Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications

A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed...

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Bibliographic Details
Published in:Molecular biotechnology 2015-10, Vol.57 (10), p.947-959
Main Authors: Oscorbin, Igor P., Boyarskikh, Ulyana A., Filipenko, Maksim L.
Format: Article
Language:English
Subjects:
Anticoagulants
Bacillus - enzymology
Bacillus smithii
Bacterial Proteins - chemistry
Bacterial Proteins - drug effects
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Bacteriology
Biochemistry
Biological Techniques
Biotechnology
Cell Biology
Chemistry
Chemistry and Materials Science
Cloning, Molecular - methods
DNA polymerase
DNA Polymerase I - chemistry
DNA Polymerase I - drug effects
DNA Polymerase I - genetics
DNA Polymerase I - metabolism
Enzyme Inhibitors - pharmacology
Enzyme Stability
Escherichia coli
Genomics
Geobacillus
Geobacillus - enzymology
Geobacillus - genetics
Geobacillus stearothermophilus - enzymology
Human Genetics
Nucleic Acid Amplification Techniques
Protein Science
Sequence Analysis, DNA
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Summary:A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli , purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA.
ISSN:1073-6085
1559-0305
DOI:10.1007/s12033-015-9886-x