Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells

Abstract

ADP-ribosylation factors (ARFs) are a family of highly conserved, 20-kDa guanine nucleotide-binding proteins that participate in protein trafficking and enhance cholera toxin-catalyzed ADP-ribosylation. ARF 2 from bovine retinal cDNA was expressed in Sf9 insect cells using recombinant baculovirus and compared to the major insect cell ARF (Sf9 ARF) and to recombinant ARF 2 expressed in Escherichia coli (E. coli rARF 2). The 150000g supernatant and particulate fractions of freeze-thawed, recombinant ARF 2 baculovirus-infected cells contained immunoreactive proteins of 20 and 21 kDa at significantly higher levels than were found in uninfected cells. Infected Sf9 cells incorporated [3H]myristate only into the 20-kDa protein. Sf9 cell recombinant ARF 2 (Sf9 rARF 2) and Sf9 ARF were separated by isoelectric focusing or ion-exchange chromatography and identified by microsequencing of HPLC-purified tryptic peptides. Sf9 ARF displayed considerable sequence identity to mammalian class I ARFs. Both Sf9 ARF and Sf9 rARF 2 stimulated in a GTP-dependent manner cholera toxin-catalyzed ADP-ribosylation. The Ka for GTP of Sf9 ARF was, however, significantly lower than that of Sf9 rARF 2 or E. coli rARF 2. Myristoylation did not significantly affect the ability of ARF 2 to enhance cholera toxin-catalyzed ADP-ribosylation or the Ka for GTP. Despite the sequence identities and the fact that both were synthesized in insect cells, the endogenous Sf9 ARF was functionally different from Sf9 rARF 2.

Analysis of Recombinant Human ADP-Ribosylation Factors by Reversed-Phase High-Performance Liquid Chromatography and Electrospray Mass Spectrometry

“…The fact that we observe glutathionation suggests that ARF4 production may induce toxic effects, which would explain the variability in ARF4 recombinant production reported by others (31). ARF4 contains two cysteines at positions 62 and 159 which should exist as free thiols according to the crystal structure of ARF1 (46,47) and are therefore potential sites for glutathionation. Cys159 is conserved among all ARFs and lies exposed on the surface of the protein.…”

“…The low endogenous levels of Group II and III ARFs make their purification from tissue sources impractical. Furthermore, attempts to obtain myrARFs using baculovirus expression yielded only partially myristoylated ARF fraction (47), and overexpression of ARFs in yeast is toxic (7).…”

Protein Expression in Drosophila Schneider Cells

“…In addition to myristoylation, we have evidence for palmitoylation of caveolin 1, a 21-kDa membrane protein, expressed in SL-3 cells (data not shown). Palmitoylation and myristoylation of proteins has also been reported in Sf9 cells (34,35). Thus, both stably transfected SL-3 and Sf9 cells infected with baculovirus are appropriate for the production of lipid-modified proteins.…”