Abstract:

In Escherichia coli, four gene products(selA, selB, selC and selD) and a selenocysteine(Sec) insertion sequence(SECIS) are required for the correct translation of UGA codons encoding Sec. Previous studies have shown that the stoichiometry of selenoproteine mRNA and elongation factor SelB affect the efficiency of Sec incorporation. Herein lies a detailed analysis of the effects of co-expressing selA, selB and selC genes under inducible promoters on the incorporation efficiency of Sec. Over-expression of either selA or selB reduced the efficiency of Sec incorporation by 61.1% or 11.6%, respectively, compared to the over-expression of the reporter vector alone did. Concomitant over-expression of selC with selA or selB completely reversed the reduce of the efficiency of Sec but still reduced the efficiency of the incorporation relative to that observed for expression of selC alone. Over-expression of selC gene alone under L-arabinose induction reduced the efficiency of the incorporation relative to that observed for co-expressing selC with selA and selB under the control of its endogenous promoter in the absence of L-arabinose. Co-expression of selA, selB and selC with selA or selB under the control of inducible promoters increased the efficiency of Sec incorporation by 69.7%. Moreover, inducing selenoprotein-related gene expression during the late exponential phase increased the efficiency of Sec incorporation by a factor of 5.4 relative to that observed for the reporter vector alone. These results suggest that the co-expression of selA, selB and selC in Escherichia coli under the control of inducible promoters is a viable and promising strategy for increasing the yields of selenoproteins.

Key words: Selenocysteine, SelA, SelB, SelC, Combinatorial expression