Expression of growth hormone-releasing factor analog Leu27GRF(1–44)OH in Escherichia coli: Purification and characterization of the expressed protein
| Autor: | Mitali Basu, Joseph F. Levine, Kramer Richard Allen, Robert M. Crowl, Elizabeth Dharm, Robert M. Campbell |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Male
Recombinant Fusion Proteins Molecular Sequence Data Restriction Mapping Biophysics Protein aggregation Biology Growth Hormone-Releasing Hormone medicine.disease_cause Biochemistry Inclusion bodies chemistry.chemical_compound Affinity chromatography Pituitary Gland Anterior Escherichia coli medicine Animals Amino Acid Sequence Cloning Molecular Molecular Biology Cells Cultured Gel electrophoresis chemistry.chemical_classification Base Sequence Fusion protein Recombinant Proteins Rats Amino acid chemistry Growth Hormone Mutagenesis Site-Directed Cyanogen bromide Plasmids |
| Zdroj: | Archives of Biochemistry and Biophysics. 286:638-644 |
| ISSN: | 0003-9861 |
| DOI: | 10.1016/0003-9861(91)90093-x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|