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Extracellular expression of porcine growth hormone releasing factor by Escherichia coli

Authors Tam, Po Ming
Issue Date 1994
Summary Recombinant porcine growth hormone releasing factor (pGRF) had been successfully expressed only as fusion products, in Escherichia coli. It has not been expressed as an independent authentic product, probably due to its small size, which makes it susceptible to proteolytic degradation. By employing the efficient E. coli expression/excretion Tat cassette, the externalized pGRF is more likely to stay intact in the cell culture medium. The pGRF gene was originally cloned in the tac excretion cassette carried on M13mp18 as a precise fusion to the ompA leader sequence. With the information on the protein hydrophobicity profile of pGRF, two regions of relatively high hydrophilicities were identified. Two mutants of pGRF potentially resistant to trypsin and chymotrypsin were generated by individually replacing the 10th Tyr residue and the 34th Arg residue of pGRF with alanine using site-directed mutagenesis. Both the wild-type and the mutant ompA-pGRF constructs were sub-cloned into expression vectors, pM and lacIQpar8. Northern blot analysis of the messenger RNAs collected from cells harboring the recombinant constructs revealed that both the wild-type and the mutant ompA-pGRF constructs were able to produce a strong transcriptional signal. Time-course experiments were then carried out to pinpoint the optimal time period for maximum yield of proteins. Subsequently, with the aid of ELISA and chromatographic methods, immunoactivity of pGRF was demonstrated in the culture supematant of an IPTG-induced E. coli clone harboring the ompA-pGRF construct.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 1994
Language English
Format Thesis
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