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4) because of the steeper temperature dependence of the uncatalyzed reaction.
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7144255201
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note
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f using the PCR with template pKMCMT-W and primers SEQT-S (5′-GTGAGCG-GATAACAATTTCACA-3′) and MJMA-N (5′-TCAG-CAATAAGCTTTTCSNNSNNSNNSNNSNNSNNC-TTAAGGATCTTATTGTCAATCTCATCAATCT- 3′) (N = A, C, G, or T; S = C or G). Cloning of this library of products into pKMCMT-NEG with restriction enzymes Nde I and Hind III gave library pKMCMT-LibA.
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22
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0029999832
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Jiang, N.3
Hilvert, D.4
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7144251548
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note
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The host strain for protein production was KA13, a CM-deficient E. coli with an isopropyl β-D-thiogalactopyranoside-inducible T7 RNA polymerase gene integrated into the chromosome.
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25
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0002498995
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S. E. Harding, A. J. Rowe, J. C. Horton, Eds. Royal Society of Chemistry, Cambridge, England
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Sedimentation equilibrium runs were performed from 3000 to 17,000 revolutions per minute with 2, 10, and 30 μM protein concentrations in phosphate-buffered saline (PBS) at 20°C. Partial specific volumes and solvent densities were calculated with the program Sednterp [T. M. Laue, B. D. Shah, T. M. Ridgeway, S. L. Pelletier, in Analytical Ultracentrifugation in Biochemistry and Polymer Science, S. E. Harding, A. J. Rowe, J. C. Horton, Eds. (Royal Society of Chemistry, Cambridge, England, 1992), pp. 90-125] and data were analyzed by nonlinear least-squares fitting with the Origin software provided by Beckman.
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(1992)
Analytical Ultracentrifugation in Biochemistry and Polymer Science
, pp. 90-125
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Laue, T.M.1
Shah, B.D.2
Ridgeway, T.M.3
Pelletier, S.L.4
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29
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7144259428
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Supplementary material
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Supplementary material is available at www. sciencemag.org/feature/data/976438.shl.
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7144228976
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note
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m were calculated from the initial rates.
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32
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7144254826
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note
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Removing six amino acids from the carboxyl terminus of MjCM increased its resistance to proteolytic cleavage without affecting the properties of the protein. Consequently, all detailed biochemical and biophysical characterizations of mMjCM were also carried out on a truncated derivative, which behaved identically to the full-length mMjCM in all aspects tested.
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33
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7144258198
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note
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We thank H. Lashuel and J. Kelly for analytical ultra-centrifugation studies. G.M. is the recipient of a Natural Sciences and Engineering Research Council of Canada 1967 Centennial Postgraduate Scholarship and an Eli Lilly Graduate Student Fellowship. Supported by the Skaggs Institute for Chemical Biology at The Scripps Research Institute.
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