High expression vectors for the production of recombinant single-chain urinary plasminogen activator from Escherichia coli.
An expression cassette containing a synonymous gene for human single-chain urokinase-type plasminogen activator (Rscu-PA) 5'-flanked by a trp promoter and the Shine-Dalgarno sequence of the xyl A operon of Bacillus subtilis and terminated by the terminators trp A and Tn10 was constructed and inserted into a pBR322 derivative to yield pBF160. When compared to pUK54 trp 207-1 containing the natural scu-PA gene without the Shine-Dalgarno sequence and terminator, the expression efficiency of pBF160 in Escherichia coli strains was improved by one order of magnitude. Replacement of the trp by the tac promoter (pBF171) did not affect expression. Inserting the Shine-Dalgarno sequence and Tn10 terminator into pUK54 trp 207-1 (pWH1320) slightly increased the expression level, whereas elimination of the Shine-Dalgarno sequence and the terminators from pBF160 with almost complete conservation of the synonymous structural gene (pBF191) significantly reduced the expression. Variation of the distance between the Shine-Dalgarno sequence and the start codon between 8 and 10 bp (pBF163) proved irrelevant. In conclusion, poor expression of mammalian genes in E. coli may result from both improperly designed regulatory elements and structural features of the coding region and therefore de-novo synthesis of the gene may be required to obtain satisfactory expression.
Grünenthal GmbH, Centre of Research, Aachen, Federal Republic of Germany.
R
R
Brigelius-Flohé
G
G
Steffens
W
W
Strassburger
L
L
Flohé
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D003062:Codon; D014568:Urokinase-Type Plasminogen Activator