Optimization of the Fermentation and Purification Process of Recombinant Human α1-Microglobulin by Pichia pastoris
Objective:To optimize the fermentation and purification process of human α1-microglobulin(A1M),an index protein of kidney injury,in order to prepare recombinant A1M with high purity and high affinity in gram scale.Methods:The optimal pH of A1M fermentation was optimized by a single factor experiment in a 1 L shake flask culture system,followed by a 5 L large-scale fermentation,and the key feeding points and fermentation time were optimized.According to the characteristics of A1M antigen,the protein purification process was established,and N-glycosylation and purity identification were carried out.Finally,A1M immunoaffinity column was prepared and its loading was evaluated.Results:The optimal initial pH of human recombinant A1M Pichia pastoris was 6.0,the expression level reached 888 mg/L in 1 L shake flask culture system,and the degree of glycosylation was relatively lowest.During the 48～120 h of 5 L scale fermentation,DO≥30％reverse correlation methanol was used to feed 4～6 mL/(h·L-1),which can effectively control the core area of DO＞20％.The 48th,72nd and 96th hours of fermentation were the key feeding points,and the expression level was 12.5 g/L.The space-time yield of fermentation from 58th to 78th hours was 160～210 mg/(L·h),and the batch of fermentation in a 5-L tank for 120 h was 49 g.The purification process of A1M was established by ultrafiltration concentration,cation exchange chromatography and desalting chromatography column,and the total yield was 18％.The coupling rate of A1M antigen affinity column was 96％,the coupling density was 2.46 mg/mL,and the loading capacity of A1M sheep polyclonal antibody was 37 mg/mL.Conclusion:The yeast fermentation and purification process of recombinant human A1 M antigen in 5 L scale was successfully established,and the high purity and high affinity A1M antigen in gram scale was prepared.
α1-MicroglobuliPichia pastoriHigh density fermentatioGlycosylation modification
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