Data of expression and purification of recombinant Taq DNA polymerase

Fang, Na; Zhong, Niannian; Yang, Yueyang; Guo, Yujian; Ji, Shaoping

doi:10.1016/j.dib.2016.08.032

Cited by 3 publications

(3 citation statements)

References 4 publications

(3 reference statements)

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“…At present, the yield of Taq DNA polymerase enzyme was about 45 -50 mg/L. The purification protocol from previous researchers [3,4,14] found traces of contaminating protein from E. coli.…”

Section: Discussionmentioning

confidence: 99%

“…The patent for Taq DNA polymerase has expired and hence many companies and organization are involved in the manufacturing of the enzyme. Different vectors have been used by different investigators to increase the yield of Taq DNA polymerase such as pTTQ18 [3], pUC18 [10], pTZ57R [11], pET 15b [12], pTrc99A [13], pET28b [14]. Similarly, different E. coli expression strains like INV1alphaF' [4], DH1, BL21 (DE3), TOP10 have been used.…”

Section: Discussionmentioning

confidence: 99%

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High-level Expression and Purification of DNA and DNase Free Taq DNA Polymerase

Mishra¹,

Mohanraj²,

Nisshanthini³

et al. 2018

AJRB

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Aim: Taq DNA polymerase from Thermus aquaticus is a key enzyme in the field of molecular biology that has been mostly used in polymerase chain reaction (PCR). Our aim is to produce standard grade Taq DNA polymerase free from censorious impurities like DNase, DNA and other contaminating proteins. Place and Duration of Study: The experiments were performed at the Molecular Diagnostic Division of Bhat Biotech India (P) Ltd., Bangalore from February 2017 to January 2018. Methodology: The recombinant Taq DNA polymerase clone was confirmed by PCR and DNA sequencing followed by BLAST analysis. The recombinant protein was in soluble form and was expressed by E. coli DH5α strain. The enzyme was extracted using the boil-lysis method and followed by purification with ion exchange chromatography and silica column chromatography to remove the contaminating protein, DNase and DNA. The yield of the protein was also calculated. Results: In our laboratory, high-quality Taq DNA polymerase was purified using ion exchange chromatography columns and silica column, with a resulting yield of about 45-50 mg/L and the activity was found to be 1.5 U/µL. Conclusion: The use of a silica column to remove the residual DNA is a remarkable step in obtaining an unequalled quality of Taq DNA polymerase.

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“…At present, the yield of Taq DNA polymerase enzyme was about 45 -50 mg/L. The purification protocol from previous researchers [3,4,14] found traces of contaminating protein from E. coli.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

High-level Expression and Purification of DNA and DNase Free Taq DNA Polymerase

Mishra¹,

Mohanraj²,

Nisshanthini³

et al. 2018

AJRB

View full text Add to dashboard Cite

show abstract

“…Following the developments in molecular biology, Taq DNA polymerase was recombinantly expressed in E. coli, (Engelke et al, 1990b;Lawyer et al, 1993;Moazen et al, 2012;Fang et al, 2016)and puri ed from the most commercially used Taq DNA polymerases consisting of recombinantly produced Taq DNA polymerase.…”

Section: Introductionmentioning

confidence: 99%

A Novel and Cost-Friendly Expression Method of Taq DNA Polymerase Using Milk Powder and Effective Utilization of the Enzyme in Diagnostic Kits

Hekim,

Kaçıran,

Akmehmet

et al. 2024

Preprint

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Taq DNA polymerase has been used in PCR-based pathogen detection kits and the demand increased during the COVID-19 epidemic. In this study, it was aimed to produce recombinant Taq DNA polymerase in a low-cost and optimized setting and test its activity in various qPCR kits. Taq DNA polymerase gene was amplified by PCR and cloned into expression vectors. Recombinant protein expression was induced using IPTG and purification was performed using heat incubation and ammonium sulfate precipitation and dialysis. Protein expression was analyzed by SDS-polyacrylamide gel electrophoresis. Enzyme activity was tested by PCR and qPCR in different buffers. Recombinant Taq DNA polymerase was purified, and purified protein and commercial Taq DNA polymerase were compared by SDS-PAGE. The PCR activity of the recombinant enzyme was shown in different commercial and in-house buffers. Enzyme was stable for at least 12 months and kept at -20ºC. Recombinant enzyme and commercial enzyme had PCR activity at dilutions up to 1/32. Milk powder, which is an economical alternative for IPTG, was tested and, use of milk powder equivalent to 3 mM lactose was as efficient as IPTG for the induction of the recombinant protein. Recombinant Taq DNA polymerase was tested in two DNA-based qPCR kits that were developed in our laboratory and was shown to perform as well as commercial enzymes. Finally, our enzyme was tested in our commercial SARS-CoV-2 diagnosis RT-qPCR kit using in-house produced primer-probe oligonucleotides and was shown to have equivalent efficacy to the commercial enzyme.

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Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection

Liu

Yang

et al. 2022

The CRISPR Journal

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Data of expression and purification of recombinant Taq DNA polymerase

Cited by 3 publications

References 4 publications

High-level Expression and Purification of DNA and DNase Free Taq DNA Polymerase

High-level Expression and Purification of DNA and DNase Free Taq DNA Polymerase

A Novel and Cost-Friendly Expression Method of Taq DNA Polymerase Using Milk Powder and Effective Utilization of the Enzyme in Diagnostic Kits

Enhanced Taq Variant Enables Efficient Genome Editing Testing and Mutation Detection

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