Construction and characterization of thermo-inducible vectors derived from heat-sensitive lacl genes in combination with the T7 A1 promoter

The lack of stringency and the cost of induction are two major disadvantages of using lac-derived vectors for recombinant protein productions. To compensate for these drawbacks, a series of thermo-inducible vectors was developed by coupling heat-sensitive lacl (laclts) with the T7 A1 promoter on a multiple-copy-number plasmid. The laclts genes were created by the introduction of Gly187-->Ser substitution along with three alternative mutation sites, Leu233-->Lys, Ala241-->Thr, and Gly265-->Asp, generated by site-directed mutagenesis into the wild-type lacl gene. With the LacZ production as a model, the induction profiles for various vectors containing distinct laclts exhibited a positive trend as the temperature increased. The fully induced level was achieved by applying the temperature shift from 30degreesC to 42, 40, or 37degreesC to the cells harboring the plasmid with the Gly187-->Ser, Ala241-->Thr, or Gly265-->Asp substitution in lacl, respectively. As a result, it produced the maximal LacZ production ranging between 46,000 and 54,000 Miller units, corresponding to a 100- to 400-fold amplification over the uninduced level. As a whole, these novel expression vectors are characterized as having tight regulation and facile inducibility, and their practical usefulness in industrial production of recombinant proteins appears promising. (C) 2002 Wiley Periodicals, Inc.