Glutathione S‐transferase can be used as a C‐terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli

Abstract

Glutathione S-transferase (GST) from Schistosoma juponicum, which is widely used for the production of fusion proteins in the cytoplasm of Escherichia coli, was employed as a functional fusion module that effects dimer formation of a recombinant protein and confers enzymatic reporter activity at the same time. For this purpose GST was linked via a flexible spacer to the C-terminus of the thiol-protease inhibitor cystatin, whose binding properties for papain were to be studied. The fusion protein was secreted into the bacterial periplasm by means of the OmpA signal peptide to ensure formation of the two disulfide bonds in cystatin. The formation of wrong crosslinks in the oxidizing milieu was prevented by replacing three of the four exposed cysteine residues in GST. Using the tetracycline promoter for tightly controlled gene expression the soluble fusion protein could be isolated from the periplasmic protein fraction. Purification to homogeneity was achieved in one step by means of an affinity column with glutathione agarose. Alternatively, the protein was isolated via streptavidin affinity chromatography after the Strep-tag had been appended to its C-terminus. The GST moiety of the fusion protein was enzymatically active and the kinetic parameters were determined using glutathione and l-chloro-2,4-dinitrobenzene as substrates. Furthermore, strong binding activity for papain was detected in an ELISA. The signal with the cystatin-GST fusion protein was much higher than with cystatin itself, demonstrating an avidity effect due to the dimer formation of GST. The quaternary structure was further confirmed by chemical crosslinking, which resulted in a specific reaction product with twice the molecular size. Thus, engineered GST is suitable as a moderately sized, secretion-competent fusion partner that can confer bivalency to a protein of interest and promote detection of binding interactions even in cases of low affinity.

Enzymatic properties of the N- and C-terminal halves of human hexokinase II

“…Many lines of evidence suggest either GST or the GST-domains of fusion proteins have the potential to mediate oligomerization both in vitro and in vivo (23,24). In particular, Tudyka and Skerra mentioned that one should remember many proteins require an intact N-terminus for their bio-chemical activity when considering the use of GST as a permanent rather than transient fusion partner (24).…”

“…In particular, Tudyka and Skerra mentioned that one should remember many proteins require an intact N-terminus for their bio-chemical activity when considering the use of GST as a permanent rather than transient fusion partner (24). Regarding hydrophobicity, previous studies have shown the first 12 N-terminal amino acids of HK II (the mitochondrial binding domain) along with the first 11 N-terminal amino acids of HK I are very hydrophobic and may influence the kinetic properties of the enzyme (12).…”

Interactions of Human Rad54 Protein with Branched DNA Molecules

“…Branch Migration Assay-The hRad54 protein (100 nM, unless indicated otherwise) was incubated with 32 P-labeled synthetic PX-junction (number 71/169/170/171) (33 nM, molecules), 32 P-labeled synthetic X-junction (number 71/170/234/ 235) (33 nM, molecules), or 32 P-labeled synthetic PX-junction (number 265/266/269/270) (20 or 30 nM, molecules) in a 90-l branch migration buffer containing 25 mM Tris acetate, pH 7.5, 2 mM ATP, 1 mM dithiothreitol, 100 g/ml bovine serum albumin, the ATP-regenerating system (10 units/ml creatine phosphokinase and 15 mM creatine phosphate), and the indicated concentrations of magnesium acetate. The reactions were carried out at 30°C or in the case of PX-junction (number 265/ 266/269/270) at 20°C.…”

New Therapeutic Strategies for Antibiotic-Resistant Select Agents

“…This property is very different from free GST, 100% of which migrated as dimers after being crosslinked (22). Therefore, primase with a GST tag is a stable monomer under reducing conditions but is capable of forming large crosslinked networks under oxidizing conditions.…”