Expression of growth hormone-releasing factor analog Leu27GRF(1-44)OH in Escherichia coli: purification and characterization of the expressed protein.
A recombinant plasmid has been constructed to direct the synthesis of Leu27GRF(1-44)OH in Escherichia coli as a fusion protein containing a hexa-His tail followed by amino acids 1-99 of interferon-gamma and a methionine residue at the N-terminal. The expression of the 18-kDa fusion protein (H6GAMGRF) was induced by isopropylthiogalactoside treatment and the protein accumulated as insoluble aggregates in inclusion bodies. The protein aggregates were solubilized in 6 M guanidine-HCl and purified directly by affinity chromatography on a Nichelate column. The growth hormone-releasing factor (GRF) moiety was released from the fusion protein by cyanogen bromide cleavage and purified to homogeneity by anion-exchange chromatography followed by reverse-phase chromatography. The identity of the GRF peak was determined by comparing its retention time with that of synthetic Leu27GRF(1-44)OH. The purified material was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and amino-acid analysis. The recombinant-derived product and the synthetic compound showed identical reactivities toward anti-GRF polyclonal antibodies and were essentially equipotent as determined by an in vitro biological assay for growth hormone-releasing activity.
Basu
M
M
Department of Protein Biochemistry, Roche Research Center Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Dharm
E
E
Levine
J F
JF
Kramer
R A
RA
Crowl
R M
RM
Campbell
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D013006:Growth Hormone / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C071002:somatotropin releasing hormone (1-44)-OH, Leu(27)-; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone