Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the methylotropic yeast Pichia pastoris

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leech Haementeria ghilianii. In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an in-frame fusion of the ghilanten-coding sequences with the region encoding the pre-pro alpha-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter. Pichia pastoris yeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5'-AOX1 locus via homologous recombination. Both strains yielded His(+) transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level of r-ghilanten than KM 71. Significant clonal variation in the expression of r-ghilanten was found among the His(+) transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales. r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions. (C) 1995 Academic Press, Inc.