Gag-derived proteins of HIV-1 isolates from Indian patients: cloning, expression, and purification of p24 of B- and C-subtypes.
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 lymphocytes (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 expression 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 subtype) 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 immunological characterization and, eventually, for diagnostic and prognostic applications.
Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India.
S
S
Gupta
K
K
Arora
A
A
Gupta
V K
VK
Chaudhary
D000595:Amino Acid Sequence; D002845:Chromatography; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D016655:HIV Core Protein p24 / Q000032:analysis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D015658:HIV Infections / Q000276:immunology / Q000821:virology; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007194:India / Q000453:epidemiology; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D004279:DNA, Viral; D016655:HIV Core Protein p24; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins