The Expression Of Alkaline-stable Protein A And Separation And Purification Process For Related Protein

Staphylococcal Protein A (SPA) has high affinity to immumoglobuins, and has been widely used in antibody detection and purification. Due to the high price of protein A affinity media, it is necessary to insert a cleaning-in-place (CIP) step into the whole purification process to remove all kinds of contaminants, extending the the life cycle of chromatography media. NaOH (0.5-1.0mol/L) appears to be the most effective cleaning agent for use in the CIP step. Currently, there are few researches on this aspect in our country. Consequently, it is urgent to establish the localization process of alkaline-stable protein A to meet the ever-increasing demand. One of the main contents of this paper is to optimize the separation and purification process of alkaline-stable protein A on the basis of its construction and expression optimization, to obtain the product with a higher purity at a lower cost. On the other hand, our laboratory has accomplished the construction and expression of Chimeric Protein AG (CPAG) which can make up for the weak binding ability of protein A for IgG3. This paper makes a further study on the separation and purification technologies of CPAG, to improve the purity and recovery, and reduce cost at the same time.The work in this thesis is as follows:(1) The construction and expression optimization of alkaline-stable protein A. The sequence of alkaline-stable protein A has been designed. The shake flask expression process included the induction of expression using0.2mM IPTG after the five-hour cultivation, and the continuation of the cultivation for another five hours.(2) The separation and purification process of alkaline-stable protein A. The optimized initial purification program included:60mM,25mM pH8.0Tris-HCl plus lysozyme for cell breakage, heating at the temperature of80℃for10min,0.13%(w/v) polyethylenimine (PEI) precipitation, and so on. The optimal conditions of DEAE chromatography:this paper achieved the elution of alkaline-stable protein A using a linear ionic strength gradient from5%B to25%B,10column volumes. After DEAE chromatography, the purity of the end product is more than93%. Finally, the alkali resistance and IgG binding capacity of alkaline-stable protein A were investigated. With the time going, protein A had significant degradation phenomenon in0.05M NaOH solution, while alkaline-stable protein A still remained relatively stable after24h. Protein A had been completely degraded after6h in0.5 M NaOH solution, while some of alkaline-stable protein A still remained intact. The electrophoresis result showed a single band. It’s evident that alkaline-stable protein A is much more stable to the alkaline conditions than protein A, and alkaline conditions have no significant influence on IgG binding capacity of alkaline-stable protein A adsorbent. The dynamic combined capacity of alkaline-stable protein A adsorbent for IgG is more than double that of protein A adsorbent with similar ligand density. The affinity constant of fixed alkaline-stable protein A for human IgG is1.2×105L/mol, the balance dissociation constants is1.31mg/mL, and the biggest theory binding capacity is72.84mg/mL.(3) The separation and purification process of CPAG. The optimized purification process comprised:the ultrasonication with the supplementary of lysozyme, heating at of temperature of80℃for20min,0.10%(w/v) polyethylenimine (PEI) precipitation,70%(v/v)ethanol precipitation, DEAE chromatography followed by butyl hydrophobic interaction chromatography. This paper achieved the elution of CPAG using a linear ionic strength gradient from10%B to25%B and10column volumes in the process of DEAE chromatography. The purity of the end product is approximately98%, and the total recovery is63%.