Expression in a cell-free system of normal and variant forms of human
antithrombin III. Ability to bind heparin and react with alpha-thrombin
RC Austin, RA Rachubinski, F Fernandez-Rachubinski and MA Blajchman
Canadian Red Cross Blood Transfusion Service, McMaster University,
Hamilton, Ontario, Canada.
Human antithrombin III (AT-III) cDNA was cloned into the cell-free
expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was
used to remove nucleotides encoding the signal peptide. AT-III messenger
RNA (mRNA) transcripts derived from this construct were translated in an
mRNA-dependent rabbit reticulocyte lysate (RRL) system containing
(35S)methionine. Immunoprecipitation of the cell-free translation mixture
with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide
which is the non-glycosylated mature form of plasma AT-III. Densitometric
scanning showed that this polypeptide constitutes greater than 90% of the
radiolabeled polypeptides produced in this system. Heparin-Sepharose
chromatography resulted in the elution of cell-free derived AT-III as a
broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III
also reacted with human alpha- thrombin. In 2 minutes approximately 20% of
the AT-III was found associated with a higher molecular weight species,
consistent with the formation of a 1:1 stoichiometric covalent complex
between alpha- thrombin and AT-III. Unfractionated heparin accelerated the
rate of formation of such complexes. When Ser394 was mutated to Leu to form
the AT-III Denver mutant, the cell-free translation product of this
mutation did not show any significant complex formation when reacted with
alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only
the putative thrombin-binding domain, was synthesized independently. This
21-Kd polypeptide did not bind heparin; however, it was cleaved by
alpha-thrombin presumably at the reactive center Arg393- Ser394. When
Ser394 was mutated to Leu the cell-free translation product of this
truncated AT-III mutation did not react with alpha- thrombin at the
reactive center. This simple cell-free approach, along with site-directed
mutagenesis, should allow for the rapid and accurate mapping of the
functional domains of human AT-III.
Volume 76,
Issue 8,
pp. 1521-1529,
10/15/1990
Copyright © 1990 by The American Society of Hematology
