Separation And Purification Process For Recombinant Protein A

Staphylococcus aureus protein A, which has the ability to bind immunoglobulins selectively, has been widely used in many fields, such as antibody detection and purification, immunocytochemistry, rapid pathogens diagnosis, and immunoadsorption therapy. Producing recombinant protein A (rPA) by genetical engineering technology is the main method to obtain large quantities of protein A products. Currently, industrialization production of rPA has been achieved abroad. However, in China, we still rely heavily on imports. In order to achieve the localization process of rPA, this paper, based on the previous prokaryotic expression and fermentation research in our laboratory, attempts to establish an efficient separation and purification process suitable for large-scale production of therapeutic-quality rPA. The work in this thesis includes:(1) The extraction and initial purification techniques for rPA were first studied. The results showed that heating at a temperature of 80℃for 10 min could remove the majority of thermolabile contaminating proteins, and 75% (v/v) ethanol precipitation could achieve rapid buffer exchange.(2) The chromatography polishing techniques for rPA were then studied. Synthesis of DEAE Sepharose CL-6B were achieved by optimizing the synthesis conditions. For DEAE weak anion-exchange chromatography, initial pH did not show any significant effects on the purity of rPA. The optimal conditions for DEAE chromatography were obtained:the column was equilibrated with buffer A (25 mM Tris-HCl, pH 8.0), and rPA was eluted using a 10 column volumes linear ionic strength gradient from buffer A to 18% buffer B (25 mM Tris-HCl,1 M NaCl, pH 8.0). For MEP hydrophobic charge induction chromatography, the binding capacity gradually increased with the ligand density of MEP media, while the selectivity did not change significantly. The pH and salt concentration of equilibration buffer showed weak effect on rPA adsorption, demonstrating salt independent rPA adsorption with MEP media. The optimal conditions for MEP chromatography were obtained:the equilibration buffer used was 25 mM Tris-HCl, pH 8.0, and rPA was eluted in a single step with 100 mM citric acid, pH 3.5.(3) Based on the above study, the complete separation and purification strategy for rPA was further established by logical selection and combination of purification techniques:rPA was purified from E.coli BL21(DE3) through heat precipitation, ethanol precipitation, DEAE weak anion-exchange chromatography and MEP hydrophobic charge induction chromatography. Bench-scale purification was continuously conducted. The purity of end product analyzed by SDS-PAGE, SEC-HPLC and RP-HPLC was greater than 98% and its total recovery was 61%. The molecule weight and pI of the end product were determined to be 32743.585 Da by MALDI-TOF-MS and 4.46 by isoelectric focusing electrophoresis, respectively. Western blot analysis showed that the purified rPA had good reactogenicity with anti-protein A antibody. Amino acid sequencing revealed that N-terminal 15 residue sequence was identical to the theoretical sequence. Biological activity assay demonstrated that the activity of purified rPA was similar to the imported rPA. Finally, the storage method for rPA was preliminarily investigated. The results indicated that the purified rPA stored in 25 mM PBS,100 mM NaCl, pH 7.4 at-80℃had good storage stability.In this paper, separation and purification process for rPA was established. The results laid the foundation for large-scale production of rPA.