Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae

The use of mass spectrometry to examine the formation and hydrolysis of the phosphorylated form of phosphoglycerate mutase

“…PGAM from S. pombe was produced using the PGK-based vector pMA91 [9] for the high level expression of recombinant GPM ~p in a transformed null mutant strain of S. cerevisiae (S150-gpm:: HIS3) [7]. The overexpressed S. pombe PGAM was purified in a similar manner to overexpressed S. cerevisiae PGAM [7]; routinely 10-15 mg of enzyme of at least 95% purity on SDS-PAGE [10] could be obtained per litre of cells.…”

Improvement of bacterial -glucanase thermostability by glycosylation

“…The (1,3-1 ,4)-/3-glucanase genes were excised from pUC 13-M as a DraI-Hind111 fragment and from pUC 13-H1 as a BsmI-SphI fragment. Following T4 DNA polymerase treatment to produce blunt ends, the DNA fragments were cloned into the filled-in BgiII site downstream from the phosphoglycerate kinase promoter of the yeast expression vector pMA91 (Mellor et al, 1983). In order to obtain efficient secretion of the hybrid enzyme from yeast cells, the B. amyloliquefaciens (AMY) (1,3-1,4>/3-glucanase signal peptide coding region was exchanged with the corresponding region from the MAC (1,3-1,4>/3-glucanase gene.…”

The ORF YNL274c (GOR1) codes for glyoxylate reductase in Saccharomyces cerevisiae

“…This longer PCR product was subsequently used to clone the ORF for FDH1. The cloned ORFs were transferred to the Bgl II site of an expression cassette containing PGK1 promoter and terminator (Mellor et al, 1983), at the Hind III site of YEplac195 yeast vector (Gietz and Sugino, 1988). The coding regions were again sequenced.…”