Skip to main content

Advertisement

SpringerLink
Log in

Expression and purification of ecdysteroid-regulated protein from Chinese mitten crab Eriocheir sinensis in E. coli

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. The goal of this study was to clone, efficiently express and purify the ecdysteroid-regulated protein (ERP) in the form of a GST fusion protein. The mature peptide-coding cDNA fragment was extracted from Chinese mitten crap (Eriocheir sinensis), and then after using PCR to obtain the open reading frame, a recombinant plasmid designated pGEX-4T-1_ERP was successfully generated and showed to efficiently express the ERP fusion protein as determined by SDS-PAGE. The resulting expressed protein was successfully purified by a combination of affinity and conventional chromatographic methods. After purification, the recombinant protein showed the expected size of 41 kDa on SDS-PAGE gels which was further confirmed by mass spectrometry and western blotting. Purification of recombinant protein was achieved by fast protein liquid chromatography. About 2.4 mg/l recombinant protein with purity more than 80 % was obtained.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. Wang HZ, Wang HJ, Liang XM, Cui YD (2006) Stocking models of Chinese mitten crab (Eriocheir japonica sinensis) in Yangtze lakes. Aquaculture 255:456–465

    Article  Google Scholar 

  2. Rudnick DA, Hieb K, Grimmer KF, Resh VH (2003) Patterns and processes of biological invasion: the Chinese mitten crab in San Francisco Bay. Basic Appl Ecol 4:249–262

    Article  Google Scholar 

  3. Sui L, Wille M, Cheng Y, Sorgeloos P (2007) The effect of dietary n-3 HUFA levels and DHA/EPA ratios on growth, survival and osmotic stress tolerance of Chinese mitten crab Eriocheir sinensis larvae. Aquaculture 273:139–150

    Article  CAS  Google Scholar 

  4. Herborg LM, Bentley MG, Clare AS, Last KS (2006) Mating behaviour and chemical communication in the invasive Chinese mitten crab Eriocheir sinensis. J Exp Mar Biol Ecol 329:1–10

    Article  CAS  Google Scholar 

  5. Torres G, Charmantier-Daures M, Chifflet S, Anger K (2007) Effects of long-term exposure to different salinities on the location and activity of Na+–K+–ATPase in the gills of juvenile mitten crab, Eriocheir sinensis. Comp Biochem Physiol A 147:460–465

    Google Scholar 

  6. Keller R (1992) Crustacean neuropeptides structures, functions and comparative aspects. Experieutia 48:439–448

    Article  CAS  Google Scholar 

  7. Burtis KC, Thummel CS, Jones CW (1990) The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins. Cell 61:85–99

    Article  PubMed  CAS  Google Scholar 

  8. Cho WL, Kapitskaya MZ, Raikhel AS (1995) Mosquito ecdysteroid receptor: analysis of the cDNA and expression during vitellogenesis. Insect Biochem Mol Biol 25:19–27

    Article  PubMed  CAS  Google Scholar 

  9. Deng WM, Bownes M (1997) Two signaling pathways specify localized expression of the broad-complex in Drosophila eggshell patterning and morphogenesis. Development 124:4639–4647

    PubMed  CAS  Google Scholar 

  10. Buszczak M, Freeman MR, Carlson JR, Bender M, Cooley L, Segraves WA (1999) Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila. Development 126:4581–4589

    PubMed  CAS  Google Scholar 

  11. Kozlova T, Thummel CS (2000) Steroid regulation of postembryonic development and reproduction in Drosophila. Trends Endocrinol Metab 11:276–280

    Article  PubMed  CAS  Google Scholar 

  12. Sun G, Zhu J, Li C, Tu Z, Raikhel AS (2002) E74, an ecdysone inducible early gene, is involved in the ecdysteroid gene regulatory hierarchy during mosquito vitellogenesis. Mol Cell Endocrinol 190:147–157

    Article  PubMed  CAS  Google Scholar 

  13. Hepperle C, Hartfelder K (2001) Differentially expressed regulatory genes in honey bee caste development. Naturwissenschaften 88:113–116

    Article  PubMed  CAS  Google Scholar 

  14. Lee KJ, Elton TS, Bej AK, Watts SA, Watson RD (1995) Molecular cloning of a cDNA encoding putative molt-inhibiting hormone from the blue crab, Callinectes sapidus. Biochem. Biophys. Res Comm 209:1126–1131

