Studies were conducted to determine the timing of maximal protein production following baculovirus infection of Sf21 cells. The 16.4kDa-tagged protein was then overexpressed in Sf21 cells following a 48-h infection with bvHisSLPI-encoding baculovirus, purified by nickel-chelating affinity chromatography under non-denaturing conditions, and analyzed by Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Purified bvHisSLPI was further characterized by enterokinase digestion to remove the polyhistidine tag from its N-terminus. In serine protease inhibition assays, purified bvHisSLPI blocked substrate cleavage by two serine proteases, chymotrypsin and cathepsin G, comparable to bacterially expressed SLPI. The baculovirus expression and affinity purification strategy described here will facilitate further studies of the structural and biological properties of this important multifunctional protein.     

Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus

The cysteine-rich peptide hepcidin is an antimicrobial peptide and iron transport regulator that has been found in vertebrates including birds, fish and mammals. To elucidate the structure and biological function of fish hepcidin, which is difficult to produce synthetically, we have cloned several plasmid constructs encoding hepcidin from Japanese flounder, Paralichthys olivaceus, and tested expression of recombinant peptides, each with an N-terminal hexahistidine (6xHis) tag, in inclusion bodies or the periplasmic space of Escherichia coli. Hepcidin expressed in inclusion bodies was reduced, and subsequently refolded using a dilution technique with a cysteine redox system. The oxidized His-hepcidin monomer was separated from protein multimers and mass spectrometry analysis showed that the peptide was of the predicted size and contained four disulfide bonds. Removal of the 6xHis tag was attempted using enzymatic cleavage by Factor Xa and tobacco etch virus (TEV) protease or chemical cleavage by hydroxylamine. The Factor Xa cleavage was unsuccessful and hydroxylamine cleavage resulted in aggregation of cleaved peptide. TEV protease cleavage was successful but immediately resulted in hexamer formation despite varying reaction conditions (redox, non-redox, pH, temperature, target protein concentration, type of buffer). However, the recombinant His-hepcidin fusion peptide monomer showed considerable antimicrobial activity. NMR-based studies showed that hepcidin contained a rare vicinal disulfide linkage at the top of a loop structure and a short beta-sheet structure encompassing residues 7-13 and 19-25 that is stabilized by three disulfide bonds.     

Purification and initiation of structural characterization of human peripheral myelin protein 22, an integral membrane protein linked to peripheral neuropathies

Gene duplications, deletions, and point mutations in peripheral myelin protein 22 (PMP22) are linked to several inherited peripheral neuropathies. However, the structural and biochemical properties of this very hydrophobic putative tetraspan integral membrane protein have received little attention, in part because of difficulties in obtaining milligram quantities of wild type and disease-linked mutant forms of the protein. In this study a fusion protein was constructed consisting of a fragment of lambda repressor, a decahistidine tag, an intervening TEV protease cleavage site, a Strep tag, and the human PMP22 sequence. This fusion protein was expressed in Escherichia coli at a level of 10-20 mg/L of protein. Following TEV cleavage of the fusion partner, PMP22 was purified and its structural properties were examined in several different types of detergent micelles using cross-linking, near and far-UV circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy. PMP22 is highly helical and, in certain detergents, shows evidence of stable tertiary structure. The protein exhibits a strong tendency to dimerize. The 1H-15N TROSY NMR spectrum is well dispersed and contains signals from all regions of the protein. It appears that detergent-solubilized PMP22 is amenable to detailed structural characterization via crystallography or NMR. This work sets the stage for more detailed studies of the structure, folding, and misfolding of wild type and disease-linked mutants in order to unravel the molecular defects underlying peripheral neuropathies.     

Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease

Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.     

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification¹. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast^2,3 but more recently has been adapted to use in mammalian cells^4-8.As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E^9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation¹⁰. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence⁸. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.     

An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni(2+) affinity chromatography, 65mg of His6-TEV was isolated with purity over 95% from 1L of culture. The enzyme activity of His6-TEV was generally characterized by using GST-EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties.