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 inframe 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.
Brankamp
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RG
Hoechst Marion Roussel, Department of Molecular Genetics, Cincinnati, Ohio 45215, USA.
Sreekrishna
K
K
Smith
P L
PL
Blankenship
D T
DT
Cardin
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000302:isolation & purification*; D000970:Antineoplastic Agents / Q000302:isolation & purification*; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007447:Invertebrate Hormones / Q000235:genetics* / Q000302:isolation & purification*; D007865:Leeches / Q000235:genetics*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D012471:Salivary Proteins and Peptides / Q000235:genetics* / Q000302:isolation & purification*
D000925:Anticoagulants; D000970:Antineoplastic Agents; D004274:DNA, Recombinant; D007447:Invertebrate Hormones; D012471:Salivary Proteins and Peptides; C063005:ghilanten