HIV-1 Protease E.coli
Retroviral protease from HIV-1 virus is an enzyme important in the life cycle of the virus. It is expressed in the infected cells as a part of Gag-Pol polyprotein from which it is autocatalytycaly released after formation of immature viral particle. The enzyme subsequently cleaves the other parts of viral polyproteins causing the maturation of the virus. In HIV-infected patients the enzyme is a subject of intensive mutagenesis and mutants resistant to applied medcines are produced as a consequence of the seletion pressure. HIV-1 protease is active as a homodimer.
Type
Active
Description
Total 99 AA. MW: 10.8 kDa (monomer), protein active as dimer
Source
E. coli
Purity
Purity as determined by densitometric image analysis: >95%
SDS-PAGE Gel
14% SDS-PAGE separation of Human HIV-1 Protease
1. M.W. marker – 14, 21, 31, 45, 66, 97 kDa
2. reduced and heated sample, 2.5 μg/lane
Formulation
20 mM Tris, 20 mM MES, 200 mM NaCl, 1mM EDTA, 10% (v/v) glycerol, 0,05% 2-mercaptoethanol, pH 6.5 – filtered (0.4 μm), frozen
Reconstitution
Defrost at ambient temperature.
Applications
Kinetic studies, Inhibitor screening, Crystallography
Shipping
On ice. Upon receipt, store the product at the temperature recommended below.
Storage/Expiration
Store protein at –80°C. Protein remains stable until the expiry date when stored at –80°C. Avoid repeated freezing/thawing cycles.
Quality Control Test
SDS PAGE to determine purity of the protein.
Active site titration by tightly binding inhibitor.
Note
Km = 15.1 μM Kcat = 30 s-1 Kcat /Km = 1981 mM-1 s-1 with peptide substrate KARVF(NO2 )VRKA (F(NO2 ) … p-nitrophenylalanine)
– Ingr M, Uhlikova T, Strisovsky K, Majerova E, Konvalinka J. Kinetics of the dimerization of retroviral proteases: the “fireman’s grip” and dimerization. Protein Sci. 2003 Oct;12 (10):2173-82
– Lindsten K, Uhlikova T, Konvalinka J, Masucci MG, Dantuma NP. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity. Antimicrob Agents Chemother. 2001 Sep;45 (9):2616-22
– Parra A, Martin-Fonseca S, Rivas F, Reyes-Zurita FJ, Medina-O’Donnell M, Martinez A, Garcia-Granados A, Lupianez JA, Albericio F. Semi-synthesis of acylated triterpenes from olive-oil industry wastes for the development of anticancer and anti-HIV agents. Eur J Med Chem. 2014 Mar 3;74:278-301
– Saskova KG, Kozisek M, Rezacova P, Brynda J, Yashina T, Kagan RM, Konvalinka J. Molecular characterization of clinical isolates of human immunodeficiency virus resistant to the protease inhibitor darunavir. J Virol. 2009 Sep;83 (17):8810-8