Prokaryotic Expression, Purification And Preliminary Identification Of High Loading Recombination Protein G

Streptococcus is a genus of Gram-positive bacteria, some of the genus are symbiotic bacteria of human body, but most of the genus can make human and many kinds of mammalians serious infection. A kind of protein was found in the cell walls of A, C and G populations of Streptococcus which can combine either the IgGs of human and many kinds of mammalians, or the human serum albumin(HSA), called Streptococcus Protein G(SPG). Compare with Staphylococcus Protein A(SPA)——a kind of IgG-binding protein which was found earlier, SPG has higher affinity and broader binding-spectrum with IgGs.The SPG molecule is composed of three regions: A, B and C, The A region contains three homologous domains: A1,A2 and A3, which are divided by two homologous domains B1 and B2; The C region also contains three homologous domains, they are C1,C2 and C3, linked by two homologous domains D1 and D2. Researches showed that the sites of binding between SPG and HSA or Fab fragment of IgG are in the homologous regions A and B, however, the binding site with Fc fragment is in the homologous region C.SPG is widely used in immunology and immunochemistry because it has high affinity with IgG. SPG was extracted from the cells of Streptococcus strain G148 for the first time by Bjorck et al. They used papain to digest the cell wall, then purified the protein by ion-exchange chromatography and gel chromatography. Later, this method had been widely used to obtain SPG, however, the yield of the method is low and the products have different molecular weight, so this method is not a good approach to obtain a great quantity of SPG. This problem can be solved by gene engineering: clone the coding gene of SPG from the genome of Streptococcus, link it to an adequate expression vector, let the gene express in E. coli or other expression systems, then purify the SPG from the protein mixture after expression. By this method, high quantity of product with single molecular weight can be obtained.This study has done some deepgoing researches on expression and purification of a functional fragment of SPG by some gene engineering methods.This study was trying to obtain a kind of protein that can be efficiently used in the purification and directional immobilization of IgG, This protein can be used to make affinity chromatography resin or antibody chip, meanwhile, it can also be used in other application area of immunology and immunochemistry. SPG has high affinity with the IgGs of many kinds of mammalians, but the A and B regions of SPG can also combine with HSA and the Fab fragment of IgG, thus, A and B regions will harm the directional immobilization of IgG and decrease the specificity of the binding between SPG and IgGs. For the above reasons, this study has only selected the C region as the object of the research. The C region is composed of three IgG-Fc-binding domains, this research has adopted all the three regions in order to make the protein which would be obtained has a high loading of IgG.The target gene was designed and constructed according to the following procedures: the coding sequence of three IgG-Fc-binding domains in the C region of SPG was selected as primary sequence, the coding sequences of 6×His tag and the cutting site of enterokinase are inducted into the upstream of the primary sequence in order to make the purification of the expression product (especially the inclusion bodies) more convenient. The product of expression will be used to make some products, such as affinity chromatography resin, so it should have to be immobilized to some carriers. Thus, the coding sequences of three Cys adaptors were inducted into the downstream of the primary sequence. Besides, two restriction sites Bam HI and Eco RI were inducted into the two terminals of the gene. The gene designed above was named as rhlpg and its product as rHLPG, that is high loading recombination protein G.The fusion gene rhlpg was synthesized, cloned and linked to the plasmid pGEX-2T, so the prokaryotic expression vetor pGEX-rHLPG was constructed. The vetors were transfered to E.coli BL21(DE3) and the strains were induced to express the target protein by IPTG. The product of expression was detected by SDS-PAGE, an evident band appeared at the predicted position(molecular weight 49 kD), indicated that the fusion gene had been successfully expressed in the E.coli cell.This study has explored four optimum conditions of expression: the density of bacteria cells when the inducer is added to the medium; the concentration of IPTG; the inductive duration and the inoculum size. The best conditions are: the optimum density of bacteria cells when the inducer is added to the medium is about 1.0(OD600 value); the optimum concentration of IPTG is 0.1 mmol/L; the optimum inductive duration is about 6.0 h; the optimum inoculum size is 2%-5%.There is a coding sequence of glutathione-S-transferase (GST) in the upstream of multiple clone site of the vector pGEX-2T, therefore, the product of expression is a fusion protein contains GST and rHLPG. Moreover, there is a 6×His tag in the protein rHLPG, so the product of expression is a fusion protein with two tags. GST is a kind of protein which is highly soluble and can be expressed in high quantity in E.coli, besides, GST is an important purification tag, can be purified by GSH-Sepharose. 6×His is a purification tag can be used in either natural or denaturalized conditions and the proteins with this tag can be purified by Ni2+-Sepharose. Some of the fusion proteins GST-rHLPG were soluble, others were insoluble inclusion bodies, the inclusion bodies were treated by the processes of denaturalization and renaturation, part of the inclusion bodies refolded and became soluble protein. Both soluble proteins and refolded inclusion bodies were purified by Ni2+-NTA-Sepharose and detected by SDS-PAGE, a single band appeared at the predicted position, indicated that the purification is good.Double immunodiffusion was used in this study to preliminarily identify the immunologic competence of the purified fusion protein GST-rHLPG. The results showed that the fusion protein GST-rHLPG can strongly combine with the IgGs of human, mouse and rabbit, indicated that the product of this study is a kind of bioactive IgG-binding protein.