Refolding of recombinant human granulocyte colony stimulating factor: effect of cysteine/cystine redox system.
Granulocyte colony-stimulating factor (G-CSF) is a multifunctional cytokine which is widely used for treating neutropenia in humans. Evaluation of alternative to expensive components of redox buffer (reduced and oxidized glutathione) is an important step in reducing the cost of production of human biotherapeutic proteins. In the present study, refolding of recombinant human G-CSF expressed as inclusion bodies (IBs) in E. coli was optimized using cysteine and cystine redox agents. The refolding to correct native form of G-CSF was assessed by reverse phase high performance liquid chromatography (RP-HPLC). The optimized concentrations of cysteine and cystine for correct refolding of G-CSF were found to be 2 mM and 1 mM, respectively. The correctly refolded G-CSF was detected as early as 4 h of incubation in renaturation buffer containing optimized concentrations of cysteine (2 mM) and cystine (1 mM) redox agents. Refolding of G-CSF in optimized redox system increased with increase in shuffling time. Overall, the results suggested the use of cysteine/cystine redox pair could be an alternative to the costlier redox pairs for successful refolding of G-CSF and possibly other human biotherapeutic proteins of importance.
Gennova Biopharmaceuticals Ltd, MIDC,Phase II, Hinjwadi, Pune 411057, India. krishna.tiwari@gennova.co.in
Krishnanand
K
Tiwari
Sunil
S
Shebannavar
Krishna
K
Kattavarapu
Santosh
S
Pokalwar
Maheshwari K
MK
Mishra
Ugam Kumari
UK
Chauhan
D003545:Cysteine / Q000494:pharmacology*; D003553:Cystine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D010084:Oxidation-Reduction / Q000187:drug effects; D058849:Protein Refolding / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry*; D013997:Time Factors
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D003553:Cystine; D003545:Cysteine