Process for the preparation of motilin-like polypeptide and expression thereof

A process for the preparation of a polypeptide of formula (I) Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-X-Gln-Glu-Lys-Glu-Arg-Asn- Lys-Gly-Gln wherein X is an amino acids residue other than Met or Asp and which method consists essentially of the following steps:

• (a) synthesizing a single-stranded DNA fragment encoding a leader polypeptide of the formula (II) Met-Thr-Met-Ile-Thr-Asn-Ser-Asn-Gln-Asn-Gln-Asn-Gln-Asn-Gln-Asn-Gln-Asn-Gl n-Ile-Phe-Met and synthesizing its complementary single-stranded fragment;
• (b) annealing the single-stranded fragments encoding the leader polypeptide made in step (a) to form a first double stranded DNA;
• (c) synthesizing a single-stranded DNA fragment encoding a polypeptide of said formula I but with sequence Asp-Gly-Ile-Leu at the C-terminus and synthesizing its complementary single-stranded DNA fragment;
• (d) annealing the single stranded DNA fragments encoding the polypeptide of formula (I) made in step (c) to form a second double-stranded DNA;
• (e) ligating said first and second double-stranded DNAs so that the first and second double-stranded DNAs are in tandem arrangement;
• (f) adding a recognition site for a specific restriction enzyme to both ends of the double stranded DNA made in step (e);
• (g) cleaving a plasmid with said restriction enzyme;
• (h) ligating the double-stranded DNA made in step (f) with the cleaved plasmid from step (g) to construct a recombinant plasmid;
• (i) transforming a prokaryotic organism with said recombinant plasmid;
• (j) cultivating the transformed prokaryotic organism such that a polypeptide is produced comprising the leader polypeptide of formula (II) fused at the N-terminus of the polypeptide of formula (I) having the sequence Asp-Gly-Ile-Leu at its C-terminus;
• (k) lysing the transformed prokaryotic organisms after the cultivation step and treating with cyanogen bromide and endopeptidase to separate the leader polypeptide from the polypeptide of formula (I);
• (l) fractionating the resulting mixture to purify the polypeptide of formula (I).