Biology Reference
In-Depth Information
activity. One example of this is found in fragment E1', a large N-terminal frag-
ment containing the complete
α
1 and
γ
1 short arms and a proximal portion of
the
1-chain domain VI, an N-terminal globule, bears an
elastase cleavage site near the domain VI/V junction but remains noncovalently
bound to the rest of the fragment. This fragment (E35) can be dissociated in 6
M
urea; however, the resulting fragment can no longer bind to the
β
1 short arm. The
α
1 integrin
(2)
.
We have recently found that eukaryotic recombinant approaches, applying
DNA technology to protein chemistry, are effective means to address issues of
tertiary structure preservation. In recent years, we have produced functionally
active recombinant laminin and laminin fragments using insect and mamma-
lian expression systems
(2
,
4-8)
. In addition, we have found that laminin frag-
ments generated in prokaryotes, while generally not functionally active, can
sometimes be useful for the preparation of specific antigens for antibody gen-
eration or characterization (
see
Note 1
). Here we present selected protocols
that we have employed for the generation of laminin G domain recombinants
using the baculovirus expression system, as well as highlighting advantages
and disadvantages of using this and other systems for the generation of recom-
binants. We have also attempted to guide the reader through lessons learned
through our trials and tribulations.
α
1
β
1.1. Choosing a Recombinant Expression System
Bacterial Expression:
Prokaryotic recombinant expression systems have
long been used to generate proteins and protein fragments for analysis. How-
ever, these systems have more limited useful application in the case of extra-
cellular matrix molecules whose carbohydrate and disulfide-bonding may be
critical for structure and function. Our experience with recombinant laminin
fragments generated in
Escherichia coli
is that they tend to be associated with
inclusion bodies and are insoluble after bacterial lysis. Whereas the recombi-
nant fragment can often be solubilized with urea or guanidine-HCl, the result-
ing solution tends to be one of inactive and, apparently, incorrectly folded
protein. Furthermore, there is a tendency for the recombinant protein to
reprecipitate when the protein is dialyzed into urea- or guanidine-free buffer.
Baculovirus Expression:
We have had considerable success using the
baculovirus expression system in insect cells to generate recombinant C-termi-
nal laminin fragments
(4-6
,
8)
. However, baculovirus generation of N-terminal
fragments, however, has been problematic for us for unclear reasons. The
C-terminal fragments have been relatively straightforward to purify with gen-
eration of up to several milligrams per liter of conditioned medium. These pro-
teins have been useful to generate function-blocking antibodies that react with
native protein with the recombinant protein capable of exhibiting normal bio-
logical activity. Post-translational modifications of glycosylation and disulfide
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