    Article  PubMed  CAS  Google Scholar 

  15. Li X, Lei Y, Gao X, Ma C, Dong S (2007) Calcium carbonate supersaturation and precipitation in Chinese mitten crab (Eriocheir japonica sinensis) larval ponds in China: mass mortality, crystal form analysis, and safety saturation index. Aquaculture 272:361–369

    Article  CAS  Google Scholar 

  16. Jiang H, Yin Y, Zhang X, Hu S, Wang Q (2009) Chasing relationships between nutrition and reproduction: a comparative transcriptome analysis of hepatopancreas and testis from Eriocheir sinensis. Comp Biochem Physiol D 4:227–234

    Google Scholar 

  17. Wen XB, Chen LQ, Zhou ZL, Ai CX, Deng GY (2002) Reproduction response of Chinese mitten-handed crab (Eriocheir sinensis) fed different sources of dietary lipid. Comp Biochem Physiol A 131:675–681

    Google Scholar 

  18. Kalinina MV, Vinnikova NA, Semen’kova EG (2008) Gonadogenesis and color characteristics of ovaries in Japanese mitten crab Eriocheir japonicus. Ontogenez 39:58–65

    PubMed  CAS  Google Scholar 

  19. Zhang Y, Buchholz F, Muyrers JPP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128

    Article  PubMed  CAS  Google Scholar 

  20. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The structure SWISS-MODEL workspace: a web-based environment for protein homology modelling. Bioinformatics 22:195–201

    Article  PubMed  CAS  Google Scholar 

  21. Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL repository and associated resources. Nucleic Acids Res 37:387–392

    Article  Google Scholar 

  22. Peitsch MC (1995) Protein modeling by E-mail bio/technology. Nat Biotechnol 13:658–660

    Article  CAS  Google Scholar 

  23. Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene (Amst.) 67:31–40

    Article  CAS  Google Scholar 

  24. Wang Q, Fang DA, Sun JL, Wang Y, Wang J, Liu LH (2012) Characterization of the vasa gene in the Chinese mitten crab Eriocheir sinensis: a germ line molecular marker. J Insect Physiol 58:960–965

    Article  PubMed  CAS  Google Scholar 

  25. Wang Q, Chen L, Wang Y, Li W, He L, Jiang H (2012) Expression characteristics of two ubiquitin/ribosomal fusion protein genes in the developing testis, accessory gonad and ovary of Chinese mitten crab, Eriocheir sinensis. Mol Biol Rep 39:6683–6692

    Article  PubMed  CAS  Google Scholar 

  26. Li W, Jin X, He L, Wang Y, Chen L, Jiang H, Wang Q (2011) Molecular cloning, characterization and expression analysis of macrophage migration inhibitory protein (MIF) in Chinese mitten crab, Eriocheir sinensis. Fish Shellfish Immunol 30:324–329

    Article  PubMed  CAS  Google Scholar 

  27. Jiang H (2011) Transcriptome analysis and reproduction related genes cloning, expression pattern in Chinese mitten crab [D]. Shanghai: East China Normal University

Download references

Acknowledgments

This work was supported by the National Ocean Public Welfare Scientific Research Project (No. 201305001), China Agriculture Research System (CARS-50-Z04) and Liaoning scientific research program of ocean and fisheries department (No. 200801). Thank the Biortus which offers me a great environment to do my research. Also thank Guoping Xiao, James Groarke and the colleagues in Biortus for excellent technical assistance.

Author information

Authors and Affiliations

  1. Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, 116023, China

    Chongbo He, Xianggang Gao & Lei Gao

  2. College of Life Science, Liaoning Normal University, Dalian, 116081, China

    Chongbo He, Panhai Chen & Le Li

  3. Wuxi Biortus Biosciences, Jiangyin, 214437, China

    Panhai Chen

Authors
  1. Chongbo He
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Panhai Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xianggang Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lei Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Le Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chongbo He.

Rights and permissions

Reprints and permissions

About this article

Cite this article

He, C., Chen, P., Gao, X. et al. Expression and purification of ecdysteroid-regulated protein from Chinese mitten crab Eriocheir sinensis in E. coli . Mol Biol Rep 40, 6987–6995 (2013). https://doi.org/10.1007/s11033-013-2818-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-013-2818-6

Keywords

Search

Navigation