Improving heterologous protein expression in E. coli using molecular chaperones from Thermus spp
| dc.contributor.advisor | Albertyn, J. | |
| dc.contributor.advisor | Litthauer, D. | |
| dc.contributor.advisor | Van Heerden, E. | |
| dc.contributor.author | Dukunde, Amélie | |
| dc.date.accessioned | 2015-10-01T09:10:26Z | |
| dc.date.available | 2015-10-01T09:10:26Z | |
| dc.date.copyright | 2011-06 | |
| dc.date.issued | 2011-06 | |
| dc.date.submitted | 2011-06 | |
| dc.description.abstract | Molecular chaperones are proteins which enable other protein molecules to fold to their native conformation and this property has been widely used to improve the solubility of proteins expressed in Escherichia coli. The effect of co-expressing of heterologous, thermophilic DnaK chaperones alongside with Thermus thermophilus DNA polymerase in E. coli was investigated in this study. A novel approach of co-expressing these proteins was also attempted. To construct the plasmid vectors, high-copy expression vectors pET22 and pET28 commercial plasmid were used to provide the backbone for the new vectors. The KJEA operon, encoding DnaK chaperone, DnaJ co-chaperone, GrpE nucleotide exchange factor and the DnaK/DnaJ assembly factor, DafA, was amplified from Thermus scotoductus SA-01 and T. thermophilus HB8 by PCR. Similarly, the arabinose-inducible promoter PBAD and its regulator protein-encoding gene, AraC, were amplified from the pBAD commercial vector. All fragments were subcloned into pGEM®-T easy before cloning them into the pET vectors. PBAD was first ligated to either TsKJEA or TtKJEA to make a promoter-DnaK fusion gene that was then subcloned into pGEM®-T easy and later cloned into pET22 and pET28, along with AraC to yield four recombinant vectors, p22TsK, p22TtK, p28TsK and p28TtK. Induction of PBAD in these vectors with 5 mg ml-1 of L-arabinose resulted in expression of DnaK chaperone proteins from only p22TsK and p28tsK, which express T. scotoductus DnaK proteins. The problem in p22TtK and p28TtK has been attributed to non-expression of AraC protein due to the long distance between AraC and its promoter region which lies in the PBAD region, and is inverted in these vectors; however, this has yet to be investigated. The T. thermophilus DNA polymerase, TthPolI was cloned into the MCS of the chaperone expression vectors. The same polymerase was also fused to a superfolder green fluorescent protein, sGFP, and cloned into the chaperone vectors. Only the pET22-series of chaperone vectors were used as cloning into the pET28 line was unsuccessful. Expression of these two proteins was initiated by induction of the T7/lac promoter with 1mM IPTG. A commercial plasmid, pKJE7, encoding E. coli DnaK, DnaJ and GrpE was also used to co-express both proteins, for comparison. Expression was achieved in all DnaK-expressing vectors as well as non-expressing negative controls. Purification of uncoupled TthPolI by affinity chromatography was possible from cell expressing DnaK and the negative controls; however, only TthPol-sGFP protein expressed from p22TsK was purified successfully, demonstrating the need for molecular chaperones when folding large proteins and the superiority, in folding activity, of the thermophilic DnaK chaperone system, in comparison to the E. coli system. Activity assays were carried out to test the processivity, thermostability and fidelity of Tth polymerases purified from these vectors. Results show that Tth Polymerase amplifies short fragments, such as 771 bp sGFP, with high fidelity and is comparable to commercial Taq polymerase. However, Tth polymerase purified from strains co-expressing the commercial DnaK proteins had a poorer activity and yielded lower product than the other polymerases. It was able to amplify 3518bp-TtKJEA operon but the yield of product was lower than that obtained from the two commercial Taq polymerases. It was also unable to amplify a 6.6 kb plasmid, p22Cyp153A6, although even the commercial Taq polymerases only produced a mixture of DNA fragments, none of which were the correct size. This problem has been linked to the thermostability of Tth polymerase, which has a half-life of 20 min while that of Taq polymerase is 40 min. This means that while, Tth might be able to amplify large fragments, as a result of its low processivity, it is soon denatured from the high temperature cycles in long-distance PCR and substantial amplicons are not generated in time but Taq polymerase, though stable, has poor affinity for longer templates and dissociates before it can complete elongation of the template. Extended incubation of Tth polymerase at 95°C inactivates it and it is unable to amplify even the relatively short sGFP template. According to literature, the poor thermostability is a property of Tth polymerase and cannot be altered or improved by molecular chaperones. | en_ZA |
| dc.description.sponsorship | Biomolecular Research Cluster Fund | en_ZA |
| dc.description.sponsorship | TIA | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11660/1319 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Escherichia coli | en_ZA |
| dc.subject | Molecular chaperones | en_ZA |
| dc.subject | Dissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2011 | en_ZA |
| dc.title | Improving heterologous protein expression in E. coli using molecular chaperones from Thermus spp | en_ZA |
| dc.type | Dissertation | en_ZA |