Stabilization of a Recombinat Plasmid in Yeast

概要

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When Saccharomyces cerevisiae AH22R^--2075 harboring pGLD p31-RcT which encodes a modified hepatitis B virus-surface antigen P31 (rHBsAg P31 mutein) coding gene and β-isopropylmalate dehydrogenase gene (LEU2) was cultivated on a large scale in a leucine-lacking medium, rHBsAg non-producing colonies appeared at high frequency. Restriction enzyme analysis and Southern blot analysis with LEU2 probe revealed that the cells from these non-producting colonies had lost the expression plasmid, pGLD p31-RcT, and had unusual 2 μm DNA containing the LEU2 gene. This was caused by homologous recombination between 2 μm DNA and pGLD p31-RcT which contains a 2 μm DNA fragment and the LEU2 gene. Stabilization of the recombinant plasmid was achieved by the following three approaches : (i) reconstruction of the expression plasmid by separeting the LEU 2 gene from the 2 μm DNA fragment on the plasmid; (ii) selection of plasmid-stabilized clones by taking advantage of the differences in growth rate between producing and non-producing clones; and (iii) addition of a high concentration of L-histidine to the medium.
社団法人日本生物工学会の論文
1992-09-25