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  • Title: Purification and characterization of the trefoil peptide human spasmolytic polypeptide (hSP) produced in yeast.
    Author: Thim L, Norris K, Norris F, Nielsen PF, Bjørn SE, Christensen M, Petersen J.
    Journal: FEBS Lett; 1993 Mar 08; 318(3):345-52. PubMed ID: 8440393.
    Abstract:
    Recombinant human spasmolytic polypeptide (r-hSP) has been produced in relatively large amounts in Saccharomyces cerevisiae. The two intronless trefoil domains of the hSP-DNA were cloned separately by PCR from human genomic DNA, and the remaining parts of the gene synthesized. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the hSP sequence. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Kex 2 processing enzyme system. The secreted r-hSP was found in a glycosylated and an non-glycosylated form. The two forms of r-hSP were purified from the yeast fermentation broth by a combination of ion-exchange chromatography and preparative HPLC. The overall yield from 8 litres of fermentation broth was 160 mg r-hSP and 219 mg glycosylated r-hSP corresponding to 50% and 34%, respectively. The structure of the r-hSP and the glycosylated r-hSP was determined by amino acid analysis and carbohydrate composition analysis as well as by peptide mapping, amino acid sequencing and mass spectrometric analysis.
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