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Alkaline Phosphatase Fusions: A Tag to Identify Mutations that Result in Increased Expression of Secreted Human Growth Hormone from Escherichia coli

Bio/Technology volume 4, pages 51–55 (1986)Cite this article

Abstract

We describe a method for the mutagenesis of a signal sequence, and the identification of mutations leading to the enhanced expression of periplasmicly secreted human growth hormone (HGH) from Escherichia coli. A plasmid was engineered, using deoxyoligonucleotide directed site specific mutagenesis, to contain two unique restriction sites flanking the signal sequence for HGH. The signal was mutagenized specifically using nitrous acid. Mutants resulting in enhanced expression of secreted HGH were detected by expressing and secreting a human growth hormone–alkaline phosphatase fusion protein possessing phosphatase activity. Such fusions show activity only when secreted. The signal sequence mutations isolated were transferred into plasmids directing the expression and secretion of native HGH and the phenotypes of enhanced expression of secreted protein were largely preserved.

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Authors and Affiliations

  1. Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California, 94080

    Mark Matteucci & Holly Lipetsky

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  1. Mark Matteucci
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  2. Holly Lipetsky
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Matteucci, M., Lipetsky, H. Alkaline Phosphatase Fusions: A Tag to Identify Mutations that Result in Increased Expression of Secreted Human Growth Hormone from Escherichia coli. Nat Biotechnol 4, 51–55 (1986). https://doi.org/10.1038/nbt0186-51

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