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

Citation
S. Gupta et al., Gag-derived proteins of HIV-1 isolates from Indian patients: Cloning, expression, and purification of p17 of B- and C-subtypes, PROT EX PUR, 21(3), 2001, pp. 378-385
Citations number
30
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN EXPRESSION AND PURIFICATION
ISSN journal
1046-5928 → ACNP
Volume
21
Issue
3
Year of publication
2001
Pages
378 - 385
Database
ISI
SICI code
1046-5928(200104)21:3<378:GPOHIF>2.0.ZU;2-C
Abstract
A simple and efficient method for expression in Escherichia coli and purifi cation 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 blo od 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 chr omatographic techniques. Purification of p17 (C-subtype) is described for t he 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 pu rified proteins, p17B and p17C, show that the proteins are properly process ed and well-folded. The immunoreactivity of both types of p17 to sera from HIV-infected individuals is comparable. (C) 2001 Academic Press.