Molecular expression system design : theoretical and experimental characterization of a novel cross-regulation system and its application as a metabolic switch
Abstract (Summary)A novel cross-regulation system was described in this work for regulated recombinant proteins expression. Molecular-level mathematical models of the lac and [lambda]P[subscript R] promoter-repressor systems were used in example calculations and the effectiveness of the cross-regulation system was evaluated by comparing with two other repressor control configurations (constitutive repressor synthesis and autogenous regulation). Simulation results suggested that this system offered the best control of transcription over a broad range of copy number in the uninduced state and also provided the highest overall transcription rate in the induced state. The validity of the cross-regulation system in an actual experimental setting was also examined. Because of their desired properties, the tac-lacI and [lambda]P[subscript L]-cI promoter-repressor systems were used to construct vectors for regulated expression of chloramphenicol acetyltransferase (CAT). The polymerase chain reaction (PCR) technique was employed to generate the lacI and cI structural genes. Functional assays were performed to ensure active products from these PCR fragments. Induction results matched well with model prediction indicated that CAT expression from the cross-regulation system is two fold higher than the control (constitutive repressor synthesis configuration) with a very similar low basal expression. By using different copy number plasmids, the cross-regulation system has been shown to be equally applicable over copy numbers from 50-150. Various factors influencing the recombinant protein yield from the cross-regulation system in E. coli were studied. It was determined that the optimum yield can be obtained by induction at 2-3 hr into the exponential growth and by using an IPTG concentration exceeding 0.5 mM. A limitation at the transcription level was determined to be the most crucial factor for CAT expression in a batch fermentation mode. A cease in the CAT production was coincided with a 10 fold decrease in the CAT mRNA level after transition into the stationary phase. This bottleneck, however, can be eliminated by extending cell growth either by using a fed-batch fermentation mode or by using an unmutagenized E. coli strain. In view of the interesting transcriptional property of the cross-regulation system, it was applied as a metabolic switch to provide a novel mean for the redirection of metabolic flux. The validity of the metabolic switch was illustrated by an alternation in the Vitreoscilla hemoglobin (VHb) and CAT expression patterns before and after induction. A practical application to alternate glycogen synthesis and degradation was examined and results indicated a five fold increase in glycogen synthesis and a 40% increase in glycogen degradation.
Advisor:James E. Bailey
School Location:USA - California
Source Type:Master's Thesis
Date of Publication:09/08/1992