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[21-leucine] human urogastrone, corresponding gene, corresponding recombinant plasmid, transformed cell and process for production thereof
| Details |
Inventors: Miyoshi, Kenichi; Sumi, Shinichiro; Hasegawa, Akira; Suzuki, Masanori;
Assignee: Wakunaga Seiyaku Kabushiki Kaisha (Osaka, JP)
Primary Examiner: Warren; Charles F.
Assistant Examiner: Fox; David T.
Attorney, Agent or Firm: Oblon, Fisher, Spivak, McClelland & Maier
[21-Leucine] human urogastrone, hUG, comprising a polypeptide represented by the specified amino acid sequence has been found to be equivalent to the known hUG which has 21-Met and is disclosed together with its syntheses. Double-stranded polydeoxyribonucleotides comprising a structural gene for expressing the hUG, a recombinant plasmid comprising a structural gene for expressing the hUG, and E. coli transformed by the recombinant plasmid are also disclosed. Thanks to the substitution of Met in the known hUG by Leu, the peptide of hUG can now be produced by a conventional or typical method in which a genetically engineered precursor fused polypeptide is cleaved by cyanogen bromide at Met in the fused polypeptide. |
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DETAILED DESCRIPTION The present invention is based on the confirmed findings: that a structural gene representing an hUG derivative in which the 21st amino acid residue comprises Leu which is not Met, that is, [21-L] hUG, can be chemically synthesized; that this structural gene can be inserted into an appropriate plasmid; that transformation of an appropriate host cell by this recombinant plasmid and production and recovery of [21-L] hUG by culturing of the transformed cell are possible; and that the formed [21-L] hUG has an activity similar to the activity of hUG (hEGF).
Accordingly, [21-L] hUG of the present invention is characterized in that it comprises a polypeptide represented by the following amino acid sequence formula: H-Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His-Asp-Gly-Tyr-Cys-Leu-His-Asp-Gly-V al-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-Asn-Cys-Val-Val-Gly-Tyr- Ile-Gly-Glu-Arg-Cys-Gln-Tyr-Arg-Asp-Leu-Lys-Trp-Trp-Glu-Leu-Arg-OH It should be understood that this structure is expressed as a primary structure for convenience's sake.
The process for the production of [21-L] hUG according to the present invention comprises the steps of (1) chemically synthesizing a structural gene of [21-leucine] human urogastrone corresponding to a polypeptide in which the 21st amino acid of human urogastrone is leucine, (2) inserting this gene into a plasmid vegetative in a selected host cell to form a recombinant plasmid vegetative in the host cell, (3) causing transformation of the host cell by the plasmid, and (4) culturing the transformed cell and recovering the formed [21-leucine] human urogastrone.
A typical example of the plasmid vegetative in the host cell is a plasmid capable of utilizing the expression system of a lactose operon, and a typical example of the origin of the lactose operon expression system and the host cell used for transformation is E.
coli belonging to the genus Escherichia.
Furthermore, the present invention also includes a duplex (i.
e.
a pair of complementary strands of) polydeoxyribonucleotide containing a structural gene capable of compressing [21-L] hUG, a recombinant plasmid comprising said structural gene, and E
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Molecular Biology 
