Open AccessMolecular Cloning of Selected Regions of Catalytic Domain of CtsK Gene in Escherichia coli – A Feasibility Study
Author(s) -
Hasanka Madubashetha,
Ruwini Cooray,
P.D.S.U. Wickramasinghe,
Lakshan Warnakula,
Nimali De Silva
Publication year - 2021
Publication title -
asian journal of biotechnology and bioresource techonology
Language(s) - English
Resource type - Journals
ISSN - 2457-0125
DOI - 10.9734/ajb2t/2021/v7i330101
Subject(s) - restriction enzyme , microbiology and biotechnology , biology , hindiii , insert (composites) , genomic dna , agarose gel electrophoresis , cloning (programming) , cathepsin k , molecular cloning , polymerase chain reaction , gene , recombinant dna , agarose , dna ligase , gene expression , biochemistry , osteoclast , in vitro , mechanical engineering , computer science , engineering , programming language
Cathepsin K (CatK), encoded by CtsK gene in human, is involved in bone remodeling through ossification. The objective of the work conducted here was to express catalytic domains of CtsK gene in bacterial expression system as an initial step, facilitating recombinant production of human CatK for downstream applications in pharmacology. Four healthy human blood samples were collected. Genomic DNA was extracted using FlexiGene® whole blood DNA extraction kit. Upon quantification of DNA through NanodropTM spectrophotometer, sufficient quantity and quality was observed. CtsK gene was amplified by Polymerase Chain Reaction (PCR) using two pairs of primers tagged with restriction endonuclease sites of Sal1 and HindIII facilitating molecular cloning and visualized by Agarose Gel Electrophoresis (AGE). Two different bands of size 545bp and 265bp were observed. The bands were dissected and gel purified using GenaxxonTM gel purification kit and sequentially double digested by restriction enzymes; SalI and HindIII. Vector PBS was also subjected to sequential double digestion using same enzymes and visualized via AGE. Double digested insert of size 265bp and vector were ligated using T4 DNA Ligase (all enzymes from PromegaTM). On another trail, ligation of the PCR product with band size 265bp to pGEM-TTM easy vector system (from PromegaTM) was also done and transformed to Top10 Escherichia coli competent cells for expression separately. Cells were grown in LB media in presence of XGAL, IPTG and Ampicillin and transformed cells were screened. In the restriction enzyme digestion and ligation setup, since the insert and vector were both double digested, it is confirmed that white colonies obtained were Escherichia coli cells were transformed with the desired recombinant vector and is therefore confirmatory. In the case of pGEM-TTM ligation, a colony PCR was done using the white colonies obtained and product size was confirmed via AGE. In conclusion, the objective of study was successfully achieved, by expressing a catalytic domain of CtsK. Developments and improvements could be made for expression of entire CatK gene and downstream production of the Cathepsin K protein for effective therapeutic purpose.