A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle.
The inhibitor cystine knot (ICK) structural motif has been found in several small proteins and peptides from plants, insects, marine molluscs, and also in human. It is defined by a triple beta-sheet that is held together by three intramolecular disulfide bonds built by six conserved cysteine residues that generate a highly rigid and stable fold. We describe a procedure for the production of ICK peptides with correct disulfide bond connectivities via expression in Escherichia coli as fusion proteins with an enzymatically inactive variant of the Bacillus amyloliquefaciens RNAse barnase. Barnase directs the fused peptide to the culture medium and the fusion protein can be isolated by combined cation exchange/reverse-phase chromatography. The ICK peptides are released from the barnase expression and purification handle either by cyanogen bromide or by protease cleavage to give pure and correctly folded cystine knot peptides.
Schmoldt
Hans-Ulrich
HU
Abteilung für Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Wentzel
Alexander
A
Becker
Stefan
S
Kolmar
Harald
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003553:Cystine / Q000235:genetics / Q000378:metabolism; D047168:Cystine Knot Motifs; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D012260:Ribonucleases / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003553:Cystine; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease