Expression of α-amylase gene by T7 expression system in S. cerevisiae and E. coli

Abstract

Bacteriophage T7 RNA polymerase actively and selectively directs transcription from a highly specific promoter that is unlikely to be found in DNA unrelated to T7. This selectivity provides the basis for gene expression system in E. coli and potentially in a variety of cell types. Here the $\alpha$-amylase gene of B. licheniformis was expressed by T7 expression system in E. coli and S. cerevisiae. To utilize T7 RNA polymerase in this expression, its gene was introduced into the chromosome in each cell. Then vectors containing $\alpha$-amylase gene under the control of a T7 promoter were constructed. E. coli BL 21(DE3) transformed with pKS01 or pKS02 was seen to grow very slowly or died. It may be due to the basal level of T7 RNA polymerase in uninduced state. And BL 21(DE3) containng pKS01 or pKS02 together with T7 lysozyme gene produced $\alpha$-amylase, and stably maintained. On the other hand in E. coli not containing T7 RNA polymerase gene, expression of $\alpha$-amylase gene behind the T7 promoter was shown. We found that T7 promoter used in our experiment contains a fragment recognized by E. coli RNA polymerase. S. cerevisiae YK71-N containing YpKS01 or YpKS02 did not produce $\alpha$-amylase, but produced significant amounts of its mRNA. This result indicates that the gene is sufficiently transcribed in yeast by the T7 expression system, but posttranslational processes were in defect.