Molecular cloning and functional expression of rabbit α2-antiplasmin

Abstract

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.

Addition of a sequence from α2-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis

“…The resulting 1788 bp amplification product was restricted with XhoI and EcoRI and the inserted between these sites in pPICZ9ssamp [ 15 ] to form pPICZ9ssHSAH 6 . PCR products A, B, and C were then generated using as a template the human α 2 AP-encoding plasmid pBAD-H 6 α 2 AP [ 29 ]. Codons 13–42 were mobilized using oligonucleotides ML 17226 (5'-ACGTCTCGAGA AAAAGAAACC AGGAGCAGGT GTCCCC-3') and ML 17225 (5'-ACGTGGTAC CGACTCCTGG GGGACTCTTC AG-3') to form product A. Codons 13–73 were obtained using ML 17226 and ML 17227 (5'-AGCTGGTACCC CTGGTGAGCC ACCAGGGAGA AC-3') in product B. Codons 13–109 were amplified using ML 17226 and ML 17228 (5'-AGCTGGTACC CCCAGCACCT TTGCAGCCTC TG-3') to yield product C. The HSA cDNA was then PCR-amplified with oligonucleotides ML 12006 (5'-CATGCGGTAC CACAAGAGTG AGGTTGCTC-3') and ML 12007 (5'-GTTGCTGCAA GTCAGGCTGC CTTAGGCTTA CACCATCACC ATCACCATTA AGAATTCCAT G-3') to produce fragment D. A, B, and C were restricted with XhoI and KpnI and D with KpnI and EcoRI.…”

Retention of thrombin inhibitory activity by recombinant serpins expressed as integral membrane proteins tethered to the surface of mammalian cells

“…Sequences encoding Met‐Gly followed by codons 3–69 of the human asialoglycoprotein receptor (ASGPR H1 subunit, or AR) or Met‐Gly followed by codons 3–98 of the human transferrin receptor (TR) were obtained by RT‐PCR of RNA isolated from the human hepatoma cell line HepG2, as previously described [13]. A single reaction for RT‐PCR was carried out for each sequence, with a OneStep RT‐PCR kit used according to the manufacturer’s instructions (Qiagen, Mississauga, Canada), and the following primer pairs: for AR, ML 18459 and ML 18458; and for TR, ML 18457 and ML 18460.…”

Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

“…A final expression plasmid was constructed to reverse the orientation of the His tag of fusion protein α 2 AP(13-73)-HSAH 6 [19]. Plasmid pBAD-H 6 -α 2 AP [27] was PCR-amplified using oligonucleotides ML 09-3874 and 17225 [27], and the 125 bp XhoI-KpnI restriction fragment of the resulting PCR product was inserted between the corresponding sites of pPICZ9ss-α 2 AP(13-73)-HSA to yield pPICZ9ss-H 6 α 2 AP(13-42)-HSA. Each completed plasmid listed above was used to transform Pichia pastoris strain X33 to Zeocin (Invitrogen) resistance as previously described [18].…”