PURPOSE:To efficiently culture the subject Escherichia coli and mass-produce the objective protein by transforming Escherichia coli with a plasmid having a replication origin of pUC plasmid, start-culturing the resultant recombinant Escherichia coli at a specific temperature and increasing the culturing temperature in the logarithmic growth phase. CONSTITUTION:A fragment obtained by cleaving a plasmid pKK223-3 containing a gene capable of coding an amino acid sequence of hirudin or a variant thereof with restriction enzymes PvuI and NruI is linked to a fragment containing a replication origin (Ori), etc., prepared by cleaving a plasmid pUC18 with restriction enzymes PvuI and PvuII, and further linked to a fragment obtained by digesting an expression vector pMKAK3 with restriction enzymes NcoI and HindIII to prepare a plasmid having a replication origin (Ori) of the pUC plasmid. The resultant plasmid is then inserted into Escherichia coli to carry out transformation. The culturing of the obtained recombinant Escherichia coli is subsequently started at a temperature within the range of 20-35 deg.C and the temperature is increased to =37 deg.C in the logarithmic growth phase. Thereby, the objective recombinant Escherichia coli is efficiently cultured.
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