Enhanced expression, secretion, and large-scale purification of recombinant HIV-1 gp120 in insect cell using the baculovirus egt and p67 signal peptides.
The expression of glycosylated and secreted recombinant mammalian proteins in baculovirus-infected insect cells is often much less efficient than that of other foreign proteins in this system. In an effort to improve the expression and secretion of such proteins we have constructed baculovirus vectors which contain the signal peptide coding regions from two baculovirus proteins, an ecdysteroid UDPglucosyltransferase (egt) and the envelope glycoprotein gp67. We used these vectors to express HIV-1 gp120, inserting the baculovirus signal peptides in place of the HIV-1 envelope signal peptide. When Sf9 cells infected with recombinant baculoviruses made from these vectors (vegt120 and vp67120) were compared with cells infected with the normal gp120 baculovirus a 6- to 20-fold increase in expression and secretion of gp120 was observed. When the HIV-1 signal peptide was used only 40% or less of the total gp120 produced in Sf9 cells was secreted. However, using the egt or p67 signal peptides, up to 70% of the total gp120 produced was secreted. Therefore, not only was more gp120 produced from these modified viruses but secretion of gp120 was more efficient. Large-scale expression and purification of egt-gp120 from a 5-liter airlift fermenter or a 6-liter spinner flask resulted in a yield of 10 to 15 mg of purified protein per liter. Using these baculovirus-derived signal peptides in baculovirus expression vectors is thus likely to aid in increasing expression and yield of heterologous secreted proteins in insect cells.
Murphy
C I
CI
Department of Molecular Biology, Cambridge Biotech Corporation, Worcester, Massachusetts 01605.
McIntire
J R
JR
Davis
D R
DR
Hodgdon
H
H
Seals
J R
JR
Young
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D015870:Gene Expression; D005822:Genetic Vectors; D014453:Glucuronosyltransferase / Q000235:genetics*; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015497:HIV-1 / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics*
D015699:HIV Envelope Protein gp120; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014453:Glucuronosyltransferase