DEVELOPMENT OF HIV-1 RECOMBINANT PROTEASE (RPR) EXPRESSION, SOLUBILITY AND PURIFICATION PROBLEMS BY TOPO-CLONING IN E. COLI BL21

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AZARNEZHAD ASAAD; HOSSEINI ARSHAD; SHARIFI ZOHREH

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Paper Information

Journal: NEW CELLULAR & MOLECULAR BIOTECHNOLOGY JOURNAL Year:2016 | Volume:6 | Issue:21 Page(s): 21-27

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Information Journal Paper

Title

DEVELOPMENT OF HIV-1 RECOMBINANT PROTEASE (RPR) EXPRESSION, SOLUBILITY AND PURIFICATION PROBLEMS BY TOPO-CLONING IN E. COLI BL21

Author(s)

AZARNEZHAD ASAAD | HOSSEINI ARSHAD | SHARIFI ZOHREH | Issue Writer Certificate

Keywords

HUMAN IMMUNODEFICIENCY VIRUSQ2

TOPO CLONINGQ2

PROTEASEQ2

RECOMBINANT PROTEINQ2

Abstract

Aim and Background: HIV PROTEASE plays an important role in the maturation of the virus and host immune response stimulation; therefore it is important as a therapeutic target and can be used in diagnostic tests. In previous studies, producing rPR has been faced with problems such as toxicity, hydrophobic and purification. The purpose of this study is the use of expression system pET102/D. TOPO to overcome the mentioned problems.Materials and Methods: PROTEASE gene was isolated from the serum of an infected individual. Cloning process was performed by TOPO CLONING system. PROTEASE gene transformed into E.coli BL21and induced with IPTG. Produced RECOMBINANT PROTEIN was purified by affinity chromatography (Ni-NTA column). Protein concentration was checked by BCA protein assay kit. RECOMBINANT PROTEIN was identified by SDS-PAGE and western-blotting.Results: HIV-1 PROTEASE gene Cloning using TOPO CLONING vector expression system pET102 / D was confirmed by PCR and sequencing. rPR concentration after purification was 60 to 85 micrograms per milliliter. rPR Expression and Immunogenicity by SDS-PAGE and Western blotting were confirmed.Conclusion: Expression and production of rPR in soluble form was a success. In used Cloning system, because of the expression of rPR in fused form with thioredoxin and histidine tags, problems of toxicity, hydrophobicity and purification were noticeably solved. According to the generated rPR, it can be used to evaluate the diagnostic tests and for designing PROTEASE inhibitors in subsequent studies.

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APA: Copy

AZARNEZHAD, ASAAD, HOSSEINI, ARSHAD, & SHARIFI, ZOHREH. (2016). DEVELOPMENT OF HIV-1 RECOMBINANT PROTEASE (RPR) EXPRESSION, SOLUBILITY AND PURIFICATION PROBLEMS BY TOPO-CLONING IN E. COLI BL21. NEW CELLULAR & MOLECULAR BIOTECHNOLOGY JOURNAL, 6(21), 21-27. SID. https://sid.ir/paper/204430/en

Vancouver: Copy

AZARNEZHAD ASAAD, HOSSEINI ARSHAD, SHARIFI ZOHREH. DEVELOPMENT OF HIV-1 RECOMBINANT PROTEASE (RPR) EXPRESSION, SOLUBILITY AND PURIFICATION PROBLEMS BY TOPO-CLONING IN E. COLI BL21. NEW CELLULAR & MOLECULAR BIOTECHNOLOGY JOURNAL[Internet]. 2016;6(21):21-27. Available from: https://sid.ir/paper/204430/en

IEEE: Copy

ASAAD AZARNEZHAD, ARSHAD HOSSEINI, and ZOHREH SHARIFI, “DEVELOPMENT OF HIV-1 RECOMBINANT PROTEASE (RPR) EXPRESSION, SOLUBILITY AND PURIFICATION PROBLEMS BY TOPO-CLONING IN E. COLI BL21,” NEW CELLULAR & MOLECULAR BIOTECHNOLOGY JOURNAL, vol. 6, no. 21, pp. 21–27, 2016, [Online]. Available: https://sid.ir/paper/204430/en

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