Expression, Purification And Functional Identification Of The Recombinant Human CREG/myc-His Glycoprotein

Objective The cellular repressor of E1A- stimulated genes (CREG), as a transcriptional regulator, was cloned by the yeast two-hybrid method by Dr. Gill in 1998. The result that CREG could inhibit the transcription of target gene by E1A protein and E2F may contribute to the binding of CREG competing with the exogenous adenovirus E1A protein and transcription factor of E2F family proteins in mammals to the promoter region of target genes. Both E1A protein and E2F can promote cellular dedifferentiation and proliferation, CREG is therefore likely to play a role in promoting differentiation and/or inhibiting cell growth by competing with E1A and E2F.Our early research had found that the expression of CREG synchronously increased when the human vascular smooth muscle cells (VSMCs) cultured in vitro converted from synthetic into differentiated phenotype induced by serum withdrawal. The negative relationship between CREG mRNA expression and the proliferative activity of VSMCs was confirmed in rat injury carotid artery. CREG, as a secreted glycoprotein reported by Dr. Veal, could bind to the transmembrane insulin-like growth factor II receptor competing with the insulin-like growth factor II and regulate cell function through both autocrine and paracrine pathway. To study the bio-function of CREG glycoprotein, in this study, the reverse transcriptionpolymerase chain reaction(RT-PCR), Western blot and affinity chromatograph by Ni-NTA were used to express and purify the recombinant hCREG/myc-his glycoprotein. Flow cytometic analysis and BrdU incorporation method were used to confirm the biological function of hCREG/myc-his which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro. The work provided prophase research basis for engineering production of hCREG protein.Methods①The ORF(open reading frame) fragment of human CREG(hCREG) with terminator codon mutation was amplificated by RT-PCR and the eukaryotic expression vector pcDNA3.1 myc-His/hCREG was constructed.②The human 293F cells were transfected with pcDNA3.1 myc-His/hCREG using Lipofectamine 2000, and stably transfected cell clones were screened by G418. The expression of recombinant secreted hCREG/myc-His protein was identified by Western blotting. The glycosylation of hCREG/myc-His protein was analyzed by PNGaseF digestion and Western blot.③The recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. The recombinant hCREG/ myc-His protein was concentrated and desalted. The concentration of recombinant protein was analyzed by SDS-PAGE and the purity was calculated by bio-informational software.④ - 8 -The effect of recombinant hCREG/myc-His glycoprotein of different concentration(0.5μg /mL,1μg /mL and 2μg /mL)on proliferation of HITASY cells was confirmed by Flow cytometic analysis and BrdU incorporation method.Results The ORF fragment of hCREG was amplificated by RT-PCR and inserted into the pcDNA3.1 myc-His vector with EcoRΙ/BamHΙdigestion. The recombinant eukaryotic expression vector (pcDNA3.1 myc-His/ hCREG) was confirmed to be constructed successfully by restricted endonuclease digestion and DNA sequencing.The lysates of 293F cells with stably transfected pcDNA3.1 myc-His/hCREG plasmid were detected by Western blot with Anti-hCREG, Anti-myc and Anti-His respectively. The recombinanted fusion protein about 30KD was identified in transfected cells by Western blot using Anti-myc and Anti-His. The recombinant fusion protein in cell lysates was identified as a glycoprotein because the molecular weight of fusion protein decreased from 30KD to 25KD after PNGaseF digestion. The recombinant hCREG protein was purified with Ni-NTA column according to 6×His affinity chromatographic theory. After the elution was concentrated with Centriprep centrifugal filter devices(10000NMWL), the concentration of recombinant protein was determined to be 1.6 mg/mL by BCA assay. The purity of recombinant protein reached 92% identified with image-J software analysis. Glycoprotein was identified using PNGaseF digestion.The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro by Flow cytometic analysis and BrdU incorporation method. The inhibition effect of recombinant hCREG protein is stronger in low-dosage treatment than in high-dosage group. There was statistical difference among the groups(P<0.05). Conclusion The construction of eukaryotic expression vector of pcDNA3.1 myc-His/hCREG and the expression and purification of recombinant hCREG/myc-His glycoprotein with biological activity provided an experiment platform for function study and engineering production of hCREG protein. The incidence of restenosis may be reduced if the stents were coated with recombinant hCREG/myc-His glycoprotein.