A novel protein refolding system using lauroyl-l-glutamate as a solubilizing detergent and arginine as a folding assisting agent.
More than 50 detergents, including acylated amino acid derivatives, were screened for their ability to solubilize and refold recombinant proteins expressed as inclusion bodies. Two model proteins, human interleukin-6 and microbial transglutaminase, were solubilized by these detergents and the solubilized proteins were rapidly diluted for testing their solubilization and refolding effectiveness. Long chain-acylated amino acid derivatives having dicarboxylic acid moieties were found to be superior to others under the conditions tested. In particular, lauroyl-l-glutamate (C12-l-Glu) showed the highest recovery of the native proteins. The effectiveness of dilution refolding was greatly improved by adding aggregation suppressive arginine into the refolding solvents. To gain understanding how this detergent works, interactions between detergents and proteins were examined using spectroscopic and native gel electrophoretic analyses, showing ideal properties for C12-l-Glu as a solubilzing agent, i.e. highly reversible nature of the detergent binding to the model globular proteins and of the conformational changes. These properties most likely have contributed to the effective protein solubilzation and refolding of inclusion bodies using C12-l-Glu and arginine.
Kudou
Motonori
M
Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562, Japan.
Yumioka
Ryosuke
R
Ejima
Daisuke
D
Arakawa
Tsutomu
T
Tsumoto
Kouhei
K
D001120:Arginine / Q000378:metabolism*; D003902:Detergents / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D015850:Interleukin-6 / Q000737:chemistry / Q000378:metabolism*; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
D003902:Detergents; D005971:Glutamates; D015850:Interleukin-6; D011994:Recombinant Proteins; D001120:Arginine; D011503:Transglutaminases; C085318:N-dodecanoylglutamic acid