Few Ling, Ling
(2008)
Expression and purification of glyceraldehyde-3-phosphate
dehydrogenase from psychrophilic bacterium.
Universiti Sains Malaysia.
(Submitted)
Abstract
Organisms that thrive in cold environments are known as psychrophiles. One of the
strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes.
Our collection of cold-tolerant microorganisms isolated· from the Antarctic region has
offered a potential source for psychrophilic enzymes. Previously our group had
successfully cloned the open reading frame for GAPDH gene from an Antarctical
bacterium known as phi9. The ORF was cloned into a pET-14b plasmid. The full
length GAPDH protein was subsequently expressed in E. coli strain BL21 (DE3),
purified as His-tag protein and confirmed to be catalytically active. Results showed
that IPTG concentration did not have any effect on protein expression and solubility
while 3 hours of induction time at room temperature (28°C) was the best conditions for
the expression and solubility of this protein. This protein was shown to be most active
at 38°C and its specific activity increased by 40% from 3.6 JlmOI/min/mg to 6.1
J.UllOifmin/mg when the temperature increased from 23°C to 38°C. This work laid the
foundation for further biochemical and structural characterizations of GAPDH from a
psychrophilic bacterium by providing a highly purified recombinant protein sample.
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