Design, synthesis and expression in different hosts of a gene coding for a small multifunctional peptide
Van der Merwe, Walda Brenda
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Platelets and coagulation both play a pivotal role in thrombosis, one of the major causes of death in Western society. It is therefore not surprising that potent inhibitors are being developed to inhibit platelet function or coagulation. In this study we developed a multifunctional peptide that would not only inhibit thrombin, but will also prevent platelet aggregation. A 29 amino acid peptide was designed comprising three inhibitory regions: 1, a part of hirudin, a potent direct antithrombin; 2, fibrinopeptide A, also an inhibitor of thrombin and 3, an amino acid sequence (arqgly- asp) essential for binding to the fibrinogen receptor on platelets to prevent fibrinogen binding. The protein sequence was reverse translated and two 60-mers with an overlap of 22 base pairs were synthesized. After filling in the ends, the gene was cloned into a yeast expression and secretion vector, pMFα8. The construct was sequenced to verify correct orientation of the gene and transformed into yeast. The expected peptide of approximately 3 kDa could not be seen on SDS-PAGE gels, nor could inhibition of thrombin or platelet aggregation be shown. However, Northern hybridization analysis revealed the presence of the mRNA. The introduction of a methionine residue at the N-terminus of the peptide and the use of protease-deficient yeast strains as expression hosts, did not improve peptide production. Another expression system, the inducible yeast expression vector pYES2, was then employed. Although no peptide could be detected, Northern blotting showed the presence of high levels of the mRNA. Optimal codons for expression in yeast were selected in the design of the gene, but the absence of a detectable level of peptide could be due to a low translation rate or due to proteolysis. Alternatively, the gene was cloned into a regulatable E. coli vector, pQE-32, and expressed intracellularly in E. coli. No band of the correct size could be detected on SDS-PAGE gels, which might be due to the extremely small molecular size of the peptide.