Biology Reference
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ized by autoradiography. As the RNA can be synthesized in vitro from
cloned cDNA, the effect of manipulating the primary amino acid sequence on
folding and assembly can be evaluated rapidly. Here we outline the procedures
for preparing SP cells, transcribing cloned cDNAs, and translation of the
RNA transcripts generated in translation systems optimized for folding
reactions. We also describe some procedures for characterizing the product of
translation, both in terms of its incorporation into the ER of the SP cells, and
folding status.
To illustrate this approach we will describe the synthesis, translocation, and
assembly of procollagen (5 , 6) , however, it should be stressed that these tech-
niques are applicable to all proteins entering the secretory pathway. The fol-
lowing data demonstrate that when added to the SP cell translation system,
procollagen RNA can be translated into procollagen chains that are translo-
cated into the lumen of the ER and fold and assemble to form interchain disul-
fide-bonded trimers. Thus, when an exogenous protease is added to the
translation reaction after translation, all the untranslocated procollagen chains
are digested leaving only chains that have been translocated into the ER lumen
( Fig. 1A , lanes 2 and 3). The material left after protease treatment is protected
from proteolysis by being segregated within the ER as can be demonstrated by
the complete digestion of this material following disruption of the ER mem-
brane by the addition of detergent ( Fig. 1A , lane 4). To demonstrate correct
folding of the synthesized procollagen chains, a time-course of translation was
carried out and the products of translation separated by SDS-PAGE under
reducing or nonreducing conditions. Proteins containing intrachain disulfide
bonds migrate with a faster relative mobility than the fully reduced protein and
proteins forming interchain disulfide bonds have a correspondingly slower
electrophoretic mobility (7) . At early time-points, the newly synthesized
procollagen chains form intrachain disulfides indicating correct folding of the
monomeric chains ( Fig. 1B , compare lane 1 with lane 7, and lane 2 with lane 8).
These monomeric chains quickly associate to form interchain disulfide-bonded
trimers ( Fig. 1B , lanes 9-12). We have also shown, by protease resistance, that
these molecules have formed a correctly aligned triple helix (5 , 8) . Thus, the SP
cell system faithfully reproduces the initial stages in the folding and assembly
of procollagen.
2. Materials
2.1. Preparation of SP Cells
1.
HT1080 cells (75 cm 2 flask of subconfluent cells).
2.
Phosphate-buffered saline (Gibco-BRL).
3.
1X trypsin-EDTA solution (Gibco-BRL).
4.
KHM buffer: 110 m M KOAc, 2 m M MgOAc, 20 m M HEPES, pH 7.2.
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