Open AccessExpression of polyoma virus middle‐T antigen in Saccharomyces cerevisiae
Author(s) -
BELSHAM Graham J.,
BARKER David G.,
SMITH Alan E.
Publication year - 1986
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1986.tb09598.x
Subject(s) - plasmid , microbiology and biotechnology , biology , saccharomyces cerevisiae , antigen , gene , yeast , expression vector , coding region , genetics , recombinant dna
The polyoma middle‐T gene, lacking its intron, was inserted into a yeast expression plasmid containing the phosphoglycerate kinase promoter. Such plasmids transformed yeast at low frequency and these transformants expressed middle‐T antigen at a level of approximately 0.1% cell protein. Furthermore, expression of this protein was frequently lost during growth in liquid culture and this loss of middle‐T was accompanied by a twofold increase in the rate of growth. The spontaneous production of a truncated middle‐T antigen, lacking the C terminus, was also observed; the expression of this protein did not inhibit the growth rate of the cells. Recovery and analysis of the expression plasmids encoding the truncated molecule showed that a single C · G base pair had been deleted from a run of nine consecutive C · G base pairs (Pyr nucleotide 1239–1247) within the middle‐T coding region. This frame‐shift mutation results in premature termination of the protein and loss of the strongly hydrophobic region of the molecule believed to be responsible for the membrane association of middle‐T antigen.