Peptide production by a combination of gene expression, chemical synthesis, and protease-catalyzed conversion.
We describe a new approach for the production of peptides using a combination of recombinant DNA technology, chemical synthesis, and proteinase-catalyzed processing. An artificial substance P-precursor is produced as a beta-galactosidase (1-459) fusion protein containing nine copies of the decapeptide sequence Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe. The fusion protein accumulates in E. coli as insoluble inclusion bodies which are easily isolated and purified. The decapeptide blocks are selectively cleaved from the insoluble fusion protein by alpha-chymotrypsin. Alternatively, a dodecapeptide ester is produced when a dipeptide ester is included in the chymotrypsin reaction mixture. This peptide ester is converted converted to substance P by papain-catalyzed acyl transfer and subsequent tryptic cleavage. These results demonstrate that peptides can be readily produced by a combination of recombinant DNA technology and proteinase-catalyzed conversion. The approach allows incorporation of groups other than natural amino acids into oligo- and polypeptides.
Schellenberger
V
V
Institute for Molecular Genetics, Georg August University, Göttingen, Germany.
Pompejus
M
M
Fritz
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002918:Chymotrypsin / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D010452:Peptide Biosynthesis; D010446:Peptide Fragments / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013373:Substance P / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000235:genetics*; D014357:Trypsin / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D004274:DNA, Recombinant; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013373:Substance P; C036877:substance P (1-7); D001616:beta-Galactosidase; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain