Effect of the mRNA secondary strucure on the expression of a bovine growth hormone cCNA in Saccharomyces cerevisiae
1998
Pennapa Manitchotpisit, Mahidol Univ., Bangkok (Thailand). Inst. of Molecular Biology and Genetics
Yeast Saccharomyces cerevisiae was used as a host to express the bovine growth hormone (bGH) cDNA. The mature bGH cDNA was clone into yeast shuttle vector YEp352. Under the control of TEFl strong promoter, the expression of bGH from yeast containing YEp352-bGH could not be dectected while the bGH mRNA from the recombinant yeasts was detectable. The problem, therefore, was focused on the translational level. Study of the secondary structure of bGH mRNA leader has shown a stem-loop structure which can inhibit the ribosome from migrating through the start codon. To reduce the structure of stem loop of bGH mRNA leader, and adapted structure was designed by changing the first codon of mature bGH from alanine to methionine. The MetbGH was generated by using sitedirected mutagenesis technique. The expression of MetbGH was increased when compared to wild type bGH. This indicated that the mRNA secondary structure plays a role in level of bGH expression. Further studies on the expression of bGH in S. cerevisiae should reduce the stability of the stem-loop structure at the mRNA leader by designing a longer open structure than that of the MetbGH or without any stem-loop at all. Consideration should also be given to the type of yeast strain which could be help increase the number of copies of plasmid while not promoting in vivo protein degradation.
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