nested-splicing by overlap extension PCR improves specificity of this standard method

Background: splicing by overlap extension [SOE] PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function

Objectives: we introduced a nested-SOE-PCR [N -SOE-PCR] in order to increase the specificity and generating mutations in a gene by SOE-PCR

Materials and Methods: genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application

Results: in comparison to the conventional SOE-PCR, the improved method [i.e. N-SOE-PCR] increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products

Conclusions: by applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE

Periplasmic expression of Bacillus thermocatenulatus lipase in Escherichia coli in presence of different signal sequences

Efforts to express lipase in the periplasmic space of Escherichia coli have so far been unsuccessful and most of the expressed recombinant lipases accumulate in the insoluble cell fraction. To evaluate the role of native and heterologous signal peptides in translocation of the lipase across the inner membrane of E. coli, the lipase gene [btl2] was cloned downstream of the native Bacillus signal peptide and also in fusion with the pelB, ansB and ansB/asp signal peptides. For this purpose, four recombinant expression vectors [pYRKP.P, pYRKP.N, pYRKP. A and pYRKP.AA] were constructed and expressed in E. coli. Osmotic shock analysis showed that recombinant lipase was overexpressed as inclusion bodies in E. coli. The lipase inclusion bodies were subsequently solublized, refolded and purified using single column ion-exchange chromatography. To evaluate localization of lipase in the cell, the purified lipases were subjected to capillary isoelectric focusing and tandem mass spectrometry. Results showed that all signal peptides were able to direct the lipase from the cytoplasm into the periplasmic space of E. coli, because the periplasmic space of E. coli is not suitable for lipase folding, the translocated lipase aggregates in this space as inclusion bodies

Optimization of secretory expression of recombinant hGM-CSF in high cell density cultivation of recombinant Escherichia coli using Taguchi statistical method

Human granulocyte macrophage colony stimulating factor [hGM-CSF] has many therapeutic applications. In this study, in order to verify the purification process, the effect of carbon source, IPTG concentration and post-induction time on the secretion of recombinant hGM-CSF into the culture medium by recombinant Escherichia coli during high cell density cultivation were evaluated by using the Taguchi statistical method. The results indicated that glucose, 1mM IPTG and a time of 6 h post-induction, represented optimum conditions. The secreted hGM-CSF, overall volumetric productivity and purified hGM-CSF were 373 mg/l, 18 mg/l/h and 63 mg/l, respectively

Overexpression of full-length core protein of hepatitis C virus by Escherichia coli cultivated in stirred tank fermentor

The mature core protein of the Hepatitis C virus [HCVC173] carrying pelB as a signal peptide [PelB::core] was overexpressed in Escherichia coli as 18% and 23.3% of the host's total protein, in flask and fermentor cultivation, respectively. A final specific yield of 25 +/- 1 mg HCVC173/g dry cell weight and an overall productivity of 51 +/- 1 mg HCVC173/l/h were obtained in the stirred-tank fermentor. The recombinant PelB::core protein was overexpressed as the inclusion body [IB] form, higher than the expected level when compared to the HCVC173, which was also showed by the analysis of secondary structure of mRNAs and calculation of the Codon Adaptation Index of the gene. The results showed that the combined effects of protein fusion and the signal sequence significantly enhanced the production of recombinant mature HCVC173 in E. coli. Therefore, the fusion form of the mature HCV core protein and the conditions defined in this study provide an alternative strategy for HCVC173 production in high cell density culture of E. coli