DOI QR코드

DOI QR Code

Soluble Expression of Recombinant Human Smp30 for Detecting Serum Smp30 Antibody Levels in Hepatocellular Carcinoma Patients

  • Zhang, Sheng-Chang ;
  • Huang, Peng ;
  • Zhao, Yong-Xiang ;
  • Liu, Shu-Yan ;
  • He, Shu-Jia ;
  • Xie, Xiao-Xun ;
  • Luo, Gou-Rong ;
  • Zhou, Su-Fang
  • Published : 2013.04.30

Abstract

Senescence marker protein 30 (SMP30), a hepatocellular carcinoma (HCC) associated antigen, was earlier shown by our research group to be highly expressed in HCC paracancerous tissues, but have low levels in HCC tissues. In order to detect anti-SMP30 antibody in serum of HCC patients, we established pET30a-SMP30 and pColdIII-SMP30 expression systems in Escherichia coli. However, the expression product was mainly in the form of inclusion bodies. In this research, we used several combinations of chaperones, four molecular chaperone plasmids with pET30a-SMP30 and five molecular chaperone plasmids with pColdIII-SMP30 to increase the amount of soluble protein. Results showed that co-expression of HIS-SMP30 with pTf16, combined with the addition of osmosis-regulator, and a two-step expression resulted in the highest enhancement of solubility. A total of 175 cases of HCC serum were studied by ELISA to detect anti-SMP30 antibody with recombinant SMP30 protein. Some 22 were positive and x2 two-sided tests all showed P>0.05, although it remained unclear whether there was a relationship between positive cases and clinical diagnostic data.

Keywords

SMP30;genetic engineering;soluble expression;molecular chaperone

References

  1. Baneyx F, Mujacic M (2004). Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol, 22, 1399-408. https://doi.org/10.1038/nbt1029
  2. de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B (2007) . Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol, 7, 32. https://doi.org/10.1186/1472-6750-7-32
  3. Diamant S, Eliahu N, Rosenthal D, Goloubinoff P (2001). Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem, 276, 39586-91. https://doi.org/10.1074/jbc.M103081200
  4. Frank B, Jennifer M, Petersen R (2010). A cell-based screen for inhibitors of protein folding and degradation. Cell Stress Chaperon, 15, 913-27. https://doi.org/10.1007/s12192-010-0200-3
  5. Goloubinoff P, Gatenby AA, Lorimer GH (1989). GroE heatshock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature, 337, 44-7. https://doi.org/10.1038/337044a0
  6. Hoffmann F, Rinas U (2004). Roles of heat-shock chaperones in the production of recombinant proteins in Escherichia coli. Adv Biochem Eng Biotechnol, 89,143-61.
  7. Huang L, Shakhnovich EI (2012). Is there an en route folding intermediate for Cold shock proteins? Protein Sci, 21, 677-85. https://doi.org/10.1002/pro.2053
  8. Kiefhaber T, Rudolph R, Kohler HH, Buchner J (1991). Protein aggregation in vitro and in vivo: a quantitative model of the kinetic competition between folding and aggregation. Biotechnology, 9, 825-9. https://doi.org/10.1038/nbt0991-825
  9. Moonsuk SC, Saxena AM, Chilukuri NW (2010). A strategy for the production of soluble human senescence marker protein-30 in Escherichia coli. Biochem Bioph Res Co, 393, 509-13. https://doi.org/10.1016/j.bbrc.2010.02.036
  10. Nishihara K, Kanemori M, Yanagi H (2000). Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl Environ Microbiol, 66, 884-9. https://doi.org/10.1128/AEM.66.3.884-889.2000
  11. Oyunsuren T, Sanduijav R, Davaadorj D, Nansalmaa D (2006). Hepatocellular carcinoma and its early detection by AFP testing in Mongolia. Asian Pac J Cancer Prev, 7, 460-2.
  12. Ran F, Gadura N, Michels CA (2010). Hsp90 co-chaperone Aha1 is a negative regulator of the saccharomyces MAL Activator and Acts early in the chaperone activation pathway. J Biol Chem, 285, 13850-62. https://doi.org/10.1074/jbc.M109.040600
  13. Sambrook J, Russell DW (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, New York.
  14. Schrodel A, de Marco A (2005). Characterization of the aggregates formed during recombinant protein expression in bacteria. BMC Biochem, 31, 10.
  15. Timasheff SN (2002). Protein hydration, thermodynamic binding, and preferential hydration. Biochemistry, 41, 13473-82. https://doi.org/10.1021/bi020316e
  16. Ventura S, Villaverde A (2006). Protein quality in bacterial inclusion bodies. Trends Biotechnol, 24, 179-85. https://doi.org/10.1016/j.tibtech.2006.02.007
  17. Wilkinson DL, Harrison RG (1991). Predicting the solubility of recombinant proteins in Escherichia coli. Biotechnology, 9, 443-8. https://doi.org/10.1038/nbt0591-443
  18. Yamaguchi M, Mori S, Kato S (1988). Calcium-binding protein regucalcin is an activator of ($Ca^{2+}-Mg^{2+}$)-adenosine triphosphatase in the plasma membranes of rat liver. Chem Pharm Bull, 36, 3532-9. https://doi.org/10.1248/cpb.36.3532
  19. Zhou SF, Mo FR, Bin YH, et al (2011). Serum immunoreactivity of SMP30 and its tissues expression in hepatocellular carcinoma. Clin Biochem, 44, 331-6. https://doi.org/10.1016/j.clinbiochem.2010.10.008
  20. Zhou SF, Xie XX, Bin YH (2006). Identification of HCC-22-5 tumor-associated antigen and antibody response in patients. Clinica Chimima Acta, 366, 274-80. https://doi.org/10.1016/j.cca.2005.10.026

Cited by

  1. MAGED4 Expression in Glioma and Upregulation in Glioma Cell Lines with 5-Aza-2'-Deoxycytidine Treatment vol.15, pp.8, 2014, https://doi.org/10.7314/APJCP.2014.15.8.3495
  2. Metabolic engineering of the fungal D-galacturonate pathway for L-ascorbic acid production vol.14, pp.1, 2015, https://doi.org/10.1186/s12934-014-0184-2
  3. Senescence marker protein 30 (SMP30) serves as a potential prognostic indicator in hepatocellular carcinoma vol.6, pp.1, 2016, https://doi.org/10.1038/srep39376
  4. Diagnostic Value of Serum SMP30 and Anti-SMP30 Antibody in Hepatocellular Carcinoma pp.1943-7730, 2018, https://doi.org/10.1093/labmed/lmy004