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

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 x 2 two-sided tests all showed P>0.05, although it remained unclear whether there was a relationship between positive cases and clinical diagnostic data.

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

“…These findings were consistent with the previous reports confirming the positive effects of molecular chaperones on solubility parameter and disaggregation. 21,22,[27][28][29] Among the molecular plasmid chaperones, PG-tf2 exhibited a fairly much more positive effect on solubility of our aggregation-prone hscFv than the other two plasmid chaperones. The efficiency of ptf16 was also higher than that observed for pGro7.…”

“…18 Another study has shown that ptf16 promotes the soluble expression of human sequence marker protein 30 (SMP30) more effectively than its counterpart pG-tf2. 28 Watanabe K.I. et.al and colleagues studied the role of different chaperones on soluble expression of human ST6Gal I (hST6Gal I), and showed that chaperon pGro7 is the efficient chaperon of choice for solubility increase of the protein.…”

MAGED4 Expression in Glioma and Upregulation in Glioma Cell Lines with 5-Aza-2'-Deoxycytidine Treatment

“…Western blotting was performed with minor modification as previously described (Cen et al, 2012;Zhang et al, 2013). In brief, total protein was extracted from untreated and 5-AZA-CdR-treated glioma cells using a Total Protein Extraction Kit (KeyGen, China) according to the manufacturer's instructions.…”

Senescence marker protein 30 (SMP30) serves as a potential prognostic indicator in hepatocellular carcinoma

“…Protein extracts from the HCC cell lines and Western blots were performed as described24. Equal amounts of proteins from HCC cell lines were subjected to western blot.…”