Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria
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)
- et al.
Serine protease inhibition by insect peptides containing a cysteine knot and a triple-stranded β-sheet
J. Biol. Chem.
(1995) - et al.
Insect immunity: two proteinase inhibitors from hemolymph of Locusta migratoria
Biochem. Biophys. Res. Commun.
(1992) - et al.
Purification and characterization of a group of five novel peptide serine protease inhibitors from ovaries of the desert locust, Schistocerca gregaria
FEBS Lett.
(1998) - et al.
Proteinase inhibitors from desert locust, Schistocerca gregaria: engineering of both P(1) and P(1)’ residues converts a potent chymotrypsin inhibitor to a potent trypsin inhibitor
Biochim. Biophys. Acta
(1999) - et al.
Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors
J. Mol. Biol.
(1996) - et al.
Complexation of two proteic insect inhibitors to the active site of chymotrypsin suggests decoupled roles for binding and selectivity
J. Biol. Chem.
(2001) - et al.
What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes?
Biochim. Biophys. Acta
(2000) - et al.
Structural and functional properties of a novel serine protease inhibiting peptide family in arthropods
Comp. Biochem. Physiol. B: Biochem. Mol. Biol.
(2002) - et al.
Cloning of a Locusta cDNA encoding a precursor peptide for two structurally related proteinase inhibitors
Insect Biochem. Mol. Biol.
(1994) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding
Anal. Biochem.
(1976)
The effect of endogenous proteinase inhibitors on the prophenoloxidase activating enzyme, a serine proteinase from crayfish haemocytes
Insect Biochem.
Purification of a protease inhibitor which controls prophenoloxidase activation in hemolymph of Locusta migratoria (Insecta)
Biochem. Biophys. Res. Commun.
Cited by (16)
Exploring the molecular complexity of Triatoma dimidiata sialome
2018, Journal of ProteomicsThe first homolog of pacifastin-related precursor in the swimming crab (Portunus trituberculatus): Characterization and potential role in immune response to bacteria and fungi
2012, Fish and Shellfish ImmunologyCitation Excerpt :Among the eight domains of PtPLC, four of them could be trypsin inhibitors with P1-P1′ Arg–Gln (PtPLC-1), Lys–Lys (PtPLC-4) and Arg–Lys (PtPLC-6, PtPLC-7), one of them was supposed to be a chymotrypsin inhibitor with P1-P1′ Leu–Met (PtPLC-5). The unconsensus P1-P1′ reactive sites of PLD domains in PtPLC were also found in PlPLC (P. leniusculus pacifastin light chain) [13,31], which is more variable than those reported in locust pacifastin-related precursors [31,33,34]. The mRNA transcripts of PtPLC could be detected in all the examined tissues, with highest expression in haemocytes, intermediate levels in gill, hepatopancreas and stomach, and lower levels in eyestalk, heart and muscle.
Functional analysis of a pancreatic secretory trypsin inhibitor-like protein in insects: Silencing effects resemble the human pancreatic autodigestion phenotype
2011, Insect Biochemistry and Molecular BiologyCitation Excerpt :Statistical differences were analyzed using one-way ANOVA with a post-hoc Tukey test. For further characterization, the LmPSTI inhibitor was produced as a recombinant protein in the bacterial expression system pMAL (New England Biolabs) (Simonet et al., 2003). This system allows the expression of the protein of interest fused to Maltose Binding Protein (MBP), enabling easy purification by affinity chromatography.
High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4′ acidic residues
2011, Journal of Biological ChemistryIn vitro activity of pacifastin-like inhibitors in relation to their structural characteristics
2011, PeptidesCitation Excerpt :The results, including the unprecedented biochemical characteristics of a designed inhibitor variant are further discussed in terms of the structure-function relationship of pacifastin-like PI. Procedures for the recombinant production and purification of pacifastin-related PIs from the desert locust (SGPI-1, SGPI-2 and SGPI-3) and from the silkworm (BMPP-C and BMPP-N) were previously reported [3,21]. In this study the same methodology was used and further optimized to obtain a higher yield.