Cloning of the Thermus thermophilus methionine aminopeptidase gene and confirmation of the enzyme functional activity

Abstract

Background. Methionine aminopeptidases (MAPs) are a class of enzymes that catalyze the removal of the N-terminal initiator methionine from a polypeptide chain. Bacterial MAPs are considered as targets for the development of broad-spectrum antibacterial drugs, and using MAPs in biotechnology necessitates the search for new MAPs and the study of their functioning and inhibition mechanisms.The aim of the study. To identify methionine aminopeptidase in the Thermus thermophilus genome (Tt-MAP) and to confirm its functional activity.Materials and methods. To identify Tt-MAP, we analyzed the Thermus thermophilus genome in the GeneBank database. Modern genetic engineering techniques (polymerase chain reaction, restriction, transformation, heterologous expression) were used to clone the putative open reading frame (ORF) encoding Tt-MAP in the pHUE vector. Various chromatography techniques (affinity, ion exchange, and size-exclusion) were used to obtain a purified enzyme preparation. The fluorogenic substrate L-methionine 7-amino-4-methylcoumarin (Met-AMC) was used to confirm the specific functional aminopeptidase activity of the enzyme.Results. An ORF encoding MAP was identified in the Thermus thermophilus bacterium genome. Oligonucleotide primers were designed based on the nucleotide sequence. The ORF was cloned in the vector, and the recombinant enzyme was produced in E. coli cells. A method for purifying the enzyme to a homogeneous state was developed using a series of sequential chromatographies, allowing up to 30 mg to be obtained from 1 liter of culture. Using the fluorogenic substrate Met-AMC, the specific functional activity of the enzyme was demonstrated (the enzyme cleaves methionine from the substrate).Conclusion. We have identified the Thermus thermophilus MAP and tested its functional activity. It has been shown that the ORF product TTHA1670 encodes a methionine-specific aminopeptidase, i. e. methionine aminopeptidase. The enzyme can be used in various fields of biotechnology and scientific research.