High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli.

ABSTRACT: We have developed a new expression vector, pcI(ts) ind(+), based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector "portable" in that it can be used to transform any strain of Escherichia coli. Hence, control over strains, induction conditions, and harvest times can be used to optimize yields of heterologous proteins. To provide a proof of concept, we show that E. coli recA(+) and recA(-) host cells transformed with pcI(ts) ind(+) modKlenTaq1 (a modified version of the large fragment of Thermus aquaticus DNA polymerase I) could be grown to high cell densities in multiple shake-flasks. A mutant v

SUBMITTER: Brandis JW 

PROVIDER: S-EPMC3040104 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature