BIOACTIVITY CHARACTERIZATION OF PURIFIED RECOMBINANT HYPOTHETICAL PROTEIN CODED BY OPEN READING FRAME-112 OF STREPTOMYCES

This study was aimed to investigate the Open Reading Frame-112 (ORF-112) gene, which encoded for a hypothetical 218 amino acids protein in Streptomyces bacteria. A complete ORF-112 gene was synthesized, with addition of a 6xHis-Tag at the N-terminal location. The synthesized DNA nucleotides were sub...

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Bibliographic Details
Published in:Iraqi journal of agricultural science 2021-01, Vol.52 (2), p.502-511
Main Authors: Al-Obaidi, R. M., Salih, G. F., Nore, B. F.
Format: Article
Language:English
Subjects:
Affinity
Affinity chromatography
Amino acids
Bacteria
Bioactive compound
Candida albicans sensitivity
hypothetical protein-ORF-112
Streptomyces
Biological activity
Chromatography
Cloning
Deoxyribonucleic acid
Diffusion
DNA
Fungi
Gel electrophoresis
Gene expression
Itraconazole
Ketoconazole
Nucleotides
Open reading frames
Plasmids
Protein expression
Proteins
Sodium lauryl sulfate
Software
Synthesis
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Summary:This study was aimed to investigate the Open Reading Frame-112 (ORF-112) gene, which encoded for a hypothetical 218 amino acids protein in Streptomyces bacteria. A complete ORF-112 gene was synthesized, with addition of a 6xHis-Tag at the N-terminal location. The synthesized DNA nucleotides were sub-cloned into bacterial expression plasmid pBAT4. The pBAT4-ORF-112 plasmid transformed in bacterial cells BL21(De3)pLysS, intended for protein over expression, induced by isopropyl β- d-1-thiogalactopyranoside (IPTG). The IMAC affinity chromatography technique was deployed for protein purifications. Highly-purified fractions of ORF-112 were achieved by using affinity Ni++-columns. The purified ORF-112 protein was tested for possible biological activity.  The SDS-PAGE analysis exhibited a soluble 30 kDa size purified ORF-112 protein which showed a slight gel shifting from the predicted size. The virulent activity test on purified fractions of ORF-112 was measured using the Disk Diffusion Test and it disclosed a clear zone formed in response to fungi Candida albicans growth. The data implies that the ORF-112 protein has an acceptable protective effect against the fungus C. albicans as compared to positive control ketoconazole (KCZ) (P < 0.05) while the protein has a significantly lower protective effect against the fungus than Itraconazole (ICZ) (P > 0.05). The results clarify the hypothetical ORF-112 protein is a novel protein with protective response effect against fungi cells C. albicans on disk-diffusion.test.
ISSN:0075-0530
2410-0862
DOI:10.36103/ijas.v52i2.1314