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3
Analysis of Laminin Structure and Function
with Recombinant Glycoprotein Expressed
in Insect Cells
Todd L. Mathus and Peter D. Yurchenco
1. Introduction
Recent developments in the application of eukaryotic recombinant protein
techniques have provided new tools with which to dissect and map functional
activities in basement membrane glycoproteins. This has been particularly
valuable in the case of laminins where the relationship between structure and
function has been difficult to establish. Several characteristics of the laminins
lie at the heart of the problem. First, the molecules are very large, each
assembled from three multidomain subunits joined in a long-coiled coil (
Fig. 1
).
Second, laminins bear substantial disulfide and carbohydrate modifications that
are crucial for proper conformation. Many, perhaps most, laminin activities
present in native laminin are lost upon heat- or chaotropic-denaturation. Native
activities are often not retained in short peptides or even in recombinant frag-
ments generated in prokaryotic cells. Furthermore, there is evidence to suggest
that synthetic laminin peptides can exhibit “cryptic” activities not found in
native protein.
Traditional ways of evaluating proteolytic fragments by biochemical and
ultrastructural techniques have provided useful information in the form of a
low-resolution functional map (
Fig. 1
). However, these methods are limited by
the relatively large size of active fragments (50 - 450 kDa) with a failure of
proteases to cleave at most interdomain junctions under conditions that pre-
serve native structure. Whereas prolonged proteolysis of the standard fragments
introduces additional cleavages in the peptide backbone, noncovalent interac-
tions frequently keep the peptide moieties together as a single unit. Denatur-
ation then is required to induce dissociation, a method that can kill native
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