Molecular cloning and functional expression of rabbit alpha2-antiplasmin.
The rabbit is frequently employed in small animal models of in-vivo coagulation and fibrinolysis, but the degree to which its plasma proteins resemble their human counterparts is incompletely known. Our aims were: to determine the nucleotide sequence of a full-length rabbit liver alpha(2)-antiplasmin (alpha(2)AP) cDNA; compare it with its human, murine, and bovine counterparts; and express it in functional form. Partial cDNAs encoding 5' and 3' portions of the alpha(2)AP mRNA were obtained by reverse transcriptase (RT)-polymerase chain reaction (PCR), using rabbit liver RNA and 'guessmer' oligonucleotides based on known sequences. This information was employed to design additional oligonucleotides for RT-PCR and rapid amplification of cDNA ends (RACE) procedures yielding overlapping clones corresponding to the entire rabbit alpha(2)AP mRNA sequence. Mature alpha(2)AP was expressed as a hexahistidine-tagged recombinant protein in Escherichia coli, purified by nickel-chelate affinity and ion exchange chromatography, and its reaction with plasmin and soluble fibrin assessed electrophoretically and compared with an analogous recombinant human alpha(2)AP. The consensus rabbit alpha(2)AP cDNA is 2,157 bp long, and encodes a 464-residue mature sequence and a 27-amino-acid presequence. The mature protein is 82% identical to its human counterpart, and its recombinant form produced denaturation-resistant complexes with both plasmin and fibrin. The region of greatest sequence divergence lies within the C-terminal extension of alpha(2)AP, unique in the serpin family of proteins to which alpha(2)AP belongs. The highly conserved nature of rabbit alpha(2)AP reinforces its role as a vital antifibrinolytic protein and supports fibrinolytic investigations in models employing this small animal.
Jobse
Brian N
BN
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
Sutherland
Jason S
JS
Vaz
Darryl
D
Bhakta
Varsha
V
Sheffield
William P
WP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D000979:alpha-2-Antiplasmin / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin