Designing of a specific fluorogenic peptide substrate for HIV-1 protease activity assay

Nguyen Thi Hong Loan, Tran Thi Thu Huyen, Dang Thi Lieu, Phan Thi Lam Hong, Phan Tuan Nghia

Abstract


Based on the specific hydrolysis sequence in the Gag-Pol protein substrate of HIV-1 protease, we designed a fluorescence resonance energy transfer (FRET) substrate (designated peptide HF) for the enzyme. peptide HF has the sequence of QXL 520-GABA-SFNFPQITK-HiLyte Flour 488-NH2. HiLyte Fluor 488/QXL 520 as a FRET pair because of high excitation/emission and good activity at the optimal pH of HIV-1 protease (pH<5) was chosen. QXL 520 was more stable when conjugated with serine residue at N-terminus via GABA group while leucine at the C-terminus was replaced with a lysine residue-HiLyte Fluor 488 conjugate.

The optimal reaction conditions for HIV-1 protease activity assay were found to include 100-200 ng HIV-1 protease, 2 µM peptide HF, 100 mM CH3COONa buffer at pH 4.7, containing 1 M NaCl, 1 mM EDTA, 1 mM DTT, 5% DMSO and 0.5 mg/mL BSA. Peptide HF could be stored at the concentration of 0.1 mg/mL in DMSO and at -80oC until use.

Under the above mentioned conditions, HIV-1 protease showed the high affinity and the effective hydrolysis for the substrate with the kinetic parameters Vmax of 4.45 nM/s, Kcat/Km = 10.89 (mM.s)-1, which were 5 times higher than the Vmax and Kcat/Km when the commercial SensoLyte® 520 substrate (Anaspec-America) was used.

 

Citation: Nguyen Thị Hong Loan, Tran Thi Thu Huyen, Dang Thi Lieu, Phan Thi Lam Hong, Phan Tuan Nghia, 2017. Designing of a specific fluorogenic peptide substrate for HIV-1 protease activity assay. Tap chi Sinh hoc, 39(1): 115-121.  DOI: 10.15625/0866-7160/v39n1.8291.

*Corresponding author: loannhbio@gmail.com

Received 1 May 2016, accepted 20 December 2016

 


Keywords


fluorogenic substrate, HIV-1 protease, HIV-1 protease inhibitor, Michaelis- Menten constant (Km), Vmax

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Published by Vietnam Academy of Science and Technology