Cloning of glyceraldehyde-3-phosphate dehydrogenase from an Antarctic psychrophilic bacterium by inverse and splinkerette PCR

Too, W.C See; Liew, Y.C.; Few, L.L.

Cloning of glyceraldehyde-3-phosphate dehydrogenase from an Antarctic psychrophilic bacterium by inverse and splinkerette PCR

2008

Too, W.C See | Liew, Y.C. | Few, L.L.

Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterium codenamed π9 was selected for the cloning of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway. Here, the cloning of an 1113 bp fragment of GAPDH gene which covers the 1002 bp open reading frame by using multiple PCR steps is described. The partial sequence of this gene was PCR amplified by using degenerate primers followed by the cloning of the flanking sequences by inverse and splinkerette PCR techniques. The success in cloning the GAPDH gene by PCR has bypassed the more time consuming genomic library construction and screening method. The full length GAPDH protein was subsequently expressed in E. coli, purified as His-tag protein and confirmed to be catalytically active. This work demonstrated the use of multiple PCR techniques to clone a gene based solely on sequence comparison. It also laid the foundation for further biochemical and structural characterizations of GAPDH from a psychrophilic bacterium by providing a highly purified recombinant protein sample. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

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