Access

You are not currently logged in.

Access your personal account or get JSTOR access through your library or other institution:

login

Log in to your personal account or through your institution.

If You Use a Screen Reader

This content is available through Read Online (Free) program, which relies on page scans. Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.

Combinatorial Mutagenesis to Restrict Amino Acid Usage in an Enzyme to a Reduced Set

Satoshi Akanuma, Takanori Kigawa and Shigeyuki Yokoyama
Proceedings of the National Academy of Sciences of the United States of America
Vol. 99, No. 21 (Oct. 15, 2002), pp. 13549-13553
Stable URL: http://www.jstor.org/stable/3073444
Page Count: 5
  • Read Online (Free)
  • Subscribe ($19.50)
  • Cite this Item
Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Combinatorial Mutagenesis to Restrict Amino Acid Usage in an Enzyme to a Reduced Set
Preview not available

Abstract

We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function. The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection. The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared. Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid. The converged sequence also implies simpler constituents for proteins in the early stage of evolution.

Page Thumbnails

  • Thumbnail: Page 
[13549]
    [13549]
  • Thumbnail: Page 
13550
    13550
  • Thumbnail: Page 
13551
    13551
  • Thumbnail: Page 
13552
    13552
  • Thumbnail: Page 
13553
    13553