Effect of promoters and signal sequences on the production of secreted HIV-1 gp120 protein in the baculovirus system.
We compared insect cell production levels of secreted HIV-1 gp120 glycoprotein encoded by five different baculovirus expression constructs. Combinations consisting of one of two baculovirus promoters (very late or hybrid late/very late) and one of three different signal sequences [human tissue plasminogen activator (tpa), human placental alkaline phosphatase (pap), or baculovirus envelope glycoprotein (gp67)] were constructed. Production of secreted gp120 from these constructs was analyzed in two enzyme-linked immunosorbent assay formats, one detecting the total amount of secreted gp120 protein and the other measuring the level of "active" gp120 (as defined by the ability to bind to CD4). We found that for all of the constructs, approximately 50 to 90% of the secreted gp120 protein was active. Furthermore, our results indicated that expression from either promoter yielded comparable production of secreted protein, despite the fact that transcription from the hybrid promoter begins at an earlier time. By contrast, the signal sequence had a much greater effect on the levels of secreted gp120: the tpa leader yielded the highest level of secreted protein, followed by the gp67 and pap sequences. This result suggests that transcription is not a limiting factor in the production of secreted gp120, but rather that downstream processing of the protein is more critical. Furthermore, these results confirm the notion that the "optimal" signal sequence is protein dependent and that an insect-derived signal sequence is not optimal in all cases.
Procept, Inc., 840 Memorial Drive, Cambridge, Massachusetts, 02139, USA.
A
A
Golden
D A
DA
Austen
M R
MR
van Schravendijk
B J
BJ
Sullivan
E S
ES
Kawasaki
M S
MS
Osburne
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005260:Female; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis* / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010920:Placenta / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera; D010959:Tissue Plasminogen Activator / Q000235:genetics; D014760:Viral Fusion Proteins / Q000235:genetics
D015699:HIV Envelope Protein gp120; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins; D000469:Alkaline Phosphatase; D010959:Tissue Plasminogen Activator