Expression Of Recombinant PSMB1and EGFR And Application In Screening Of Drug In In Vitro

Proteasome is a kind of multi-subunit protease complexes in eukaryocytes, whichplays an important role in ubiquitin-proteasome pathway. Recombinant proteasomesubunit could be used in vitro to screen possible proteasome inhibitors. In this research,recombinant plasmid of pET28a-PSMB1was transformed into E. coli BL21(DE3)strains and expressed in soluble form. The recombinant protein was purified throughaffinity chromatography to reach a purity of more than95%. The purified product wasused in BIAcore analysis to screen proteasome inhibitors. The detailed methods andresults are as follows:Recombinant plasmid of pET28a-PSMB1was constructed by inserting humanproteasome catalytic subunit(PSMB1) cDNA(726bp) into the prokaryotic expressionvector pET28a(+), and transforming the plasmid into E. coli BL21(DE3) cells forexpression. After overnight induction (1mM IPTG,20℃), an expected protein bandwith molecular weight of27KD was observed on SDS-PAGE gel. The recombinantprotein was then purified through affinity chromatography to reach a purity of morethan95%. The amino acid sequence of the recombinant protein was validated byNanoLC-MS/MS. In vitro BIAcore analysis result showed that the recombinantPSMB1exhibited bind affinity with different nature compounds. The binding affinitybetween PSMB1and10μM celastrol was more than27RU. In conclusion, weestablished an expression, purification method of proteasome subunit PSMB1andapplied this method to screen possible proteasome inhibitors in vitro.Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein thatdistributes widely in human tissues and cell membranes. It plays an important role inmolecular target therapy of tumor. Recombinant EGFR, could be used to screen naturecompounds which might have binding affinity with EGFR.In this study, we expressed the extracellular EGFR cDNA in three differentexpression systems(prokaryotic, mammalian cells and insect cells) to try to obtain the bioactive recombinant protein. The detailed methods and results are as follows:1. EGFR expressed in Prokaryotic expression systemsIn this research, the recombinant plasmid of pET28a-EGFR was constructed byinserting human extracellular EGFR cDNA(1857bp) into the prokaryotic expressionvector pET28a(+). Recombinant plasmid pET28a-EGFR was transformed into E. coliBL21(DE3), after induction of different concentrations of IPTG and temperature, nospecific protein band was found.2. EGFR expressed in mammalian cells (CHO) expressionThe recombinant plasmid of pcDNA3.1/myc-His(-)A-EGFR was constructed byinserting human extracellular EGFR cDNA(1857bp) into the mammalian expressionvector pcDNA3.1/myc-His(-)A, recombinant plasmid pcDNA3.1/myc-His (-) A-EGFRwas transfected CHO cells. There was also no significant difference between thetransfected and the control group.3. EGFR expressed in insect cells (sf9)The recombinant plasmid of pFastBacHTb-EGFR was constructed by insertinghuman extracellular EGFR cDNA(1857bp) into the insect cells expression vectorpFastBacHTb, by homologous recombination, the transfer plasmid pFastBacHTb-EGFR was inserted into the virus genome. After transfected sf9cells with therecombinant viral DNA, we collected the P3virus solution through virus amplificationto analyze the expression level of the recombinant protein. Western Blotting resultsshowed two protein bands, one at130KD, and the other at17KD.