Skip to main content Accessibility help

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

  • Liu Yuan (a1), Meng Guo-Quan (a1), Zhou Jian-Ping (a1), Zhang Teng (a1), Feng Juan (a1) and Ren Zheng-long (a1)...


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.


Corresponding author

*Corresponding author. E-mail:


Hide All
Aubert, D, Chevillard, M, Dorne, AM, Arlaud, G and Herzog, M (1998) Expression patterns of GASA genes in Arabidopsis thaliana: the GASA4 gene is up-regulated by gibberellins in meristematic regions. Plant Molecular Biology 36: 871883.
Ben-Nissan, G, Lee, JY, Borohov, A and Weiss, D (2004) GIP, a Petunia hybrida GA-induced cysteine-rich protein: a possible role in shoot elongation and transition to flowering. Plant Journal 37: 229238.
Berrocal-Lobo, M, Segura, A, Moreno, M, Lopez, G, Garcia-Olmedo, F and Molina, A (2002) Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection. Plant Physiology 128: 951961.
Betton, JM and Hofnung, M (1996) Folding a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies. Journal of Biological Chemistry 271: 80468052.
de la Fuente, JI, Amaya, I, Castillejo, C, et al. (2006) The strawberry gene FaGAST affects plant growth through inhibition of cell elongation. Journal of Experimental Botany 57: 24012411.
Furukawa, T, Sakaguchi, N and Shimada, H (2006) Two OsGASR genes, rice GAST homologue genes that are abundant in proliferating tissues, show different expressions patterns in developing panicles. Genes & Genetic Systems 81: 171180.
Herzog, M, Dorne, AM and Grellet, F (1995) GASA, a gibberellin-regulated gene family from Arabidopsis thaliana related to the tomato GAST1 gene. Plant Molecular Biology 27: 743752.
Kotilainen, M, Helariutta, Y, Mehto, M, et al. (1999) GEG participates in the regulation of cell and organ shape during corolla and carpel development in Gerbera hybrida. Plant Cell 11: 10931104.
Medina-Escobar, N, Cardenas, J, Valpuesta, V, Munoz-Blanco, J and Caballero, JL (1997) Cloning and characterization of cDNAs from genes differentially expressed during the strawberry fruit ripening processes by a MAST–PCR–SBDS method. Analytical Biochemistry 248: 288296.
Messens, J and Collet, JF (2006) Pathways of disulfide bond formation in Escherichia Coli. International Journal of Biochemistry and Cell Biology 38: 10501062.
Roxrud, I, Lid, SE, Fletcher, JC, Schmidt, ED and Opsahl-Sorteberg, HG (2007) GASA4, one of the 14 member Arabidopsis GASA family of small polypeptides, regulates flowering and seed development. Plant and Cell Physiology 48: 471483.
Schunmann, PHD, Smith, RC, Lang, V, Matthews, PR and Chandler, PM (1997) Expression of XET-related genes and its correlation to elongation in leaves of barley (Hordeum vulgare L.). Plant Cell and Environment 20: 14391450.
Segura, A, Moreno, M, Madueno, F, Molina, A and Garcia-Olmedo, F (1999) Snakin-1, a peptide from potato that is active against plant pathogens. Molecular Plant–Microbe Interactions 12: 1623.
Taylor, BH and Scheuring, CF (1994) A molecular marker for lateral root initiation: the RSI-1 gene of tomato (Lycopersicon esculentum Mill.) is activated in early lateral root primordia. Molecular & General Genetics 243: 148157.
Wigoda, N, Ben-Nissan, G, Granot, D, Schwartz, A and Weiss, D (2006) The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity. Plant Journal 48: 796805.
Zhou, JP, Yang, ZJ, Bai, LL, et al. (2006) Construction of a full-length cDNA library of Gymanadenia conopsea by modified oligo-capping. Acta Botanica Boreali-Occidentalia Sinica 26: 18741877 (in Chinese with English abstract).



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed