Recombinant Expression Of Protein L Mutant Of Peptostreptococcus Grandis And Its Preparation Technology For Affinity Chromatography Packing

Protein L is a kind of cytoderm surface protein produced by Peptostreptococcus magnus.It can specifically bind the variable region of light chain on antibody Kappa.As a ligand of affinity chromatography packing,it can be used to purify antibody fragments lack of Fc.At present,there are few affinity chromatography packings for sale.Most of them are imported and expensive which leads to a 45%-75%share of production cost to purify antibody fragments downstream.Meanwhile,there is large room to improve the alkali resistance and capacity of the existing Protein L affinity chromatography packing.This study targets on the fermentation expression,separation,purification and immobilization of protein L based on the independently designed recombination mutant r Protein L,sets up an efficient and stable r Protein L affinity chromatography packing process and establishes the foundation of producing high-performance r Protein L affinity chromatography domestically.First of all,this study carried out strain screening and fermentation optimization.The expression of two different r Protein L industrial strains containing different signal peptides were compared to verify that the STII signal peptide had a stronger ability to promote the secretion of r Protein L to periplasmic space.Thus,DMR582 was decided to be the expression strain.The fermentation culture composition was determined by screening eight different types of fermentation culture.The fermentation unit of r Protein L can reach 4.7 g/L by optimizing parameters like induction condition.In the second place,the processes of extraction and separation and purification of r Protein L were established.By comparing between heating extraction,low-pressure homogenate and PEI extraction,PEI extraction was decided to be the best method to extract r Protein L in periplasm.The preprocessing parameters of the periplasm extraction like p H and temperature were optimized,adjusting periplasm extraction p H to be 4.0 when precipitating impurity proteins and adjusting supernatant p H to be 6.0before 60?1h water bathing.The purity of r Protein L increased to 70.6%after preprocessing and collection efficiency of the preprocessing increased to 91%.The structure was verified preliminarily using HPLC external standard method to determine sample concentration and using high resolution mass spectrometry to determine r Protein L.The dimer was restored to monomer after TCEP processing ultrafiltered and concentrated r Protein L.TCEP concentration was studied and was decided to be 7 mmol/L.In the end,the immobilization of r Protein L was developed and self-made dynamic adsorption capacity and alkali resistance were assessed.The immobilization carrier type,reaction p H,Na Cl concentration,packing ratio,r Protein L final concentration in the reaction system and reaction time were further studied.The results were as follows:(1)Use Epoxy-activated Bestarose 6B as immobilization carrier.(2)Adjust reaction p H to 8.8,add Na Cl concentration to 1.5 mol/L.(3)Adjust r Protein L verses packing ratio to be 60mg/m L packing,resulting r Protein L final concentration to be 10 mg/m L in a 10 h reaction.The dynamic binding capacity(DBC_10%)of self-made r Protein L affinity filler for three kinds of antibody Fab samples under 10%penetration was determined.The results showed that the DBC_10%could reach more than 30 mg/m L under 1 min contact time,and the purification recovery of Fab was more than 90%.Alkali resistance result showed that when using 100 mmol/L Na OH as regenerative cleaning fluid after 50 times of CIP(Cleaning-in-place),the final DBC_10%reached 73.9%of its initial capacity and when using 15 mmol/L Na OH as regenerative cleaning fluid after 100 times of CIP,the final DBC_10%reached 89.3%of its initial capacity,which was close to the level of the on sale Capto L of Cytiva.