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Organic & Biomolecular Chemistry
Page 1 of 4
DOI: 10.1039/C7OB00844A
Organic & Biomolecular Chemistry
COMMUNICATION
Genetic incorporation of 4-fluorohistidine into peptides enables
selective affinity purification
Christine M. Ringa, Emil S. Iqbala,b, David E. Hackera,b, Matthew C.T. Hartmana,b*, and T. Ashton
Received 00th January 20xx,
Accepted 00th January 20xx
Croppa*
DOI: 10.1039/x0xx00000x
Due to the lowered pKa of 4-fluorohistidine relative to histidine,
peptides and proteins containing this amino acid are potentially
endowed with novel properties. We report here the optimized
synthesis of 4-fluorohistidine and show that it can efficiently
replace histidine in in vitro translation reactions. Moreover,
peptides containing 6x-fluorohistidine tags are able to be
selectively captured and eluted from nickel resin in the presence of
his-tagged protein mixtures.
The incorporation of non-canonical amino acids into proteins is
a powerful tool for the manipulation of protein function and
binding.
One such non-canonical amino acid is 4-
fluorohistidine. Because the van der Waals radius of fluorine is
only slightly larger than that of hydrogen,1 fluorohistidines can
be considered isosteric with histidine. Indeed fluorohistidines
can serve as substrates for histidyl-tRNA synthetase (HisRS), a
feature has be capitalized on for the in vivo production of
proteins containing these residues. Bann and co-workers have
demonstrated that through the use of E. coli auxotrophs, both
2- and 4-fluorohistidine can be incorporated into proteins.2,3
This can serve as a tool for the use of 19F NMR to study protein
conformational changes.3,4 In addition, fluorohistidines can
serve as mechanistic probes due to the fact thatwhile the size
of the molecule is not substantially altered, the electronics of
the two systems (fluorohistidine vs. histidine) are changed.
Addition of the electronegative fluorine lowers the pKa from ~6
for histidine to ~2 for 4-fluorohistidine.5 This reduction in pKa
can allow for elucidation of a histidine’s role in a protein
function or manipulation of protein function at variable pH,6
and as we demonstrate here, selective affinity enrichment at
lowered pH.
Scheme 1. Synthesis of 4-fluorohistidine.
The hexa-histidine tag is one of the most ubiquitous
methods of protein purification. With the small size of the hexa-
histidine tag, high affinity to nickel resin, low cost, and use of
easily-regenerated resin, capturing recombinant protein with a
hexa-histidine tag is appealing. There are certain situations,
however when the hexa-histidine tag is a disadvantage. In the
Protein Synthesis using Recombinant Elements (PURE) system,
E. coli translation proteins (elongation, initiation, and
termination factors, and aminoacyl-tRNA synthetases) are
expressed and purified via hexa-histidine tags.7 These
components are then combined along with necessary amino
acids, ribosomes, and mRNA to biosynthesize peptides or
proteins.7 While this system is widely used for in vitro protein
expression, it has also found a niche in experiments exploring
the limits of the translation apparatus with noncanonical amino
acids (ncAAs).8-20 These experiments typically utilize an mRNA
template that encodes for a short, FLAG-tagged peptide and use
35S-methionine incorporation and MALDI-TOF respectively for
analysis of yield and purity. The FLAG-tag presents three
difficulties in this context: (1) the high cost of the Anti-FLAG
affinity resin, (2) the high negative charge of the FLAG peptide
that reduces MALDI ionization, and (3) the requirement for
a. Virginia Commonwealth University, Department of Chemistry, 1001 West Main
Street, Richmond, Virginia 23284-2006, United States. Email: tacropp@vcu.edu
b. Massey Cancer Center, Virginia Commonwealth University, 401 College Street,
Richmond, Virginia 23298-0037, United States. Email: mchartman@vcu.edu
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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