A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse alpha-amylase, is described. Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain. To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored. Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged and not completely corrected. Further assay showed that the viability and mitotic ability of the NI-C strain were increased. While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse alpha-amylase, we observed that transformed colonies grew more quickly and secreted more alpha-amylase than general yeast strains. A further test showed that the NI-C strain was able to use mouse alpha-amylase as a positive selection marker to form transformed colonies on nitrogen-starved plates that contain starch as the sole carbon source. The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation system.

• PMID: 10091324
• DOI full text
• PubMed
• PubTator

Reference Type

Journal Article | Research Support, Non-U.S. Gov't

Authors

Chen DC, Chen SY, Gee MF, Pan JT, Kuo TT

Primary Lit For

Additional Lit For

Review For