title: Recombinant approach for the production of HIV fusion inhibitor Enfuvirtide using Escherichia coli.
abstract: The use of antiretroviral drugs is gaining importance in the recent past for the treatment of human immunodeficiency virus infection. Enfuvirtide (T20) is one of the fusion inhibitors, inhibits the fusion between the virus and healthy target CD4 cells. The treatment with T20 involves very high therapeutic dose. In addition to its high dose, production of T20 by synthetic methods is expensive and cumbersome. We report an alternative recombinant approach for the production of the T20 peptide through a novel short fusion-tag expression system. This expression system consists of the hydrophobic region of growth hormone (GH) as the fusion tag, a factor Xa cleavage site upstream to the T20. The fusion protein was expressed in Escherichia coli as inclusion bodies. We also report here, a simple and an efficient down-stream strategy for the purification of recombinant T20 peptide (rT20). Our study is the first to demonstrate a novel approach using GH fusion tag, ensured the peptide stability, for the production of rT20 which yields more than 250mg/L with 98% purity. The biological activity of the rT20 is comparable to its synthetic counterpart. Thus, this novel approach could be an alternate method of choice for production of therapeutically important small peptides.
authors.affiliation: Virchow Research Center, Hyderabad 500 043, AP, India; Center for Biotechnology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500 085, AP, India.
authors.forename: Ravikanth Reddy
authors.initials: RR
authors.lastname: Kosana
authors.affiliation: Virchow Research Center, Hyderabad 500 043, AP, India.
authors.forename: Chitra
authors.initials: C
authors.lastname: Bajji
authors.affiliation: Virchow Research Center, Hyderabad 500 043, AP, India.
authors.forename: Radha Madhavi
authors.initials: RM
authors.lastname: Kanumuri
authors.affiliation: Department of Biotechnology, Government Degree College for Men, Kadapa, AP, India.
authors.forename: Kalpana
authors.initials: K
authors.lastname: Panati
authors.affiliation: Center for Biotechnology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500 085, AP, India.
authors.forename: Lakshmi Narasu
authors.initials: LN
authors.lastname: Mangamoori
authors.affiliation: Virchow Research Center, Hyderabad 500 043, AP, India.
authors.forename: Muralikrishna Reddy
authors.initials: MR
authors.lastname: Tummuru
authors.affiliation: Department of Zoology, Yogi Vemana University, Kadapa 516 003, AP, India. Electronic address: nvramireddy@gmail.com.
authors.forename: Venkata Ramireddy
authors.initials: VR
authors.lastname: Narala
mesh_terms: D000077560:Enfuvirtide; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D023581:HIV Fusion Inhibitors / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D012995:Solubility
chemical_list: D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000077560:Enfuvirtide; D012967:Sodium Dodecyl Sulfate
keywords: Enfuvirtide; Fusion protein expression; Recombinant T20; SDS solubilization