ABSTRACT
Granulocyte colony-stimulating factor (G-CSF) is a multi-functional 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.
Subject(s)
Cysteine/metabolism , Cysteine/pharmacokinetics , Cystine/metabolism , Cystine/pharmacokinetics , Granulocyte Colony-Stimulating Factor , Oxidation-Reduction , Protein RefoldingABSTRACT
Se presenta el caso de un niño de dos años de edad cistinosis clásica, manifestada por síndrome de Fanconi (glucosuria, aminoaciduria y fosfaturia), raquitismo, talla baja, presencia de cristales de cistina en córnea y daño glomerular progresivo; demostrándose en le biopsia renal por medio de microscopia electrónica el acúmulo de los característicos cristales hexagonales de cistina. Debido a que la frecuencia de esta patología en nuestro país es muy baja, y el cuadro clínico de nuestro paciente es representativo de la variante infantil nefropática, decidimos realizar este informe