Abstract
Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo.
Highlights
From the Department of $Cellularand Molecular Biology and SBiologicalChemistry, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486
Two short peptides from V8 digests of the native leech antiplatelet protein (LAPP) Recently, a tetrapeptide was described that blocks platelet were used as primers to generatea polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides
In the preceding paper (11)we describe the identification and isolation of a protein, LAPP,’ from Haementeria officinalis leeches that
Summary
1:; :-; screen the cDNA library in order to eliminate any possible sequenceerrors.,the Xgt cDNAexpression library previously constructed from poly(A)+RNA isolated. GCC CAC CAI WA C A I GCC GCI 5i;l GCC GrJ GAT from leech salivary glands (12)was screened for the presence of LAPP cDNAs using the probe generatebdy PCR amplification with primer pair1,2c. Screening by hybridization using this probe initially identified numerous positipvleaques. Ten plaques were isolated from the screeningof the library. DNA isolated from these clones was digested with the restriction endonucleases Not and SalI and electrophoresed in a 1.5% agarose gel. Inserts sized about 0.7 kb were seen in fourof the 10 clones, theothersix gave smallerorno visible bands. Subequent subcloning, sequencing, and translation of two of the 0.7-kb cDNAinserts revealed anuninterruptedopen reading frame of 441 nucleotides (Fig. 3).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
