Characterization of the basement membrane glycoprotein entactin synthesized in a baculovirus expression system.
A cDNA clone encoding the entire open reading frame of mouse entactin was constructed. This cDNA was inserted into Autographa californica multiple nuclear polyhedrosis virus and expressed in Spodoptera frugiperda, Sf9, insect cells. The recombinant entactin was produced in large quantities under the control of the polyhedrin promoter. Amino terminus sequence analysis indicated that the signal peptide in the preprotein was correctly cleaved. Polyclonal antibodies prepared against either the naturally occurring or recombinant entactin cross-reacted with both molecules in the native and denatured states. The recombinant molecule promoted cell attachment and exhibited a calcium- and temperature-dependent self-aggregation. These properties reflect those of the naturally occurring mouse molecule. Although entactin is an extracellular matrix component that is normally exocytosed, the vast majority of the molecules were localized inside the cell by immunoelectron microscopy where the molecules formed insoluble aggregates.
Tsao
T
T
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Hsieh
J C
JC
Durkin
M E
ME
Wu
C Y
CY
Chakravarti
S
S
Dong
L J
LJ
Lewis
M
M
Chung
A E
AE
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification; D007150:Immunohistochemistry; D007304:Insect Viruses / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D015183:Restriction Mapping
D006023:Glycoproteins; D008562:Membrane Glycoproteins; C029967:nidogen