Isolation of recombinant partial gag gene product p18 (HIV-1Bru) from Escherichia coli.
The membrane-associated structural protein, p18, of the human immunodeficiency virus (HIV-1), has been expressed in Escherichia coli. The recombinant protein was purified by cation-exchange chromatography on S Sepharose followed by cation-exchange high-performance liquid chromatography (HPLC) on Sulfoethyl Aspartamide. The isolation of 28.7 mg of recombinant p18 from 16.71 of cell culture represents an overall yield of ca. 20%. Recombinant p18 was characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, reversed-phase HPLC, amino acid composition and amino acid sequence analysis of the N-terminus. Edman degradation of peptides generated by trypsin or Staphylococcus aureus V8 proteolytic digestion, including the C-terminus, confirmed the amino acid sequence to be that predicted from the cDNA. A C-terminally cleaved form of recombinant p18, p18LM, was separated in the cation-exchange HPLC step and was partially characterized in parallel with the intact molecule. By Western blotting it was shown that recombinant p18 in addition to the cleaved form p18LM is recognized by a monoclonal antibody which was generated against the natural protein from HIV-1.
Kolbe
H V
HV
Transgene SA, Strasbourg, France.
Jaeger
F
F
Lepage
P
P
Roitsch
C
C
Lacaud
G
G
Kieny
M P
MP
Sabatie
J
J
Brown
S W
SW
Lecocq
J P
JP
Girard
M
M
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag; D015488:HIV Antigens / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids; D011492:Protein Hydrolysates / Q000032:analysis; D011994:Recombinant Proteins / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D014764:Viral Proteins / Q000302:isolation & purification*; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015488:HIV Antigens; D011492:Protein Hydrolysates; D011994:Recombinant Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C061169:p18 gag protein, Human immunodeficiency virus 1