基于GEM展示技术内含肽介导的犬α干扰素快速纯化系统的建立

CONSTRUCTION OF SPLIT INTEINS MEDIATED NOVEL PURIFICATION SYSTEM FOR RECOMBINANT PROTEIN BASED ON GEM DISPLAY TECHNOLOGY

本研究分别构建含有断裂型内含肽(inteins)C端与犬α干扰素融合基因(C*-CaIFN-α)、断裂型内含肽N端与锚钩蛋白(protein anchor,PA)融合基因(NC1A-PA3)的重组表达质粒,利用IPTG诱导表达。通过GEM颗粒(gam-positive baterial enhancermatrixparticles)与PA3之间的特异性结合,将NC1A-PA3蛋白捕获到GEM颗粒上,再利用内含肽的N端与C端的结合,获得GEM-PA3-NC1A-C*-CaIFN-α。在DTT或EDTA存在情况下,内含肽C末端自我剪切,获得纯化的犬α干扰素重组蛋白CaIFN-α。通过微量细胞病变抑制方法测定CaIFN-α蛋白的活性。结果表明,两种重组蛋白均有可溶形式表达,GEM/inteins纯化系统效果良好;纯化的犬α干扰素CaIFN-α对水疱性口炎病毒(Vesicular stomatitis virus,VSV)的抗病毒活性为3.0×106U/mL。本研究建立的基于GEM展示平台的蛋白纯化系统,将非层析标签与断裂内含肽系统相结合,为快速有效地纯化重组蛋白提供了一种方法。

The objective of this study was to develop a simple and low cost method for rapid purification of the target protein. Using an engineered DnaE inteins from Nostoc punctiforme, we developed a split inteins mediated system for protein purification based on GEM display technology. Two recombinant E.coli strains were constructed, of which one expressed the fused gene of the C-terminal of split inteins and CaIFN-α named C*-CaIFN-α, and another expressed the N-terminal of split inteins and the anchor gene PA named PA3-NC1 A. PA3-NC1 A protein covalently combined with GEM particles according to the anchor protein PA3. Then the recombinant protein C*-CaIFN-α was incubated with GEM-PA3-NC1 A, and GEM-PA3-NC1 A-C*-CaIFN-α was collected through centrifugation according to the specific combination of the N-terminal and C-terminal of the split inteins. Purified CaIFN-α protein were obtained from GEM-PA3-NC1 AC*-CaIFN-α by C-terminal cleavage of split inteins in presence of DTT or EDTA. The activity of CaIFN-α protein was measured by MDCK/VSV method. Results showed that the two recombinant CaIFN-α proteins were expressed in a soluble form and the GEM/inteins purification system worked for its purpose. The anti-VSV activity of the purified CaIFN-α protein was up to 3.0×106 U/mL. This system developed here provided a new tool for rapid and efficient purification of recombinant proteins.