Patent ID: 6194200
Filing Date: 2001-02-27
Classification: C07K,C12N

Abstract:
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--TwhereinP 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 S.D.;G consists essentially of a second DNA sequence encoding the mature polypeptide; andT 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.