Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli.
Amongst the various endogenous growth factors, epidermal growth factor (EGF) plays an important role in normal wound healing of tissue such as skin, cornea and gastrointestinal tract. Various studies have proved that supplementing recombinant human EGF (rhEGF) results in significant augmentation of wound healing. In the present work, a high level expression system with poly-arginine sequences was used for the production of recombinant human EGF (rhEGF) as inclusion bodies. The inclusion bodies were solubilized and the protein was refolded by using expanded-bed adsorption chromatography. The renatured protein was digested with appropriate concentration of trypsin and subsequently the digested rhEGF is purified by passing through ion-exchange chromatography (Toyopearl-SP) to obtain a biologically active protein. This process is the shortest process with reduced number of steps of purification, eliminates the usage of preparative reversed phase HPLC (RP-HPLC) for final purification, which is an expensive technique. The purified protein was analyzed by RP-HPLC, showing a purity > 99% and size exclusion chromatography profile shows that there are minimal aggregates, with 99% renatured active protein. The purified rhEGF showed a specific activity of 5 x 10(5) IU/mg protein, in comparison with NIBSC standard (1st International Standard of rDNA-derived EGF, Code 91/530). The process has been successfully adopted at 100 L fermentation scale and the rhEGF based formulation has been commercialized with brand name REGEN D, with excellent clinical results.
Sharma
K
K
Bharat Biotech International Limited, Genome Valley, Shameerpet, Turkapally, Hyderabad, AP 500078, India.
Babu
P V Cherish
PV
Sasidhar
P
P
Srinivas
V K
VK
Mohan
V Krishna
VK
Krishna
Ella
E
D001483:Base Sequence; D002845:Chromatography; D004815:Epidermal Growth Factor / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D004815:Epidermal Growth Factor