Amgen, Inc., DL Hare, Amgen, Inc.
produces protein epidermal growth factor urogastrone
DESCRIPTION OF VECTOR COMPONENT:
Name of vector: unknown
Intact vector size:
Type of vector: plasmid
Vector end: EcoRI
Vector end: SalI
Host range: Escherichia coli
Features (with orientation and position when available):
DESCRIPTION OF INSERT COMPONENT:
Gene symbol: EGF
Genomic copy number: unique
Gene name: epidermal growth factor
Contains complete coding sequence?: U
Chromosome: 4; Localization: 4 q25
Type of DNA: synthetic
Insert 5' end: EcoRI
Insert 3' end: SalI
Insert size (kb): 1.75
Insert lengths(kb): 1.75
Gene product: epidermal growth factor(urogastrone) [EGF]
Alleles: A1, B1, B2, A2
Target Gene: epidermal growth factor
Restriction digests of the clone give the following sizes (kb): EcoRI/SalI--3.3, 1.75; EcoRI--5.1; SalI--5.1; BamHI--4.9, 0.2.
Runaway vector contains a temperature-sensitive mutation in the copy-control gene. Maintained at a low copy number below 34C; replication increases 50-fold at temperatures greater than 34C. Growth at 37C is lethal.
The 1.75 kb EcoRI/SalI fragment contains a trp promoter/regulator and the EGF/urogastrone coding region. The complete coding sequence of the mature peptide, plus a "pro" sequence of 8 aa, is contained in a 0.2 kb XbaI/SalI fragment.
Directs the synthesis of a peptide of 61 aa, including an 8-residue "leader" sequence.
Expresses the urogastrone analog product on the order of 50 mg per O. D. liter as determined by SDS-PAGE. The microbially expressed product is biologically active.
Constructed from oligonucleotides selected to reflect the codon usage of Escherichia coli. Contains a HinfI site interrupting Asp3, an SphI site interrupting Cys31, and an NruI site after Tyr44.
Banks AR, Hare DL. Manufacture and expression of genes for urogastrone and polypeptide analogs thereof. US Patent 4,764,593 dated Aug 16 1988