Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag

Abstract

We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP6), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His6-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP6 to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

“…Cloning, Expression, and Characterization of Endo-S2-cDNA sequence encoding Endo-S2 (38 -819) was cloned into a pET22b-CPD vector, which adds to the C terminus of the expressed protein the cysteine protease domain (CPD) of the Vibrio cholerae MARTX toxin and a ϫ10 histidine tag (35). We have recently reported that a high-level, soluble expression of Endo-F3 and its mutants could be achieved using this vector (32).…”

“…Site-directed Mutagenesis and Expression and Purification of Recombinant Endo-S2-cDNA encoding amino acids 38 -819 of Endo-S2 from S. pyogenes NZ131 (serotype M49) was amplified by PCR and cloned into the pCPD-Lasso vector (a pET22b-CPD derivative) (35). For saturation mutagenesis of the Asp-184 residue, a forward primer, 5Ј-CGTAAATTCGTGCT-CAATNNNAATATCTAGTCCATCGACACCACGATCA-GTT-3Ј, and a reverse primer, 5Ј-AACTGATCGTGGTGTCG-ATGGACTAGATATTNNNATTGAGCACGAATTTACG-3Ј, were used.…”

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

“…For Sen1 Hel expression, we used a fusion protein with a cleavable C‐terminal His tag coupled to Vibrio cholerae MARTX toxin cysteine protease domain (His 8 ‐CPD) (Shen et al , 2009). His 8 ‐CPD tagged Sen1 Hel (1,095–1,904) and its mutant derivatives were purified from Escherichia coli BL21 (DE3) STAR pRARE (Stratagene) cells grown in TB medium.…”

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

“…Wild-type full-length NES was previously cloned into the cysteine protease domain (CPD) fusion protein expression system developed by Shen et al and optimized by our lab (7,21). Loop deletion mutants were made through site-directed mutagenesis to remove hairpin loop 1 (residues 77 to 82) and hairpin loop 2 (residues 150 to 157) and replace each with a linker composed of one glycine and one serine.…”