Purification and characterization of the trefoil peptide human spasmolytic polypeptide (hSP) produced in yeast

FEBS Lett. 1993 Mar 8;318(3):345-52. doi: 10.1016/0014-5793(93)80543-4.


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.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / analysis
  • Base Sequence
  • Carbohydrates / analysis
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • DNA / genetics
  • Glycosylation
  • Growth Substances / chemistry
  • Growth Substances / genetics
  • Humans
  • Intercellular Signaling Peptides and Proteins
  • Mass Spectrometry
  • Molecular Sequence Data
  • Mucins*
  • Muscle Proteins*
  • Neuropeptides*
  • Peptide Mapping
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / isolation & purification*
  • Plasmids
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Trefoil Factor-2
  • Trefoil Factor-3


  • Amino Acids
  • Carbohydrates
  • Growth Substances
  • Intercellular Signaling Peptides and Proteins
  • Mucins
  • Muscle Proteins
  • Neuropeptides
  • Peptides
  • Recombinant Fusion Proteins
  • TFF3 protein, rat
  • Tff2 protein, rat
  • Trefoil Factor-2
  • Trefoil Factor-3
  • pancreatic spasmolytic polypeptide
  • DNA