Construction and characterization of thermo-inducible vectors derived from heat-sensitive lacI 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 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.
Chao
Yun-Peng
YP
Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan. ypchao@fcu.edu.tw
Chern
Jong-Tzer
JT
Wen
Chin-Sheng
CS
Fu
Hongyong
H
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D007763:Lac Operon / Q000235:genetics*; D056968:Lac Repressors; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D011994:Recombinant Proteins; D012097:Repressor Proteins; D001616:beta-Galactosidase