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Overexpression of an
abiotic-stress inducible plant protein in the bacteria
Escherichia coli
Aryadeep Roychoudhury1, 2* and Supratim Basu1
1Department
of Botany, Bose Institute, 93/1, Acharya Prafulla Chandra
Road, Kolkata-700009, West Bengal, India.
2Department
of Botany, Plant Molecular Biology and Biotechnology
Laboratory, University of Calcutta, 35, Ballygunge Circular
Road, Kolkata- 700 019, West Bengal, India.
*Corresponding author. E-mail:
aryadeep.rc@gmail.com.
Abbreviations:
cDNA, complementary DNA; FL, full length; GST,
glutathione S-transferase;
IPTG, isopropyl thiogalactoside; ORF, open
reading frame; PCR, polymerase chain reaction; and
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis.
Accepted
15 August, 2008 |
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The aim of our work was the overexpression of the abiotic
stress-inducible dehydrin protein, namely RAB16A, from rice
in the BL21 strain of Escherichia coli. The Rab16A
transcript of 0.5 Kbp was amplified from the total RNA of
the salt-tolerant indica rice cultivar Nonabokra by RT-PCR
and cloned into the expression vector pGEX-3X. The 47 kDa
protein, expressed as GST: RAB16A fusion protein, after 2 mM
IPTG-mediated induction, was collected as S10 fraction and
purified through glutathione-sepharose affinity resin.
Immunoblot analysis with the maize dehydrin antiserum showed
cross-reaction with the above band, but not with GST protein
alone, showing functional expression of the heterologous
RAB16A protein in the bacterial system.
Key
words:
Fusion protein, Glutathione-sepharose, GST: RAB16A, pGEX-3X.
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