Abstract
Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.
Highlights
Full length cDNAs coding for a 14-kDa&galactoside binding lectin have been isolated from HL-60 cells and human placenta
We report here the preliminary characterization of the purified protein from HL-60 cells, the first cloning and sequencing of full length cDNA clones corresponding to a 14-kDa human lectin from both HL-60 cells and human placenta, and the expression of active recombinant lectin in Escherichia coli
Taking into account ethxetensive amino acid sequence homologies observed previously between some animal lectins [4] we designed two 14-mer oligonucleotide probes, LP59 and LP60, as described under “ExperimentalProcedures.” Screening about lo6 recombinant phages with the LP59 probe, we identified and plaque-purified 17 positive clones
Summary
Placenta extracts trophoresis, electroblotting onto apolyvinylidinedifluoride purified on lactose-Sepharose showed a major 14-kDa species membrane, stainingwith Coomassie Blue, and sequencing the and significantlymore of a 30-kDaspecies thanseen in stained bands [15].Because the amino terminus of the 14-. Taking into account ethxetensive amino acid sequence homologies observed previously between some animal lectins [4] we designed two 14-mer oligonucleotide probes, LP59 and LP60, as described under “ExperimentalProcedures.” Screening about lo recombinant phages with the LP59 probe, we identified and plaque-purified 17 positive clones. The ATG start codon fit Kozak’s criteria for effective initiation [24] Both the5’- and 3”untranslated regions were unusually short (50 and 53 base pairs, respectively), but of aboutthesame size as the corresponding sequences of the chicken and rat lectin cDNAs[25, 26]. The longest clone, when compared to the HL-60 clone 11,was missing the five terminal nucleotides of the 5’untranslated region and continued3‘ to include the sequence used to prime thceDNA synthesis
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