中国生物工程杂志

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 48^th, 72^nd and 96^th hours of fermentation were the key feeding points, and the expression level was 12.5 g/L. The space-time yield of fermentation from 58^th to 78^th 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 A1M antigen in 5 L scale was successfully established, and the high purity and high affinity A1M antigen in gram scale was prepared.