Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle.
Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37 degrees C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCl, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.
Iwakura
M
M
Research Institute for Polymers and Textiles, Ibaraki.
Obara
K
K
Kokubu
T
T
Ohashi
S
S
Izutsu
H
H
D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003902:Detergents / Q000494:pharmacology; D013007:Growth Hormone-Releasing Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000737:chemistry / Q000187:drug effects; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D012995:Solubility / Q000187:drug effects; D012997:Solvents / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D014508:Urea / Q000494:pharmacology
D000085:Acetates; D003902:Detergents; D012997:Solvents; D014508:Urea; D013007:Growth Hormone-Releasing Hormone; D013762:Tetrahydrofolate Dehydrogenase; D019342:Acetic Acid