Cloning, Expression And Preliminary Purification Of Porcine β₂-Adrenergic Receptor

This study was aimed to provide high purityβ₂-adrenergic receptor (β₂-AR) for receptor chromatography model which we established to screen the effective ingredients in drugs. Therefore, we used genetic engineering technology to clone the full-lengthβ₂-AR gene sequences in porcine liver tissues and express these gene sequences in E. coli in form of fusion proteins, which were further separated and purified preliminarily. That has laid foundation for the large-scale preparation ofβ₂-AR.(1) According to the full-length cDNA sequences of porcineβ₂-AR published in GenBank, a pair of specific primers were designed respectively. RT-PCR reaction and PCR reactions were carried out separately in order to amplify target genes simultaneously with pig liver total RNA and genomic DNA as template respectively. And then the target genes were linked to the pUC18 vector in vitro, which was further transformed into competent E. Coil DH5αto screen positive clones. PCR, double digestion and sequencing results showed that both of them had amplified1257 bp target gene fragment encoding 418 amino acids. GenBank sequence of results included in the sequence of porcineβ₂-AR than for the former to 99.52% homology, amino acid are the same as 99.04%; the latter to 99.44% homology, amino acid are the same as 98.80%. Both this study and then the sequencing results for the ratio of its 99.84% homology, amino acid are the same as 99.52%.(2) In the process of designing primers, restriction site EcoRâ… was added to the upstream primer and Salâ… restriction site was added to the downstream primer. The full-lengthβ₂-AR fragment was cut off from the cloning vector pUC18-β₂-AR by using EcoRâ… / Salâ… and was linked to the pET32a vector after double enzyme digestion in vitro, which was transformed into competent E. Coil BL21(DE3) for expression in E.coli induced by EPTG. SDS-PAGE detection of the whole cell protein was performed before and after the IPTG induction. Compared with before induction, there is a clear 66.5 kD band after induction, indicating that the receptor proteins had been expressed in form of fusion proteins. Recombinant strains of were induced at different IPTG concentrations and under different culture time. The results of SDS-PAGE and TLC analysis showed that the concentration of IPTG was about 2.0 mmol/L, about 4.0 h induction time when the highest amount of fusion protein expression, about 17.1%;(3) Because of the integration ofβ₂-AR receptor with 6×His-tag, His tag can combine with Ni²⁺selectly and strongly. We carried out the initial separation and purification of proteins in Ni²⁺-Sepharose Fast Flow affinity chromatography, eluted peaks though SDS-PAGE analysis. The results showed that a clear 66.5 kD goaled band appeared on peak 2, purity was about 50%.