Overexpression of HIV-1 proteins in Escherichia coli by a modified expression vector and their one-step purification.
A synthetic expression system, pMH, was constructed for high-level expression and rapid purification of HIV-1 proteins in Escherichia coli, by introducing two synthetic sequences. The first sequence permitted a high-level expression via a second ribosome-binding site plus an A/T-rich region. This minicistron between the 5' untranslated region of the mRNA and the protein-coding region minimized the formation of local secondary structures and favored ribosome binding. The second region coded for six histidines, which allowed for easy purification of the proteins. Purification was based on interaction of the protein with metal ions: immobilized metal affinity chromatography. The p17 and p24 structural genes of HIV-1 were cloned into this modified expression vector. Expression of the structural proteins represented up to 30% of the total protein in E. coli, i.e., 40 mg of highly purified protein could be obtained per liter of culture.
Cheynet
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V
Unité Mixte 103 CNRS, BioMérieux, Ecole Normale Supérieure de Lyon, France.
Verrier
B
B
Mallet
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F
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D015683:Gene Products, gag / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors; D015488:HIV Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016655:HIV Core Protein p24 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012270:Ribosomes / Q000378:metabolism; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015488:HIV Antigens; D016655:HIV Core Protein p24; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1