Gag-Derived Proteins of HIV-1 Isolates from Indian Patients: Cloning, Expression, and Purification of p24 of B- and C-Subtypes Sanjay Gupta, Kajal Arora, Amita Gupta, and Vijay K. Chaudhary 1 Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India Received November 1, 1999, and in revised form April 17, 2000 A simple and efficient method for hyperexpression in Escherichia coli and purification of capsid protein, p24, of human immunodeficiency virus type 1 (HIV-1) of both B- and C-subtypes is described. DNA-encoding p24 of C-subtype was cloned from C-subtype gag sequence which was obtained by PCR amplification using DNA extracted from peripheral blood mononuclear lympho- cytes (PBMLs) of an HIV-1-infected patient from India. DNA-encoding p24B protein was amplified directly by two-step PCR using genomic DNA obtained from PBMLs of an HIV-infected individual. A T7 promoter-based ex- pression system was optimized for hyperexpression of p24 in the soluble form. Both p24 (B- and C-subtype) were purified to near homogeneity using conventional chromatographic techniques. Purification of p24 (C sub- type) was described for the first time with yield of 53 mg from 1 liter of culture. The yield of p24 (B-subtype) was 67 mg from 1 liter of culture, which was severalfold better than reported earlier. The immunoreactivity of both types of p24 to sera from HIV-infected individuals was comparable. This report describes a simple, highly efficient, and reproducible method for obtaining large quantities of highly pure p24 of both B- and C-subtype, which can be used for structural, biochemical, and im- munological characterization and, eventually, for diag- nostic and prognostic applications. © 2000 Academic Press The core of human immunodeficiency virus-1 (HIV- 1), 2 the causative agent of acquired immune deficiency syndrome is made of Gag-derived proteins. Gag is a 55-kDa precursor protein, which during the process of virion maturation, is cleaved to give several proteins, namely, the matrix protein (MA, p17), the capsid pro- tein (CA, p24), a basic nucleocapsid protein (NC, p7), and peptides p6, p2, and p1. Besides Gag, p24 and p17 have gained importance because they not only provide the shape and structure to HIV, but are also important in infection, replication, assembly, and release of viri- ons (1). Further, the presence of antibodies to Gag proteins in infected persons has been correlated with low level of HIV virion. The p24 antigenemia along with a decline in the level of antibodies to p24 has been considered a useful prognostic marker of infection (2). Determination of p24 antigen as well as anti-p24 anti- bodies has diagnostic value. Therefore, most diagnostic kits to detect HIV antibodies use p24 as an integral component in addition to envelope glycoproteins. It has also been suggested that inhibition of capsid formation can be an effective therapeutic strategy (3); therefore, there have been efforts to understand intra- and inter- molecular interaction in Gag proteins (4,5). However, a high mutation rate of HIV, evolution of a large number of different subtypes within HIV, and susceptibility of different populations/ethnic groups to different sub- types make it difficult to arrive at a consensus for the prototype strain for diagnostic and therapeutic studies. HIV-1 has been characterized in several (A to K) sub- groups and within subgroups there are further varia- tions (6). Indian isolates have been placed in subtype C-based on sequencing analyses (7–9), but there are reports of the presence of subtype B also, though at a frequency of about 8% (10). This pattern is in variance with HIV-1 spread elsewhere; e.g., in the United States and Europe mainly subtype B is prevalent while Afri- can subtypes mainly constitute A, C, and D subtypes. Thus, it is very important to analyze the local strains at the sequence level and characterize their products at 1 To whom correspondence should be addressed. Fax: 91-11- 6885270 and 91-11-6886427. E-mail: dbtudsc@nde.vsnl.net.in. 2 Abbreviations used: HIV, human immunodeficiency virus; IPTG, isopropyl thiogalactopyranoside; PBML, peripheral blood mononu- clear lymphocytes; PBS, 20 mM phosphate buffer, pH 6.7, containing 140 mM NaCl; CD, circular dichroism; PBST, 0.05% Tween 20 in PBS; HRP, horseradish peroxidase. Protein Expression and Purification 19, 321–328 (2000) doi:10.1006/prep.2000.1266, available online at http://www.idealibrary.com on 321 1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.