Abstract
Abstract This dissertation studied the recombinant protein expression in Escherichia coli based on two factors: development of a novel protein expression system and fusion protein. Firstly, a new protein expression system in E.coli was constructed. GlnAP2 element had been proven to be an effective and inducible promoter by exogenous acetate in the E. coli glnL/pta double mutant host. Based on this feature, a single-copy expression vector was constructed via coupling the glnAP2 promoter-regulated T7 RNA polymerase gene and the T7 promoter controlled lacZ gene onto the bacterial artificial chromosome. After induction with 20 mM potassium acetate, the glnL/pta double mutant E. coli host harboring this single-copy plasmid produced 47,500 Miller units of □-galactosidase activity. This high level expression corresponding to 27 % of the total cell proteins was comparable to the one which was determined to be 64,300 Miller units and 41 % of the total cell proteins on the commercial multi-copy expression vector, pET-14b, in the E. coli Tuner (DE3) strain. Moreover, this single-copy expression vector was stably maintained for at least 150 generations even in the presence of inducers. In contrast, the multi-copy expression vector was extensively lost after induction. These results suggest that this single-copy expression system developed here has a high potential for high-level heterologous protein production for industrial uses. Secondly, we designed a new fusion protein for the assay of HA. The gene expression plasmid pET-Lmluc for the fusion protein of the link module from human TSG-6 (product of tumor necrosis factor (TNF)-stimulated gene-6) and luciferase from Renilla reniformis was constructed. The fused gene was expressed in E. coli and the resulted insoluble Lm-luc fusion protein was purified and refolded to recover both the hyaluronan (HA) binding capability and the luciferase activity. HA concentration as low as 1 ng/ml was assayed by using the indirect enzymatic immunological assay (EIA) with the refolded Lm-luc fusion protein.