Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon α-2b
| Autor: | Muhammad Nadeem, Amina Arif, Kaleemuddin Mohammed |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Physiology Science Biology medicine.disease_cause Microbiology Aminopeptidase Article law.invention 03 medical and health sciences chemistry.chemical_compound law Interferon Genetics medicine Molecular Biology Escherichia coli chemistry.chemical_classification Fen Methionine Tetrapeptide Methionine aminopeptidase E. coli human interferon in vitro in silico Cell Biology In vitro 030104 developmental biology Enzyme chemistry Biochemistry Recombinant DNA General Agricultural and Biological Sciences medicine.drug |
| Zdroj: | Volume: 42, Issue: 3 240-249 Turkish Journal of Biology |
| ISSN: | 1303-6092 1300-0152 |
| DOI: | 10.3906/biy-1801-83 |
| Popis: | Escherichia coli is an extensively used host for the production of recombinant proteins, making its N-terminal methionine aminopeptidase (MAP) an attractive candidate for studies on posttranslational protein processing. The present study describes the recombinant production and properties of MAP from the DH5α strain of E. coli. The soluble and active enzyme was produced in E. coli BL21 (DE3) RIL - codon plus cells under a T7 promoter system and purified by anion-exchange chromatography. It exhibited a molecular weight of 29,200.94 Da by MALDI-TOF analysis. The purified enzyme showed specific activity of 1.64 U/mg with methionylp-nitroanilide and 1.51 U/mg with synthetic tetrapeptide substrate `MGMM' in a discontinuous HPLC-based assay. In vitro studies showed the processing of up to 36% of Met-INFα-2b in 40 min. In silico studies revealed that the ES-complex formation between the enzyme and interferon has a ΔG -683.07 kJ/mol. Molecular docking results showed that the processed INFα-2b has greater binding affinity with IFNAR2 receptor as indicated by ΔG -784.53 kJ/mol, significantly lower than that of methionine containing INFα-2b (ΔG -717.63 kJ/mol). These findings emphasize the functional superiority or better efficacy of N-terminal methionine processed recombinant interferon. |
| Databáze: | OpenAIRE |
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