High level expression of the deleted and mutated urokinase-scFv fusion gene in Escherichia coli.
The fusion gene of a specific anti-human fibrinogen D-dimer scFv and low molecular weight single chain urokinase (scu-PA-32K) was restricted, spliced, and digested with exonuclease Bal31 to obtain a series of deletion mutants. Study of their expressions in E.coli revealed that the key sequence which reduced its expression level resided in the fragment from 841 to 851 bp, in which a tandem of AGG codons (encoding arginine, rarely used in E.coli) existed. By means of PCR mediated site-directed mutagenesis, we altered these two AGG codons to CGT codons, which could be more efficiently translated in E.coli, and the expression level turned out to be about 30% of the total bacterial proteins while that of the natural gene was only 2%-3%.
Wang
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X
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, China.
Yu
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W
D001483:Base Sequence; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005340:Fibrinogen / Q000276:immunology*; D017353:Gene Deletion; D015870:Gene Expression; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen; D014568:Urokinase-Type Plasminogen Activator