Characterization of nuclear pore protein p62 produced using baculovirus.
Nuclear pore glycoproteins are essential components of the nuclear import apparatus in eukaryotes. In vertebrates, the most abundant of these glycoproteins is a molecule called p62. Like other O-linked N-acetylglucosamine glycoproteins, p62 is normally modified in the cytoplasm and cannot be overexpressed and conveniently collected in a secreted form. We devised an efficient scheme for expression and purification of recombinant p62 from Sf9 cells that may have general applicability for this class of glycoproteins. The purified rat p62 bound to wheat germ agglutinin, consistent with modification by O-linked N-acetylglucosamine. Carbohydrate analysis, in conjunction with amino acid analysis, revealed that baculovirus-expressed rat p62 contains 5-6 mol of N-acetylglucosamine/mol of p62. As observed by circular dichroism, purified p62 expressed in the baculovirus system or in Escherichia coli share essentially the same secondary structure. Purified glycosylated rat p62 will be critical in determining the role of N-acetylglucosamine in both nuclear transport and assembly of the nuclear pore complex.
Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
S M
SM
Bailer
W K
WK
Berlin
C M
CM
Starr
J A
JA
Hanover
D000117:Acetylglucosamine / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015394:Molecular Structure; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera
D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C049312:nuclear pore protein p62; D000117:Acetylglucosamine