Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria

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Abstract

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5) and 11 additional locust peptides were identified by cDNA cloning. Furthermore, the light chain of the 155-kDa heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of 6 conserved cysteine residues (Cys-Xaa9-12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa2-3-Gly-Xaa3-4-Cys-Thr-Xaa3-Cys) with the locust inhibitors. So far, for most of the PLD-related peptides the biological functions remain obscure. To obtain sufficient amounts of material to perform physiological experiments, we have optimised the production of SGPI-1-2 via a bacterial (Escherichia coli) expression system. The cDNA sequences encoding these peptides were inserted in the pMAL-2pX vector, downstream of the gene encoding the maltose-binding protein (including a signal peptide). As a consequence, both peptides were expressed as fusion proteins (2–3 mg/l) and targeted to the periplasmic space. Following a one-step affinity purification, both fusion proteins were successfully cleaved by Factor Xa and after a methanol extraction, it took only one additional RP-HPLC run to purify both peptides to homogeneity. Finally, the formation of the disulphide bridges and the biological activity of the recombinant peptides were verified by mass spectrometry and a spectrophotometric protease inhibitor assay, respectively.

Section snippets

Materials

The “pMAL Expression and Purification Kit,” including the expression vector pMAL-2pX, the amylose resin, Factor Xa, and the MBP2* protein (see below) were purchased from New England Biolabs.

Construction of fusion plasmid

Based on the reported cDNA sequence encoding SGPP-1 [18], two sets of primers were designed to selectively amplify two DNA-sequences, each of which codes for one of the inhibitors, SGPI-1 and SGPI-2. To allow a directional insertion of these PCR fragments in the polylinker of the pMAL-p2X vector, two

Production, purification, and cleavage of fusion proteins

To produce sufficient amounts of SGPI-1 and SGPI-2 (Overview: Table 2), we have inserted the corresponding genes in the polylinker of the pMAL-p2X vector, down-stream of the MalE gene (Fig. 1). This vector also contains a short sequence coding for the recognition site (IEGR) of the serine protease, Factor Xa, located just 5 to the polylinker site (Fig. 1). Since the MalE gene encodes the MBP, including the pre-MBP signal peptide, the expressed fusion proteins are exported to the periplasmic

Discussion

We report here on the biosynthetic production of two serine protease inhibitors, SGPI-1* and SGPI-2*, as recombinant fusion proteins in E. coli. By a combination of only two purification steps, namely maltose-affinity chromatography followed by reversed-phase HPLC, both peptides could be purified to homogeneity. The yield of the purified fusion proteins was typically in the range of 2–3 mg/l culture and due to the high effectiveness of the cleavage reaction with Factor Xa, 100–200 μg (per litre

Acknowledgements

The authors especially thank S. Van Soest, J. Gijbels, M. Van Der Eeken, J. Puttemans, M. Christiaens, L. Vanden Bosch, and R. Jonckers for technical and administrative assistance. The authors gratefully acknowledge the Belgian “Interuniversity Poles of Attraction Programme” (IUAP/PAI P5/30, Belgian State, Prime Minister’s Office-Federal Office for Scientific, Technical and Cultural Affairs) and the “FWO-Vlaanderen” for financial support. J.Vd.B. was Senior Research Associate of the FWO and G.S

References (24)

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