Gag-derived proteins of HIV-1 isolates from Indian patients: Cloning, expression, and purification of p17 of B- and C-subtypes

摘要

A simple and efficient method for expression in Escherichia coli and purification of matrix protein, p17, of human immunodeficiency virus type 1 (HIV-1) of both B- and C-subtypes is described. DNA sequences encoding p17 of B- and C-subtype were cloned from respective gag sequences. The gag sequences were obtained by PCR amplification using DNA extracted from peripheral blood lymphocytes of an HIV-1 infected patient from India. A T7-promoter-based expression system was optimized for expression of p17 in soluble form, p17 (B- and C-subtype) was purified to near homogeneity using conventional chromatographic techniques. Purification of p17 (C-subtype) is described for the first time with yield of 7.7 mg from a 1-liter culture. The yield of p17 (B-subtype) is 14.7 mg from a 1-liter culture, which is severalfold better than that reported earlier. N-terminal sequencing and CD spectra of the purified proteins, p17B and p17C, show that the proteins are properly processed and well-folded. The immunoreactivity of both types of p17 to sera from HIV-infected individuals is comparable. (C) 2001 Academic Press. [References: 30]