Recombinant human interferon regulatory factor-1 (IRF-1) protein expression and solubilisation study in Escherichia coli
| Autor: | Kumar Gautam, Kumar Nitish, Prakash Krishna, Kumar Mishra Santosh, Kashav Tara |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Recombinant Fusion Proteins lac operon medicine.disease_cause law.invention 03 medical and health sciences 0302 clinical medicine Interferon law Genetics medicine Escherichia coli Humans Cloning Molecular Molecular Biology Glutathione Transferase Molecular mass Chemistry General Medicine Fusion protein Recombinant Proteins 030104 developmental biology IRF1 Biochemistry Solubility 030220 oncology & carcinogenesis Recombinant DNA Interferon regulatory factors medicine.drug Interferon Regulatory Factor-1 |
| Zdroj: | Molecular biology reports. 45(5) |
| ISSN: | 1573-4978 |
| Popis: | Interferon regulatory factor-1 (IRF-1) is a tumor suppressor gene, which encodes a mammalian transcription factor that serves various vital functions in a cell, such as cell cycle regulation, immunomodulation, and antiviral response. We report full-length human IRF-1 cDNA cloning and expression in E. coli/BL21 cells with complete solubilisation of recombinant protein. We cloned the gene by the RT-PCR technique using ORF-specific primers followed by expression of recombinant IRF-1 in E. coli under GST fusion system. The profound expression of recombinant protein was observed after inducing with 0.5 mM IPTG for 3 h at 37 °C. We observed few degradation products of low molecular mass along with full-length fusion protein. We successfully minimized the formation of low molecular mass degradation products of GST-huIRF-1 protein at 16 °C. Simultaneously, we achieved the expression of recombinant protein in soluble fraction of E. coli/BL21 cells at 20 °C with higher yield, which is crucial to the study of the biological functions of any protein. We further confirmed it by the immunoblotting technique using anti-IRF-1 and anti-GST antibodies under the induction of E. coli cells harboring the IRF-1 recombinant plasmid after sonicated and fractioned fractions. This work will serve as a platform for characterizing the recombinant protein that may pave the way to understand molecular mechanism of tumour suppression caused by this molecule. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink