Abstract
Thermostable DNA polymerases are being widely used in polymerase chain reaction for diagnosis, cloning and DNA sequencing purposes. We have cloned a gene from Pyrobaculum calidifontis encoding a thermostable DNA polymerase and expressed in Escherichia coli BL21(DE3). The gene product was produced in soluble and highly active form. However, the expression was low, which resulted in a poor yield of the recombinant enzyme. In order to get higher expression, we introduced a silent mutation at second codon by replacing the rare codon (AGG), for arginine, with an E. coli preferred codon (CGT). This resulted in nearly a 2-fold higher production of the recombinant protein. We further optimized the inducer concentration and time of cultivation after induction. Under optimized conditions nearly 3-fold higher production of the recombinant protein was observed. Furthermore, we changed the expression host to E. coli Rosetta DE3 cells. However, there was no increase in the expression level, rather it was slightly decreased. Such silent mutations and use of an alternate expression host can be used to enhance heterologous expression of other genes too.
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Abbreviations
- PCR:
-
polymerase chain reaction
- Taq :
-
Thermus aquaticus
- tRNA:
-
transfer RNA
- mRNA:
-
messenger RNA
- dNTP:
-
deoxynucleoside triphosphate
- dATP:
-
deoxyadenosine triphosphate
- dGTP:
-
deoxyguanosine triphosphate
- dCTP:
-
deoxycytidine triphosphate
- dTTP:
-
deoxythymidine triphosphate
- SDS:
-
sodium dodecyl sulphate
- PAGE:
-
polyacrylamide gel electrophoresis
- Tm:
-
melting temperature
- IPTG:
-
isopropyl β-D-1-thiogalactopyranoside.
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Ahmad, S., Ali, S.F., Azim, N. et al. Studies on enhancement of production of recombinant DNA polymerase originated from Pyrobaculum calidifontis. Biologia 76, 3579–3586 (2021). https://doi.org/10.1007/s11756-021-00887-7
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DOI: https://doi.org/10.1007/s11756-021-00887-7