| The interferons are Cytokines with antiviral, antiproliferative and imrnnnomodulatory activities. Among three major distinguished types, interferon-γ (IFN- γ) is more hailed for their specific properties in inhibition of cell growth and modulation of immune functions. Now the recombinant DNA techniques make it possible to mass express recombinant IFN-γ in E. coli; however, this may result in the formation of inclusion bodies, and the recovery of biologically active products becomes significant.Molecular chaperone GroEL-GroES is a ubiquitous class of proteins in E. coli that play an essential role in protein folding by helping other polypeptides reach a proper conformation or cellular location without becoming part of the final structure. As the minimal mechanism of GroEL-mediated protein folding, minichaperones, fragments encompassing the apical domain of GroEL, can facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL.Here, we reported the minichaperone sht GroEL191-345 mediated refolding of recombinant human IFN- γ (rhIFN-γy) produced in E. coli. SDS-PAGE was performed with Laemmli's Tris-glycine buffer system for protein analysis. Protein concentrations and rhIFN-γ total activity were determined by a modification of Bradford's .method and cytopatic effect (CPE) method, respectly.Firstly, the colorimetric CPE method has been optimized to measure the biological activity during the refolding of rhIFN-γ inclusion bodies.The dosage and the absorbance of crystalline violet, the extraction time of the solvent, plate effect and marginal effect were investigated.Secondly, we successfully transferred a batch culture from a 500ml shaldng flask to a 3.7L bioreactor and explored the soluble expression and efficient purification of recombinant sht GroEL191-345, and 216.2 mg/L homogeneous protein was obtained. Control parameters: operating volume 2.1L, inoculation percent 2.5%, temperature 33℃, the stirred speed 350 r/mih and the airflow rate 200 L/h.Thirdly, we optimized the refolding condition of rhIFN-γ assisted by sht GroEL191-345. Compared with the conventional method, such as dilution and dialysis, the presence of sht GroEL191-345 in the refolding buffer not only enhanced the specific activity, but also raised the refolding efficiency. With the initial protein concentration of 100μg/mL, the protein yield and specific activity, of rhIFN- γassisted by sht GroEL191-345 was 2.2 and 3 folds of that under spontaneous condition respectively. Optimal operating parameters in refolding of rhlFN-γy assisted by sht GroEL191-345 were as follows: refolding temperature 15 ℃, refolding time 4 h, pH 7.7, initial concentration of IFN- y 100-200μg/mL and the mol ratio of sht GroEL191-345 versus IFN- y 1:1-2:1. Furthermore, the immobilization of sht GroEL191-345 on NHS-activated sepharose fast flow made it possible to be recycled. The protein yield and the specific activity of rhlFN- y were 46.29% and 9.05 x 106 IU/mg even the initial protein concentration was up to 400μg/mL.Finally, preliminary results were obtained in the refolding of rhlFN- y assisted by artificial chaperone system (CTAB and P-CD). Optimal operating parameters in refolding of rhlFN- y assisted by CTAB and P-CD were as follows: refolding temperature 15℃, refolding time 4 h, pH=7.7, urea concentration 1.5mol/L, CTAB concentration 100μmol/L, the mol ratio of P-CD versus CTAB 18:1 and the point of adding beta-CD 30 min after the refolding process began. With the initial protein concentration of 100 μg/mL, the protein yield and specific activity of rhlFN- y assisted by CTAB and p-CD was 29.76% and 6.34 x106 IU/mg correspondingly.
|
PDF Full Text Request