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

• Published in: Applied biochemistry and microbiology Vol. 50; no. 4; pp. 359 - 367
• Main Authors: Dmitrochenko, A. E, Turiyanskaya, O. M, Gilep, A. A, Usanov, S. A, Yantsevich, A. V
• Format: Journal Article
• Language: English
• Published: Moscow Springer-Verlag 01.07.2014; Pleiades Publishing; Springer Nature B.V
• Subjects: affinity chromatography; amino acid sequences; Amino acids; 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; Enzyme Preparation; Mass Spectrometric Analysis; Uracil; Apply Biochemistry; Polymerase Chain Reaction
• ISSN: 0003-6838; 1608-3024
• DOI: 10.1134/S0003683814030041

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.