Cell-surface expression and purification of human CD4 produced in baculovirus-infected insect cells.
CD4 is an integral membrane glycoprotein that acts as the cellular receptor for human immunodeficiency virus (HIV). A cDNA encoding full-length CD4 was inserted into the genome of Autographa californica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of CD4 as evaluated by flow cytometry and immunoblot analysis. Recombinant CD4 expressed on the surface of infected insect cells was immunologically indistinguishable from human CD4 when using 11 different anti-CD4 monoclonal antibodies. The extraction of infected cells by phase-transition separation with Triton X-114 followed by immunoaffinity chromatography yielded a single protein detected by NaDodSO4/PAGE using silver staining. N-terminal sequence analysis of the purified recombinant protein showed that CD4 produced in Sf9 cells is efficiently cleaved from the precursor protein. Immunoblot analysis under nondenaturing conditions showed that the purified protein reacted with the anti-CD4 monoclonal antibody Leu-3a. The potential use of the recombinant membrane-associated CD4 in anti-HIV therapy is discussed.
Webb
N R
NR
Department of Entomology, Texas A & M University, College Station 77843.
Madoulet
C
C
Tosi
P F
PF
Broussard
D R
DR
Sneed
L
L
Nicolau
C
C
Summers
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D015967:Gene Expression Regulation, Viral; D005796:Genes; D005814:Genes, Viral; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification; D014158:Transcription, Genetic; D015678:Viral Structural Proteins / Q000235:genetics
D015704:CD4 Antigens; D011994:Recombinant Proteins; D015678:Viral Structural Proteins