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note
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Loci slr0473 and slr0474 were amplified by PCR with purified Synechocystis sp. PCC6803 genomic DNA, both individually and as an operon, with primers that allowed them to be cloned into the pASK75B expression vector (23). Expression of Strep-Tagged fusions of Cph1 and Rcp1 in E. coli strain DH5α was done according to the manufacturer's instructions (Biometra Inc.).
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17
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15144358688
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note
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2-terminal residues 1 to 514. The PCR product was cloned into pASK75B and the Strep-Tagged fusion protein N514 was expressed in E. coli (see 10).
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18
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15144345705
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Unpublished data
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Unpublished data.
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15144341433
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note
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Maltose binding protein (MBP) fusions with Strep-Tagged wild type (WT) and D68A mutant of Rcp1, generated by site-specific mutagenesis (24), were obtained by subcloning into the Bam HI site of pMAL-c2, expressed in E. coli, and purified according to the vector manufacturer's instructions (New England BioLabs). Strep-Tagged Cph1 and N514 mutant (10, 12) were purified with a homemade streptavidin-Sepharose matrix (25).
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24
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0028596145
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M. Perego et al., Cell 79, 1047 (1994).
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Hua, J.1
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35
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15144348258
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note
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, He; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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39
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15144344345
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note
-
32P]ATP (8000 cpm/pmol), 2.4 μg of Cph-PCB adduct or 1 μg of N514-PCB adduct in Pr or Pfr form, and 2 μg of MBP-Rcp1 (WT and D68A). Reactions were initiated by adding ATP, mixtures were incubated 30 min at 30°C, and reactions were stopped by adding SDS sample buffer (4).
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40
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15144348020
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note
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We are indebted to E. Campbell for providing us with purified Synechocystis DNA, K. C. McFarland for figure design, A. Skerra for core streptavidin, and D. Kehoe for critical review of the manuscript. Supported in part by National Science Foundation grant MCB-9604511 to J.C.L. and NIH National Center for Research Resources award 1 P41 RR06009 to the Pittsburgh Supercomputing Center.
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