Appeal No. 1995-4867
Application 07/993,482
31. An expression cassette for the secretion of a disulfide bond-containing
polypeptide in a biologically active, mature form from an E. coli host cell into the culture
medium, said cassette having DNA segments operably linked with each other as follows:
P--S.D.--met--L--G--T
wherein
P consists essentially of a promoter having a -35 consensus regulatory sequence
from the trp promoter and a -10 consensus regulatory sequence from the lac promoter;
S.D. consists essentially of a Cro gene Shine-Dalgarno sequence;
met consists essentially of a codon for an initiating methionine;
L consists essentially of a first DNA sequence encoding an E. coli alkaline
phosphatase signal sequence, wherein the codons for the amino acids of said signal
sequence are modified using codon degeneracy so that the nucleotides encoding said
signal sequence approximate those of the native nucleotide sequence associated with
said D.S.;
G consists essentially of a second DNA sequence encoding the mature
polypeptide; and
T consists essentially of a transcriptional termination region having a
guanine:cytosine-rich region capable of forming a stem-loop structure in the corresponding
RNA transcript, followed by a string of thymines; said S.D. being operably linked
downstream from said P and upstream from said met, said L being operably linked
downstream from said met and upstream from said G, and said T being operably linked
downstream from said G.
The references relied upon by the examiner are:
Crowl 4,582,800 Apr. 15, 1986
Maniatis et al. (Maniatis), Molecular Cloning, “A Laboratory Manual,” pp. 226-27,
408-13, 417, 422-23, 433, 507-20 (1982).
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