Regular Article
Expression and Purification of a Spider Silk Protein: A New Strategy for Producing Repetitive Proteins

https://doi.org/10.1006/prep.1996.0060Get rights and content

Abstract

Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner. This strategy should have general utility in the controlled construction of repetitive proteins composed of identical or different repeat units. The protein from the 16-unit repeat was produced inEscherichia coliat levels up to 10 mg/g wet wt of cells although yields of 1–2 mg/g were more typical. The protein was easily purified with high recovery using an affinity column. The purified protein had the predicted amino acid composition and N-terminal sequence after cleavage of a leader sequence. The methodology described will allow production of sufficient quantities of protein for basic structure/function studies including production of synthetic fibers.

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1

To whom correspondence should be addressed at the Department of Molecular Biology, Box 3944, University of Wyoming, Laramie, WY 82071-3944. Fax: (307) 766-5098; E-mail: [email protected].

2

Current address: Cardiovascular Research Institute, Box 0130, University of California, San Francisco, CA 94143-0130.

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