Expanding the DNA-recognition repertoire for zinc finger proteins beyond 20 amino acids

Journal of the American Chemical Society

Volumn 125, Issue 17, 2003, Pages 4960-4961

  Jantz, Derek ^a   Berg, Jeremy M ^a  

^a JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACID; DNA; DNA BINDING PROTEIN; NUCLEIC ACID BINDING PROTEIN; ZINC;

ARTICLE; CRYSTALLOGRAPHY; DNA BINDING; PROTEIN BINDING; PROTEIN INTERACTION; PROTEIN STRUCTURE; STRUCTURE ANALYSIS;

EID: 0037473557     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja029671i     Document Type: Article

References (24)

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18. d for the protein-unlabeled probe complex could be determined. By carrying out 12 back-titrations in parallel using each of the point variants in the 5′-GAG triplet, the specificities conferred by each of the proteins' C-terminal fingers could be determined quantitatively.
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20. The C-terminal 23 amino acids of QNK-QDK-RHX (where X is Arg, Gln, or Cit) were synthesized using standard Fmoc chemistry. The N-terminal amino acid of this peptide was the second metal-liganding cysteine in the third finger to allow for subsequent ligation. All peptides were purified by reversed-phase HPLC and confirmed by MALDI mass spectrometry prior to ligation.
21. Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6705-10.
22. (b) A region encoding amino acids 1-65 of QNK-QDK-RHR, corresponding to the N-terminal two fingers, was cloned into the PTYB2 vector (New England Biolabs) for expression as a C-terminal intein fusion. Glu65 was mutated to a glycine to avoid the possibility of racemization at the point of ligation, and a C-terminal His-tag was cloned in place of the chitin-binding domain in PTYB2 to improve expression. The fusion protein was expressed in BL21 cells and purified over a nickel column. Following elution with imidazole, purified finger 3 peptide was added in the presence of 200 mM mercaptoethanesulfonic acid, and the ligation reaction was allowed to proceed overnight at room temperature. Ligated three-finger protein was then purified by reversed-phase HPLC and quantitated spectrophotometrically. All three proteins used in this report were made in this semisynthetic fashion.
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