Self‐cleavage of fusion protein in vivo using TEV protease to yield native protein

Abstract

Overproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additional amino acid residues than the native proteins. Here we describe a new method for productions of native proteins with original amino termini in vivo via intracellular self-cleavage of the fusion protein using tobacco etch virus (TEV) protease. Our design allows one to simultaneously clone any gene into multiple fusion protein vectors using two unique cloning sites (i.e., SnaBI and XhoI) without restriction digestion, and then rapidly identifies those constructs producing soluble native proteins. This method will make the fusion protein approach more feasible for protein drug research.Keywords: tobacco etch virus protease; sticky-end PCR; fusion protein approach The fusion protein approach has been widely applied in modern biology and protein science research. More than 20 carrier proteins or affinity tags are available now for this approach to produce soluble heterologous proteins in various host organisms (Sambrook and Russell 2000). Although the use of these carrier proteins has resulted in successful overexpression of many heterologous proteins, each must be tested empirically, and certainly may not possess maximal solubility. Moreover, each expression scenario requires a specific vector. Recloning of the passenger protein gene into each of specific vectors is extremely labor-intensive. Recombinational cloning methods (Liu et al. 1998;Hartley et al. 2000) and sticky-end PCR cloning strategy (Shih et al. 2002;Wang and Wang 2004) were applied to minimize efforts required for alternate expression, and also allowed one to develop a high-throughput system to screen for soluble recombinant proteins.Because of concerns about the impact of carrier proteins or affinity tags on the structure or activity of a passenger target protein, it is ordinarily desirable to remove them. Typically, passenger target protein is separated from the fusion carrier by site-specifically proteolysis after affinity chromatography. It is this step that was considered to be the Achilles' heel of fusion protein approach, particularly in applications such as structural biology or protein-drug production. It is relatively common to encounter a situation in which fusion carriers cannot be processed effectively because of the steric hindrance at the cleavage site. Tedious

N-Lysine Propionylation Controls the Activity of Propionyl-CoA Synthetase

“…Cells were harvested and MBP-H 6 -PncA purified as described for purification of GST-H 6 -Pat. PncA eluted at ϳ30% buffer B. MBP-H 6 -PncA-containing fractions were pooled and H 6 -rTEV protease (26) added to reach a 1:50 H 6 -rTEV protease:MBP-H 6 -PncA ratio; H 6 -rTEV protease was purified as described (27). The cleavage reaction mixture was incubated at room temperature for 3 h and dialyzed overnight against two liters of buffer A at 4°C.…”

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

“…Though we previously reported the in vitro proteolytic action of PPV NIa protease on recombinant substrates (Zheng et al, 2008), no direct evidence show that PPV NIa protease can carry out intracellular cleavage for self-containing artificial polyprotein within E. coli, as TEV NIa protease did (Shih et al, 2005;Chen et al, 2010). Here we constructed an expression cassette in which EGFP protein was expressed downstream of PPV NIa protease and site F, therefore the polypeptide composed of Tag-Protease-site F-EGFP (designated as P-sG) was transcribed within E. coli (Fig.…”

“…New methods based on co-expression strategy offered alternative way to facilitate the process of protein expression and protein-protein interaction, by using compatible vectors (Huppa and Ploegh, 1997;Fabian et al, 1998;Hanzlowsky et al, 2006), multiple promoters (Belyaev and Roy, 1993;Scheich et al, 2007), polycistrons (Selleck et al, 2005;Neumann et al, 2007;Bieniossek et al, 2009), fusion linkers (Clements et al, 2000) in both prokaryotic and eukaryotic systems. One advantageous application was that TEV (Tobacco Etch Virus) NIa protease carried out site-specific cleavage on polyprotein to yield multiple native proteins within E. coli using a single promoter (Shih et al, 2005;Chen et al, 2010). TEV and PPV (Plum Pox Virus) belong to Potyviridae family with similar proteolytic function (Urcuqui-Inchima et al, 2001), therefore we speculated that PPV NIa protease can act as a powerful tool in vivo to process co-expressed substrate and then facilitate the verification of protein-protein interaction.…”

Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker

“…PreScission protease leaves a dipeptide overhang (GP) after cleaving the sequence LGVLFQ/GP, and TEV protease leaves a single amino acid overhang (G) after cleaving the consensus sequence EXXYXQ/G [11]. In addition, the relatively low activity or specificity of some enzymes used to cleave fusion proteins can result in tedious and expensive experiments, making this approach unappealing for clinical or bioindus-trial purposes [12].Here, we offer an alternative purification system that addresses the issues of low specificity and of leaving overhangs on the target protein. By incorporating a caspase-3 cleavage recognition sequence into a GST-fusion protein, we have made a complete purification system that utilizes a well characterized enzyme.…”

“…PreScission protease leaves a dipeptide overhang (GP) after cleaving the sequence LGVLFQ/GP, and TEV protease leaves a single amino acid overhang (G) after cleaving the consensus sequence EXXYXQ/G [11]. In addition, the relatively low activity or specificity of some enzymes used to cleave fusion proteins can result in tedious and expensive experiments, making this approach unappealing for clinical or bioindus-trial purposes [12].…”