Design, synthesis and expression in different hosts of a gene coding for a small multifunctional peptide
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Date
1994-01
Authors
Van der Merwe, Walda Brenda
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
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.
Description
Keywords
Peptides -- Synthesis, Molecular cloning, Dissertation (M.Sc. (Microbiology and Biochemistry))--University of the Free State, 1994