Method for the production of cystine-knot proteins

摘要

The present invention relates to a method of producing a biologically active recombinant cystine-knot protein comprising (a) solubilisation of inclusion bodies comprising said cystine-knot protein produced in a bacterium in the presence of a chaotropic agent; (b) renaturation of the solubilized cystine-knot protein in batch or by pulse addition of said solubilized cystine-knot protein to a refolding buffer essentially devoid of NaCl having a pH value of 7.5 to 9.5 and preferably of 8 to 9 comprising (ba) an aggregation suppressor in a final concentration of at least 0.5 mol/L; (bb) a mixture of reduced and oxidized glutathione wherein the ratio of reduced to oxidized glutathione is equal or above 1:10; and (bc) a solubilizing chaotropic agent in a non-denaturing concentration, wherein said renaturation is carried out at a temperature between 0 DEG C and 20 DEG C; (c) dialysis of the renatured cystine-knot protein against or dilution of the renatured cystine-knot protein into a buffer having an alkaline pH value comprising a solubilising non-ionic agent in a non-denaturing concentration; (d) binding to a heparin matrix of the product obtained in step (c) at a conductivity between 15 and 25 mS/cm; and (e) elution of the renatured biologically active cystine-knot protein.