Studies On Protein Refolding By Ultrafiltration Technology Coordinating With Gigaporous Microspheres

Ultrafiltration is one of the methods for protein refolding in vitro.However,the aggregation and precipitation of protein during refolding,resulting in serious membrane fouling.And the difficulty in optimizing the refolding process,there are relatively few reports on protein refolding using ultrafiltration.Aiming at the above problems of ultrafiltration refolding,the following three aspects of research work have been carried out in this paper,and satisfactory experimental results have been obtained.Firstly,"parameter scanning ultrafiltration technology",which is used to optimize protein ultrafiltration separation process,was introduced into the protein ultrafiltration refolding process to realize rapid optimization of lysozyme refolding.After single factor optimization,the optimum process for lysozyme refolding by using PES ultrafiltration membrane with 10k Da molecular weight cut-off was as follows:initial lysozyme concentration 0.1 mg/m L,urea concentration 2 M,refolding buffer solution p H 8.5,without adding Na Cl,refolding temperature 25oC,refolding time 8 h.Under these conditions,the refolding yield of lysozyme was 63.8%.Secondly,gigaporous microspheres,which can promote protein folding,were introduced into the process of ultrafiltration refolding,and a protein refolding method based on gigaporous microspheres combined with ultrafiltration technology was established.The effects of microsphere size,pore size and the grafting amount of poly(NIPAM-co-BMA)brushes on lysozyme refolding were investigated.The microspheres with the best refolding effect(particle size 76 um,pore size 320 nm,grafting amount 35.6 mg/m~2)were determined.In addition,the optimum dosage of the microspheres was 7 mg/m L,and the effect of repeated use of the microspheres for 5 times on the refolding of lysozyme was not obvious.Compared with the ultrafiltration refolding alone,the optimum initial concentration of lysozyme increased from 0.1 mg/m L to 0.4 mg/m L with the addition of gigaporous microspheres,and the degree of membrane fouling was greatly reduced.Finally,through particle size analysis before and after denaturation of five proteins(superoxide dismutase,lipase,laccase,bovine serum albumin and glucose oxidase)and ultrafiltration membrane retention experiment,we choose polyethersulfone ultrafiltration membrane with 100 k Da molecular weightcut-off to carry out the integrated operation of ultrafiltration renaturation separation of bovine serum albumin.The results showed that bovine serum albumin(BSA)after refolding(83%of the initial amount)in the permeate had the correct spatial secondary structure and the remaining BSA(11%of the initial amount)in the retentate did not have the correct spatial secondary structure.This indicates that BSA has basically realized the integrated operation of ultrafiltration refolding separation.