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

Abstract

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 lacI (lacIts) with the T7 A1 promoter on a multiple-copynumber plasmid. The lacIts 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 lacI gene. With the LacZ production as a model, the induction profiles for various vectors containing distinct lacIts exhibited a positive trend as the temperature increased. The fully induced level was achieved by applying the temperature shift from 30 degrees C to 42, 40, or 37 degrees C to the cells harboring the plasmid with the Gly187-->Ser, Ala241-->Thr, or Gly265-->Asp substitution in lacI, 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.