ABSTRACT
To increase the productivity and yield of recombinant protein in continual perfusion processing, Every amino acid consumption rate in continual perfusion culture of engineering CHO cell line which expressed recombinant TNFRp75: Fc fusion protein were analyzed. Then rational amino acids were accordingly added to improve its comprehensive utilizing. At the same time, glucose supply was controlled to make the concentration of glucose below 0.5 g/L for ameliorating the toxicity of lactate accumulation in order to decrease the perfusion rate. The result showed that the productivity of recombinant protein was 3.1 times (388mg/L) and the total yield was 4. 7 times (244. 4g) that of control cultures after nutrient compensation and metabolism control in 30 liter working volume, and the fermentation period was prolonged one week longer. The sialic acid content and bioactivity in vitro of recombinant TNFRp75: Fc were not changed after nutrient compensation and glucose control supply. Nutrient compensating and metabolic control in continual perfusion fermentation could significantly increase the productivity and yield of recombinant TNFRp75:Fc, and thus reduced relative industrialization costs.
ABSTRACT
To construct the combined site-directed random mutation library of recombinant human Lymphotoxin (rhLT) for in vitro molecular evolution study, and to study the structure and function relationship. The random point mutations at the sites of 46,106 and 130 were individually generated by overlap PCR amplification with the random nucleotide primers. The three point mutations were combined and cloned into pMD-18T vector to construct the combined mutation library. DNA sequencing was used to evaluate the diversity and randomness of the mutation sites. The combined mutation library was re-engineered, inserted in prokaryotic expression vector pBV220, transformed and expressed in Escherichia coli strain DH5alpha. The biological activity of some of the mutants was tested in 1929 mouse fibroblast cells. As much as 1.5 x 10(5) clones were obtained, which represents 4.5 times of the complete mutation libraries at 99% confidence. Sequencing 50 clones revealed no obvious bias in the nucleotide and amino acid mutations at the sites. Among the 30 expressed samples underwent for the bioassay, 70% (21 samples) were inactive, 23.3% (7 samples) had lower activity than rhLT, the remaining 6.7% (2 samples) had higher activity than rhLT. The combined site-directed random mutation library of rhLT has been constructed successfully. In combination with phase display, the library is ready for in vitro molecular evolution study.