Expression, Purification And Characterization Of Recombinant Protein A Of IgG-binding Domain

Staphylococcal protein A(Protein A or SPA)is a membrane-bound protein from Staphylococcus aureus that binds close to the Cγ2/Cγ3 interface of the Fc fragment of IgG molecule without interrupting its antigen-binding ability. This special immunological characteristics making it a kind of extremely useful immunological tool, SPA is widely used in the field of purification and analysis of IgG antibodies in scientific research. Adopting a certain way to make SPA cross-linked to agarose gel or dextran gel, can make affinity chromatography packing with high-capacity, easier to use . These chromatography columns can be reused dozens of times, but the activity and purity of antibody are not affected. SPA on the bacteria can combine to the known standard serum specific antibody IgG Fc-fragment, it can be used for diagnosing some unknown antigen. The SPA molecule can be linked with fluorescein, enzyme (HRP, ALP, and GO, etc.), colloidal gold and other markers to make a labeled antibody, used for antibody detection in Western blot, ELISA tests. In addition, SPA can also be used in detection of immune complexes, tumor cell surface antigen, in vitro complement activation function tests, cell surface markings ,and so on.The traditional way to get SPA was the direct isolation from the cell wall of the fermentation Staphylococcus aureus bacteria, since the pathogenicity of Staphylococcus aureus, it was not conducive to the necessary security proteins, and SPA in the content of the cell was not high, it was difficult to meet the needs of large-scale production using this method. In recent years, with the development of genetic engineering technology, construct recombinant SPA, and express it in engineered bacteria has become an effective method.SPA is a single chain polypeptide, which do not contain cysteine, so it has no disulfide bonds.The Wild-type SPA gene encods 488 amino acids(Not including the signal peptide sequence in the N terminal). The molecular weight of SPA is about 42 kD. It contains five homologous domains, and each domain is a three-αhelix bundle consisting of 57–60 residues. The five domains in the SPA can be bound to the Fc fragment of IgG independently, and the B, D, A domains has the best stability and IgG binding activity. For these reasons, in this study we selected the gene sequences of B, A, D domain as the target gene, to build a new SPA derivatives with better stability and stronger IgG adsorption capacity with 690 bp sequences. In the N-terminal and C-terminal sequences, there were designed Bam HI and Eco RI restriction sites so that it can be digested and linked to the pUC57 cloning vector and pGEX-2T expression vector. In the upstream of EcoRI restriction site, had six histidine, could express 6×His tag with high affinity nickel ions of Ni affinity column for recombinant protein purification. The designed sequences linked with pUC57 vector and cloned into E coli DH5αto construct recombinant clone bacterial. In order to express the target protein efficiently, we used E. coli expression system in this study as a expression tool, it has the advantages of safety, clear genetic characteristics, simple, short growth cycle, easy access to high expression, and so on. To make the recombinant protein with better solubility and stability, we used pGEX-2T fusion expression vector in this study, which could express a molecular weight of 26 kD glutathione- S-transferase (GST) itself. This fusion gene expression vector can be cloned with pGEX-2T and expressed into E. coli to produce GST fusion protein with the SPA, to increase the expressed protein more soluble; Secondly the expressed GST fusion protein label could make the purification method more simple, it could use glutathione affinity chromatography to isolate fusion protein by one biological step; Furthermore the GST fusion protein could use coagulation factor X to obtain a single foreign gene expression products. As the designed 690bp sequence of the recombinant gene could be expressed as a 28 kD recombinant protein, and the molecular weight of GST was 26 kD, so the molecular weight of SPA and the GST fusion protein GST-SPA should be 54kD.SPA gene was amplified and digested by restriction enzyme, then linked with pGEX-2T expression vector to construct recombinant plasmid pGEX-SPA, introduced into E coli. the expression was induced by IPTG, SDS-PAGE electrophoresis results showed that the bacterial expression products produces a 54kD specific protein band as expected.It should be the recombinant protein GST-SPA. For optimizing the expression conditions: different cell OD600 value different temperature, different concentrations of IPTG and different induction time,are use of to get the best protein expression conditions for the pGEX-SPA fusion gene in E. coli BL21. The results showed that OD600 0.8, 0.6mmol / L of IPTG, 37℃3h are best conditions for the GST-SPA fusion protein to be highly expressed in E coli BL21.Part of the expression product of GST-SPA fusion protein presented in the supernatant of recombinant bacteria after ultrasonic treatment, some recombinant protein GST-SPA formed inclusion body in the precipitate after ultrasonic treatments which need to be dissolved, denatured, renatured and then purified. Because the recombinant proteins had no cysteine, it didn't form a disulfide bond structure , so we could use 8mol / L urea to dissolve the inclusion bodies, restored the natural activity of the recombinant protein by dialysis method to decrease the concentration of urea.The recombinant protein SPA had two purification tags: GST tags and 6×His tags that we could use glutathione affinity chromatography or metal ion Ni-affinity chromatography to purify the expressed protein. In study we selected GST-Sepharose 4B affinity chromatography system, using 10 mmol/L glutathione solution to elute the recombinant protein, SDS-PAGE electrophoresis results showed that the glutathione elution samples produces a specific single protein bond, with the molecule weight of 54kD which means we got highly purified GST-SPA fusion protein monomer after one step purification. The recombinant SPA we got in this experiment could form visible white immune responseprecipitation line with the rabbit anti-human IgG at the appropriate protein concentration ratio level, indicated the recombinant SPA had IgG binding characteristics.This study had successfully constructed the GST-SPA fusion expression system, expres sed it in E. coli and purified GST-SPA fusion protein, By identification we confirmed that this protein had strong IgG binding activity. This recombinant protein SPA had low molecular weight, and the D, A, B three domains of the recombinant protein had a little difference in affinity with Fc fragment of the antibody. It could be connected with the media to build SPA-affinity chromatography as a tool for separation and purification of IgG. By the use of SPA's immune adsorption, adsorpted plasma proteins, we can reduce the patient's hemoglobin to the desired level, treat the patients of organ transplantation with accelerated graft rejection (accelerated acute rejection, AccAR), Clinically. What's more, this transformated SPA molecules can also be coupled with a variety of report molecules to prepare label antibody for the immune diagnosis, improve the accuracy and sensitivity of antibody detection such as staining, western blot, ELISA testing and the other . Re-established SPA in this study had better stability and strong IgG adsorption capacity to meet the needs of modern production. This opened up a new way to produce recombinant SPA by the use of genetic engineering technology, as a feasible and effective way to get large-scale production of protein A. This recombinant SPA not only provided a new tool for the immunological study, but also paved the better fondation for its applications in the medicine, biology, cytology and microbiology in the future.