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.
Gupta
S
S
Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India.
Arora
K
K
Gupta
A
A
Chaudhary
V K
VK
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