High throughput purification of recombinant human growth hormone using radial flow chromatography.
Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. Using fed-batch fermentation process, around 670 mg/L of r-hGH was produced at a cell OD600 of 35. Cell lysis followed by detergent washing resulted in semi-purified inclusion bodies with more than 80% purity. Purified inclusion bodies were homogenous in preparation having an average size of 0.6 microm. Inclusion bodies were solubilized at pH 12 in presence of 2M urea and refolded by pulsatile dilution. Refolded protein was purified with DEAE-anion exchange chromatography using both radial and axial flow column (50 ml bed volume each). Higher buffer flow rate (30 ml/min) in radial flow column helped in reducing the batch processing time for purification of refolded r-hGH. Radial column based purification resulted in high throughput recovery of diluted refolded r-hGH in comparison to axial column. More than 40% of inclusion body protein could be refolded into bioactive form using the above method in a single batch. Purified r-hGH was analyzed by mass spectroscopy and found to be bioactive by Nb2 cell line proliferation assay. Inclusion body enrichment, mild solubilization, pulsatile refolding and radial flow chromatography worked co-operatively to improve the overall recovery of bioactive protein from inclusion bodies.
Singh
Surinder M
SM
Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Sharma
Aparna
A
Panda
Amulya K
AK
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D019382:Human Growth Hormone / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000648:ultrastructure; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D011994:Recombinant Proteins; D019382:Human Growth Hormone