Author(s): Bitter GA, Egan KM
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Abstract The promoter region from the cloned glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Saccharomyces cerevisiae (Musti et al., 1983) has been characterized. A 653-bp TaqI restriction fragment with a 3' border 24 bp upstream from the ATG initiation codon was isolated and demonstrated to contain all sequences necessary for promoter function in vivo. This DNA segment was converted to a portable promoter by cloning it into M13mp9, and the entire nucleotide sequence of the portable promoter was determined. Two generalized yeast expression vectors have been constructed utilizing the GPD portable promoter. The expression vectors include the yeast 2 mu origin of replication and amplification functions, such that the plasmids are maintained at high copy number in ciro yeast hosts. These vectors direct synthesis of a consensus alpha-interferon (IFN-alpha Con1) as 1\% of total cell protein. Hepatitis B surface antigen (HBsAg) was also expressed from these vectors. The 5' end of the HBsAg gene was replaced with a synthetic DNA segment which restored the deleted GPD untranslated leader and utilized optimal yeast codons for the first 30 amino acids. The partially synthetic gene resulted in a 10- to 15-fold increased expression level from GPD vectors yielding HBsAg polypeptide as 2-4\% of total cell protein.
This article was published in Gene
and referenced in Journal of Bioremediation & Biodegradation