A facile and efficient method to achieve LacZ overproduction by the expression vector carrying the thermoregulated promoter and plasmid copy number.
On the basis of the runaway-replication vector, an expression plasmid was developed to achieve tight regulation as well as high-level expression of cloned genes by thermal control of the promoter together with the plasmid copy number. To demonstrate the feasibility of this approach, the lacZ gene was fused with the heat-inducible promoter on the vector, and the result showed that protein production levels in the Escherichia coli strain harboring the recombinant plasmid could be varied in response to various degrees of heat shock. The maximal soluble LacZ ranging between 45 000 and 50 000 Miller units was obtained as the recombinant strain received a 30 --> 40 degrees C stepwise upshift, and it accounted for a 450-fold amplification over an uninduced level. Further analyses by SDS-PAGE indicated the maximal protein production (including soluble and insoluble forms) in the bacteria reaching approximately 30% total cell protein. In addition, two approaches were demonstrated to be very useful in enhancing the total soluble LacZ production on a fermenter scale. One was to shuttle the culture between two fermenters connected in series and set at different temperatures. The other resorted to the use of two-step temperature alteration in a batch fermenter, namely, raising the temperature to 40 degrees C for a certain period of time followed by reducing the temperature to 37 degrees C. Overall, it illustrates the remarkable features of the expression system with stringent regulation, high-level production capacity, facile induction, and high stability, and the usefulness of this system for recombinant protein productions is promising.
Chao
Yun-Peng
YP
Department of Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung, Taiwan, ROC. ypchao@fcu.edu.tw
Wen
Chih-Sheng
CS
Wang
Jen-You
JY
D019149:Bioreactors / Q000382:microbiology; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000502:physiology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism*; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D024861:Genetic Enhancement / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D007763:Lac Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D001616:beta-Galactosidase