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Molecular cloning and expression of rabbit antithrombin III
WP Sheffield, AB Brothers, MJ Wells, MW Hatton, BJ Clarke and MA Blajchman
Canadian Red Cross Blood Transfusion Service, McMaster University,
Hamilton, Ontario, Canada.
A cDNA containing the complete open-reading frame encoding rabbit
antithrombin III (AT-III) was isolated from a rabbit liver cDNA expression
library, using a specific antibody as a probe. Sequence analysis showed 84%
identity between the deduced amino acid sequences of the rabbit and human
proteins. A previously described cell-free expression system was used to
verify the identity of the clone. The full-length cDNA was inserted into an
expression vector, and messenger RNA (mRNA) transcripts generated. In vitro
translation of these transcripts, in the presence of [35S]methionine, in an
mRNA-dependent rabbit reticulocyte lysate system resulted in the synthesis
of a 51-Kd polypeptide, as shown by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). This nonglycosylated protein was capable of
forming SDS-stable complexes with human alpha-thrombin. Complex formation
was significantly enhanced following the deletion of nucleotides encoding
the signal peptide, and the resultant generation of a 47-Kd nonglycosylated
mature protein product. When the template DNA giving rise to this product
was internally truncated, two rabbit AT- III deletion mutants were
generated that lacked the ability to interact with thrombin, but retained
the ability to bind heparin. Cell-free expression plasmids encoding the
human and rabbit AT-III mature molecules were manipulated to produce two
interspecies fusion proteins. For the first, human codons were used to
replace rabbit codons from residue 369-433, while in the second human
codons replaced rabbit codons from residue 217-433. Both fusion proteins
exhibited less efficient thrombin-complexing ability than the original
cell-free- derived mature rabbit AT-III. Thus, portions of AT-III molecules
from the two species, despite their high degree of homology, are not
interchangeable. Knowledge of the structure of rabbit AT-III, combined with
the availability of the rabbit cDNA, will permit defined experimentation
aimed at understanding antithrombin III structure relative to its function
in vivo.
Volume 79,
Issue 9,
pp. 2330-2339,
05/01/1992
Copyright © 1992 by The American Society of Hematology
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