Downstream Processing of Recombinant Proteins in E coli
From this study it is clear that hence for the high recovery of the active protein molecule, solubilization and refolding parts must be of high precision. Inclusion bodies consist of polypeptides of the recombinant protein. They are the inactive secondary –like structures. Thus, the isolation and purification of the protein are simple. The activity of the unfolded protein can be brought out by using mild solubilizing conditions. This will help in the high recovery of the bioactive protein than that compared to solubility using high chaotropic agent. The following Downstream processing steps can be used for the production of the recombinant protein from the inclusion bodies The recombinant E.coli is grown in LB medium with antibiotics such as kanamycin or ampicillin or chloramphenicol based on the plasmid. The flasks are shaken at rpm around 150 -250 and the temperature is maintained at 37 degree Celsius. After the cells have reached the log phase, This study outlines that IPTG is added and further shaken for 4-6 hours. The cells are centrifuged and re-suspended in the 50 mM sodium phosphate buffer. The cells are lysed using the homogenizer or sonicator. The cell suspension is centrifuged and filtered using 0.45 µm polyethersulfonate membrane. Many protein-specific methods are available for the increased solubility of the recombinant proteins in E.coli. To recover the soluble proteins, strong denaturants like urea, guanidinium hydrochloride are used. The solubilization is carried out under reducing conditions. These inclusion bodies must be washed well before solubilization. Solubilizing agents such as thioredoxin are known to improve the solubility of the proteins. Gene fusion techniques are equally good for the separation of the proteins. Maltose binding protein and Glutathione-S- transferase are found to bind well with the protein and can be removed by using the affinity chromatography techniques. Refolding is performed using dilution or diafliltration in buffers of low.