title: Validation of a Burkholderia pseudomallei hypothetical protein and determination of its translational start codon using chromosomal integration of His-Tag coding sequence.
abstract: In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
authors.affiliation: Universiti Sains Malaysia, Minden, Malaysia. hokchai@yahoo.com
authors.forename: Hokchai
authors.initials: H
authors.lastname: Yam
authors.forename: Ainihayati
authors.initials: A
authors.lastname: Abdul Rahim
authors.forename: Ooi
authors.initials: O
authors.lastname: Gim Luan
authors.forename: Razip
authors.initials: R
authors.lastname: Samian
authors.forename: Uyub
authors.initials: U
authors.lastname: Abdul Manaf
authors.forename: Suriani
authors.initials: S
authors.lastname: Mohamad
authors.forename: Nazalan
authors.initials: N
authors.lastname: Najimudin
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016957:Burkholderia pseudomallei / Q000235:genetics* / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D018387:Codon, Initiator; D006639:Histidine / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D001426:Bacterial Proteins; D018387:Codon, Initiator; D011994:Recombinant Proteins; D006639:Histidine