Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli.
A method for enhancing the secretion of heterologous proteins in Escherichia coli by optimizing, as opposed to simply maximizing, the translational level of a given protein is described. Random alteration of the translational initiation region (TIR) of the Heat-Stable Enterotoxin II (STII) signal sequence resulted in a library of vectors with varied translational strengths. Subsequent screening of this library using E. coli alkaline phosphatase as a reporter led to the selection of several TIR variants covering a 10-fold range of translational strength. These TIR variants, in combination with several previously generated variants, are shown to dramatically improve the secretion of a number of heterologous proteins. In fact, the heterologous proteins tested required a narrow translational range for optimal high-level secretion into the periplasm. Interestingly, the secretion of two native E. coli proteins was unaffected by TIR strength when tested over an identical range. The dependence of secretion on a narrow translational level demonstrates its critical role in the secretion of heterologous proteins in E. coli.
Simmons
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LC
Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080, USA. simmons@gene.com
Yansura
D G
DG
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000096:biosynthesis; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D004269:DNA, Bacterial / Q000235:genetics; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017930:Genes, Reporter; D014644:Genetic Variation; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D033903:Periplasmic Proteins; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; C039076:Eco protein, E coli; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; C034151:heat stable toxin (E coli); D000469:Alkaline Phosphatase