Publication: Construction of human choline kinase beta gene containing c-terminal glycine-serine linker in pet-14B plasmid
| dc.contributor.author | Kunasagaran, Dhanesh Narain | |
| dc.date.accessioned | 2026-09-09T04:05:36Z | |
| dc.date.available | 2026-09-09T04:05:36Z | |
| dc.date.issued | 2026-01 | |
| dc.description.abstract | Choline kinase beta (ChoKβ), encoded by the chkb gene, catalyses the first step of phosphatidylcholine biosynthesis and is essential for muscle integrity and mitochondrial architecture; loss-of-function mutations cause megaconial congenital muscular dystrophy. Despite its importance, few experimentally validated chkb expression constructs incorporate rationally designed C-terminal glycine-serine linkers in high-expression vectors such as pET-14b, limiting structure–function studies and protein engineering of ChoKβ. This study aimed to develop and preliminarily evaluate a recombinant pET-14b-chkb plasmid encoding human ChoKβ with a C-terminal glycine-serine linker for biochemical and structural analysis. The reference chkb coding sequence was analysed in silico, restriction sites for directional cloning were identified, and a reverse primer was designed to introduce a C-terminal glycine–serine linker while generating NdeI/BamHI-compatible ends. Using Phusion High-Fidelity DNA polymerase, the linker-containing chkb fragment was amplified by PCR, digested with NdeI and BamHI, and ligated into an NdeI/BamHI-digested pET-14b backbone. Ligation products were transformed into competent E. coli XL1-Blue and DH5α. Ampicillin-resistant transformants were obtained only in XL1-Blue for one ligation reaction, suggesting strain- and/or condition-dependent cloning efficiency; however, colony PCR screening was compromised by contamination in the negative control, preventing definitive confirmation of the recombinant pET-14b-chkb-linker construct. In silico analysis using Protein-Sol predicted similarly low solubility scores for wild-type ChoKβ and the glycine-serine linker variant, suggesting limited impact of the linker on solubility in E. coli. AlphaFold-based structural modelling and Ramachandran analysis indicated that the glycine-serine extension forms a solvent-exposed, intrinsically disordered tail while preserving the native ChoKβ catalytic core, dimer interface and overall fold, with the linker projecting away from the active site and dimer interface in the dimer model. Collectively, this work supports structural compatibility of a C-terminal glycine–serine linker with ChoKβ and provides a methodological basis for optimisation of cloning, expression, and functional characterisation of ChoKβ-based fusion proteins. | |
| dc.identifier.uri | https://erepo.usm.my/handle/123456789/24962 | |
| dc.language.iso | en | |
| dc.title | Construction of human choline kinase beta gene containing c-terminal glycine-serine linker in pet-14B plasmid | |
| dc.type | Resource Types::text::thesis::bachelor thesis | |
| dspace.entity.type | Publication | |
| oairecerif.author.affiliation | Universiti Sains Malaysia |