Comparative expression of four different metallothionein genes in escherichia coli
Student thesis: Master's Thesis
Four different metallothionein (MT) genes - the mtnA from Synechococcus vulcanus; the CUP-1 from Saccharomyces cerevisiae; the Candida glabrata MT-1; and the mouse MT-1 were PCR-cloned into the pBS cloning vector. For comparative expression in E. coli, the four MT genes were subsequently subcloned into three different types of expression vectors pTrc99A, pGEX-2T or pGEX-3X, and pET-14b and the recombinant plasmids were then transformed into E. coli DH5a (and into E. coli BL21(DE3)pLysS for recombinant pET-14b clones). Expression of the recombinant MT proteins in the E. coli host cells were monitored by SDS-PAGE. Physiological effects on expression of heterologous MTs in E. coli including Cd biosorption capacity and tolerance levels to Cd and Cu on solid media were determined. Subcloning of the four MT genes into pTrc99A yielded four recombinant plasmids - pAC14 contains the mtnA gene; pAC24 contains the CUP-I gene; pAC34 contains the C. glabrata MT-1 gene; and pAC44 contains the mouse MT-1 gene. However, expression of the expected MTs was not detectable by SDS-PAGE analysis. This may have been due either to the rapid digestion of the recombinant MTs by host proteases or cell toxicity due to the presence of large number of -SH groups in the cysteine residues of the MT protein. In an attempt to stabilize expression of the cysteine-rich MT proteins, the four MT genes were subsequently subcloned into the pGEX vectors to produce fusion proteins of the MT and glutathione S-transferase (GST). Four pGEX recombinant clones: pAC16 (mtnA); pAC26 (CUP-I); pAC36 (C. glabrata MT-1); and pAC46 (mouse MT-1) were constructed. Expression of the corresponding MT fusion proteins by the four recombinant pGEX clones were detected by SDS-PAGE. In addition, all four clones showed an increase in Cd (15-28% increase) and Cu (7- 20% increase) resistance and Cd biosorption capacities (31-43% increase). The three eukaryotic MT-expressing E. coli clones accumulated more Cd than pAC16 which expressed the prokaryotic synechococcal MT fusion protein. To study the phenotypic effects of intact forms of the four MT protein, the MT genes were subcloned into the PET-14b expression vector and introduced into a protease-deficient E. coli host strain B2 1 (DE3)pLysS. Four recombinant clones were obtained: pAC17 (mtnA); pAC27 (CUP-I); pAC37 (C. glabrata MT-1) and pAC47 (mouse MT-1). Among the four clones, only pAC17 was found to express the expected size mtnA protein as shown by SDS-PAGE. The pAC17 clone also showed an increase in Cd biosorption (61% increase compared with control cells containing PET- 14b alone). To investigate the possibility of expressing the MT proteins in sludge bacteria, the recombinant MT-pTrc99A, MT-pGEX and MT-pET- 14b piasmids containing the in-frame MT genes were electroporated into Pseudomonas aeruginosa PbH4-1, a bacterial strain isolated from activated sludge of the Shatin Sewage Treatment Plant. Transformants were screened by colony hybridization using the four MT genes as DNA probes. However, no detectable signal was obtained from the transformant colonies. It was possible that the recombinant MT genes could not be maintained inside the Pseudomonas host cells because the E. coli plasmid vectors (pTrc99A, pGEX and pET-14b) may not be able to replicate in Pseudomonas. To stably maintain the target MT genes in the Pseudomonas cells, a gene construct which contains a host-specific origin of replication, promoter and termination signal and a short stretch of homologous DNA to facilitate integration of the target MT into the host chromosome is proposed.
- Metallothionein, Escherichia coli