Structurally unique recombinant Kazal-type proteinase inhibitor retains activity when terminally extended and glycosylated (original) (raw)

2005, Protein Expression and Purification

Recombinant derivatives of the Kazal-type serine proteinase inhibitor GmSPI2 (36 amino acid residues), which is a component of insect silk, were prepared in the expression vector Pichia pastoris. The rhSPI2 had a C-terminal hexahistidine tag attached to the GmSPI2 sequence, rtSPI2 was extended with GluAlaAla at the N-terminus, and rfSPI2 included this N-terminal extension and a C-terminal tail of 22 residues (myc epitope and hexahistidine). A portion of the secreted rfSI2 was O-glycosylated with a trimannosyl or hexamannosyl. The native inhibitor was active slightly on trypsin and highly on subtilisin and proteinase K. The extended C-terminus in rhSPI2 and rfSPI2 enhanced activity on the two latter enzymes and rendered rfSPI2 active on elastase and pronase, but abolished the inhibition of trypsin. The glycosylation of rfSPI2 reduced its inhibitory activity to a level comparable with the native inhibitor. The rtSPI2 with tripeptide extension at the N-terminus and no C-terminal modiWcation was clearly less active than the native inhibitor. None of the tested compounds inhibited -chymotrypsin and the non-serine proteinases.  2 Abbreviations used: AOX1, alcohol oxidase I; BMGY, medium containing 1% yeast extract; 2% peptone; 1.34% yeast nitrogen base, 4 £ 10 ¡5 % biotin, 1% glycerol, 100 mM potassium phosphate, pH 6.0; BMMY, as above but with methanol instead of glycerol; BmSPI2, Bombyx mori silk proteinase inhibitor 2; bp, nucleotide base pairs; GmSPI2, G. mellonella silk proteinase inhibitor 2; MALDI-TOF, matrix-assisted laser desorption/ionization-time-of-Xight; nt, nucleotide(s); Mf 1, mating factor 1; Ni-NTA, nickel-nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; rhSPI2, rfSPI2, and rtSPI2 recombinant fusion protein containing the GmSPI2 sequence; SDS, sodium dodecyl sulphate; YPG, medium containing 1% yeast extract, 2% peptone, and 2% glucose.