effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics

An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori...

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Bibliographic Details
Published in:Applied biochemistry and microbiology 2014-07, Vol.50 (4), p.359-367
Main Authors: Dmitrochenko, A. E, Turiyanskaya, O. M, Gilep, A. A, Usanov, S. A, Yantsevich, A. V
Format: Article
Language:English
Subjects:
affinity chromatography
amino acid sequences
Biochemistry
Biomedical and Life Sciences
culture media
Deoxyribonucleic acid
diagnostic techniques
Diagnostics
dialysis
DNA
E coli
energy
enzyme activity
enzymes
Escherichia coli
Escherichia coli K12
genes
Glycosylation
histidine
ionic strength
Life Sciences
mass spectrometry
Medical Microbiology
Microbiology
nucleotide sequences
polymerase chain reaction
protein synthesis
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Summary:An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-terminal sequence. Using this vector and the E. coli DH5α, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 × 10³ units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60°C. Storage during 1 year at −20°C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 ± 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.
ISSN:0003-6838
1608-3024
DOI:10.1134/S0003683814030041