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Expression, purification and identification of gibberellin-induced cysteine-rich protein of Gymanadenia conopsea

Published online by Cambridge University Press:  29 January 2010

Liu Yuan ,
Meng Guo-Quan ,
Zhou Jian-Ping ,
Zhang Teng ,
Feng Juan  and
Ren Zheng-long
Liu Yuan
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Meng Guo-Quan
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Zhou Jian-Ping
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Zhang Teng
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Feng Juan*
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Ren Zheng-long
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

Primers bearing restriction enzyme sites for EcoR I and Hind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of the gcgasa gene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation into Escherichia coli BL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.

Keywords

Gymanadenia conopseagibberellin-induced cysteine-rich proteinprokaryotic expression
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 3 , December 2009 , pp. 271 - 276
Copyright
Copyright © China Agricultural University 2009

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