ABSTRACT
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-erminal sequence. Using this vector and the E. coli DH5alpha, 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 x 10(3) 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 degrees C. Storage during 1 h at 20 degrees 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.
Subject(s)
Escherichia coli K12/genetics , Escherichia coli Proteins/genetics , Plasmids/chemistry , Polymerase Chain Reaction/standards , Recombinant Fusion Proteins/genetics , Uracil-DNA Glycosidase/genetics , Amino Acid Sequence , Base Sequence , Escherichia coli K12/enzymology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Gene Expression , Hot Temperature , Hydrogen-Ion Concentration , Indicators and Reagents , Kinetics , Mass Spectrometry , Molecular Sequence Data , Plasmids/metabolism , Protein Stability , Protein Unfolding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Thermodynamics , Uracil-DNA Glycosidase/chemistry , Uracil-DNA Glycosidase/metabolismABSTRACT
The SPR assay for human cytochrome P450 51A1's (CYP51A1) ligand screening was developed. Assay has been validated with known azole inhibitors of cytochrome P450s. The studied azoles selectively interacted with human cytochrome P450 51A1, which showed the highest affinity towards ketoconazole. The efficiency of the SPR assay was showed with 19 steroid and triterpene compounds, which were not investigated as potential ligands of CYP51A1.
