Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria.
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-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-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.
Simonet
Gert
G
Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute, KU Leuven, Naamsestraat 59, B-3000 Leuven, Belgium.
Claeys
Ilse
I
Huybrechts
Jurgen
J
De Loof
Arnold
A
Vanden Broeck
Jozef
J
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005822:Genetic Vectors; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D004791:Enzyme Inhibitors; D019476:Insect Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; C113210:LOC126284306 protein, Schistocerca gregaria