Purification and characterization of the trefoil peptide human spasmolytic polypeptide (hSP) produced in yeast.
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.
Thim
L
L
Department of Protein Chemistry, Pharmaceuticals Research, Novo Nordisk, Novo Alle, Bagsvaerd, Denmark.
Norris
K
K
Norris
F
F
Nielsen
P F
PF
Bjørn
S E
SE
Christensen
M
M
Petersen
J
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006031:Glycosylation; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3
D000596:Amino Acids; D002241:Carbohydrates; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009077:Mucins; D009124:Muscle Proteins; D009479:Neuropeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C099037:TFF3 protein, rat; C000604008:Tff2 protein, rat; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3; C034407:pancreatic spasmolytic polypeptide; D004247:DNA