Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain

Abstract

A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8(T), that allow E. coli to grow at high rates at 4 degrees C (maximum growth rate, 0.28 h(-1)). The expression of a temperature-sensitive esterase in this host at 4 to 10 degrees C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37 degrees C (32,380 versus 190 micromol min(-1) g(-1)). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5 degrees C).

FIG. 1.

SDS-PAGE analysis of cell extracts of E. coli cpn and cpn⁺ strains. From left to right, the lanes contained molecular weight markers (MW), E. coli cpn cells grown at 37°C, E. coli cpn cells grown at 15°C, E. coli cpn cells induced at 37°C and then 15°C, E. coli cpn cells induced at 37°C and then 4°C, E. coli cpn cells grown at 37°C, E. coli cpn cells grown at 4°C, and pure esterase. The asterisks and arrowheads indicate the positions of Cpn60/Cpn10 and EstRB8, respectively. Each lane contained 10 μg of protein.

FIG. 2.

Analysis of the secondary structure by far-UV circular dicroism. Far-UV circular dicroism spectra at 4 to 50°C for pure EstRB8 from E. coli bearing pBK1CpnEst grown at 4°C (•), 20°C (○), 30°C (▾), or 37°C (▿) were recorded, and the relative ellipticity at 220 nm was measured. The conditions used are described in Materials and Methods. The ellipticity at 220 nm obtained with the EstRB8 produced in E. coli grown at 4°C was assumed to be 100%. The three sets of data corresponding to EstRB8 purified from E. coli expressing plasmid pBK1CpnEst or pBK1Est at 20, 30, and 37°C were statistically equivalent, and only the data for pBK1CpnEst are shown.