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

Abstract

Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S-transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the Nterminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag. KeywordsCaspase-3; Fusion protein; Protein expression; ProteolysisIn the past thirty years, a number of protein purification systems have been developed using fusion proteins to aid in the efficient purification and recovery of recombinant proteins from crude cell extracts or culture media. These systems incorporate amino-or carboxy-terminal proteins or peptides referred to as "tags." Many of the tags can be removed subsequent to binding to, or while concurrently bound to, a high affinity matrix. Matrices for binding the tags have generally incorporated immobilized metal for binding poly-histidine sequences, immobilized glutathione for binding glutathione S-transferase (GST),1 poly-alanine affinity matrices, antigenic epitopes to bind monoclonal antibodies, biotinylated resins for binding avidin or strep-avidin, carbohydrate-binding proteins, or even complete enzymes immobilized in a matrix for substrate binding [1]. While tags provide many advantages for aiding in the rapid recovery, stabilization, and increased scale of protein expression, many tags interfere with protein structure and function and must be removed after purification of the fusion protein. Removal of the tags usually occurs by cleavage of the fusion protein by a specific protease, such as thrombin. It is sometimes problematic, however, to remove the entire carrier protein sequence without leaving residual amino acids on the target protein. For example, the proteases most frequently used to remove carrier proteins are thrombin, factor Xa, and enterokinase. Factor Xa and enterokinase cleave C-terminal to their recognition sequences, leaving no residual amino acids. Thrombin, however, cleaves the amino acid sequence LVPR/GS between R and G, which leaves two amino ...

Rapid Folding and Unfolding of Apaf-1 CARD

“…44 Apaf-1 CARD was expressed and purified from Escherichia coli BL21(DE3) pLysS cells harboring plasmid pApaf-1CARD+GS as described. 30 An extinction coefficient (ε 280 ) of 5120 M −1 cm −1 was used to determine protein concentration. Caspase-3 was purified as described.…”

“…28,29 Our clone of Apaf-1 CARD contains two extra residues at the amino terminus, a GS sequence that arises from the cloning vector. 30 These residue positions are labeled -2 and -1, respectively ( Figure 1). Since the side-chain of M1 forms part of the hydrophobic core, 27 it was important to determine whether the two residues affected the structure Figure 1.…”

Fusion partners can increase the expression of recombinant interleukins via transient transfection in 2936E cells

“…The fusion partner follows the 6ÂHis tag, and a caspase-3 cleavage site (DEVD) allows for clean cleavage of the tag without any leftover residues on the downstream protein of interest. 5 A control construct employed a GAS tripeptide linker in lieu of a fusion partner between the 6ÂHis tag and the caspase cleavage site. Five interleukins which had proven historically difficult to produce were selected to test in this system and were transiently transfected in a 24-well block using standard procedures.…”

Robust production of a peptide library using methodological synchronization

“…This fact is of particular importance in production of peptide libraries where additional amino acids at either the N- or C-termini can have significant effects on the overall properties of the peptide. Since any protease can be used, the overhanging amino acids from the protease cleavage site can be matched with the desired peptide sequence, or a protease that does not leave any overhang can be selected [37]. The facts that this method is not dependent on use of any particular affinity tags or on utilizing matching affinity tags also limit sub-cloning steps that are necessary to use the library production method.…”