Analysis of Laminin Structure and Function with Recombinant Glycoprotein Expressed in Insect Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 29

isomerization can be found in those proteins that are secreted. However, it
should be realized that the N-linked carbohydrate adducts are of a simple man-
nose-rich type rather than the complex-type found in mammalian glycoproteins
(8) . Methodological details are described in detail ( see Subheading 3.1. ).
Mammalian Expression: The growing number of commercially available
vectors with high-efficiency promoters has made it possible to express reason-
able amounts of recombinant laminins and other basement membrane glyco-
proteins (typically ~1 g/mL). Vectors driven by the CMV (cytomegalovirus)

promoter have been used to express nidogen and laminin, both intact and frag-
ments (1-3 , 7) . Laminin and laminin fragments expressed in this manner contain
complex N-linked oligosaccharide and have been found to have native func-
tion. In essence, this progress has opened the door for the future of the study of
laminin structural-functional relationships by demonstrating the potential for
purifying to homogeneity recombinant laminins with point-specific mutations,
alterations, or truncations that will perhaps be attributable to disease.

µ

1.2. Strategy for Designing
and Generating Laminin Recombinants
It is thought that the domains within a multidomain protein fold largely
independently of each other. Because of concern that partial domains or motifs
may fail to fold properly and may be more susceptible to degradation, we
encourage a consideration of the known or predicted domain structure to guide
construct design. Additionally, the choice of expression system used to pro-
duce a recombinant should be determined by the purpose for creating the
recombinant; that is, why should this recombinant be made and/or what will it
be used for? Consider the following when choosing an expression system to
make any recombinant: level of expression and yield, time requirements, ease
of manipulating the system, costs, and end use of the recombinant. Bear in
mind that this is not an all-inclusive list of factors that should be considered.
We outline below, detailed protocols for the production of laminin G domain
recombinants in insect cells using the baculovirus expression system. This sys-
tem has been very useful in our laboratory and satisfies the majority of the
above criteria.

2. Materials
2.1. Baculovirus Expression System: Generation and Purification
of Recombinant Glycoproteins
1. Spodoptera frugiperda cells (Invitrogen, San Diego, CA; B825-01) ( see Note 2 ).

2. pVL1393 baculovirus expression vector (Invitrogen; V1392-20) ( see Note 3 ).

3. BaculoGold Transfection Kit (Pharmingen, 21100K) ( see Note 4 ).