Optimization of conditions for high-level expression and purification of human recombinant consensus interferon (rh-cIFN) and its characterization.
Recombinant human consensus interferon (rh-cIFN) is an artificially engineered interferon (IFN) developed by recombining and reordering the protein sequences that exist in standard IFN. This recombination resulted into a drug that has the potential to work better than natural, standard IFN. In this study, we described optimized conditions for high-level expression and recovery of biologically active consensus IFN from inclusion bodies (IBs). A synthetic gene coding 166 amino acids of consensus IFN was cloned under the T7 promoter. Escherichia coli strain BL21DE3Plys was used to transform expression construct. For high-level expression, shake-flask fermentation conditions were standardized. For isolation of IBs, the sonication method was optimized. A variety of chaotropic agents including guanidine hydrochloride, urea, SDS, and detergents were studied for solubilization of IBs. For renaturation of solubilized denatured protein by the dilution process, parameters of dilution factor, temperature, and l-arginine were optimized. A one-step chromatography method was developed for high-yield purification of consensus IFN. rh-cIFN was characterized by SDS-PAGE, Western blot, and high-performance liquid chromatography. Purified protein has a molecular weight of 19.5 kDa and specific activity was 2.0 × 10(8) as determined by the cytopathic inhibition assay. This study concludes that by using optimized conditions, we obtained a yield of 100 mg/L of biologically active rh-cIFN, which is highest ever reported according to available data.
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Nadeem
N
Ahmed
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Hamid
H
Bashir
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Ahmad Usman
AU
Zafar
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Mohsin Ahmad
MA
Khan
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Saad
S
Tahir
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Faidad
F
Khan
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Muhammad Islam
MI
Khan
Centre for Applied Molecular Biology, Ministry of Science and Technology, Lahore, Pakistan.
Muhammad
M
Akram
National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Tayyab
T
Husnain
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D016898:Interferon-alpha; D011994:Recombinant Proteins; C050739:interferon alfacon-1
biological activity; biopharmaceutical; biophysical characterization; bioprocessing; chromatography; expression and purification; rh-cIFN