Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli.
Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.
Patra
A K
AK
Product Development Cell, Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
Mukhopadhyay
R
R
Mukhija
R
R
Krishnan
A
A
Garg
L C
LC
Panda
A K
AK
D000818:Animals; D019149:Bioreactors; D002021:Buffers; D002455:Cell Division; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020673:Protein Renaturation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D002021:Buffers; D011994:Recombinant Proteins; D019382:Human Growth Hormone