Gag-derived proteins of HIV-1 isolates from Indian patients: cloning, expression, and purification of p17 of B- and C-subtypes.
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.
Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India.
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S
Gupta
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Arora
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Gupta
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Chaudhary
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D005822:Genetic Vectors; D015488:HIV Antigens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D015658:HIV Infections / Q000276:immunology / Q000821:virology*; D015497:HIV-1 / Q000737:chemistry / Q000145:classification* / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007194:India; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015488:HIV Antigens; D007106:Immune Sera; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1