Chemistry Reference
In-Depth Information
Introduction of Azide or Alkyne Tags
Similarly, many efforts have been made to introduce azide or alkyne, another pair
of popularly used chemical tags, into proteins. besides bertozzi's metabolic incorporation of azide or alkyne groups to
cell surface proteins using glycan synthetic pathways, Tirrell and co-workers also demonstrated the use of azido-amino
acids to introduce azide groups to the cell surface of
E. coli
using its metabolic pathways [133]. The Schultz group again
incorporated
p
-azidophenylalanine and
p
-propargyloxyphenylalanine to proteins using orthogonal TyrRS/tRnA
CuA
pairs that genetically encode these unnatural amino acids in yeast [277]. A similar approach was performed to introduce
alkynyl groups to proteins in
E. coli
[278]. using a genetic engineering approach, Cazalis et al. generated a thrombo-
modulin mutant with a C-terminal azido-methionine in
E. coli
, allowing site-specific Pegylation of the thrombomodu-
lin mutant via Staudinger ligation while maintaining the enzymatic activity [279]. davis and co-workers expressed a
mutant of TIM barrel protein SSβg bearing an azidohomoalanine chemical tag in a methionine auxotrophic strain of
E.
coli
and proved the use of the chemical tag by formation of glycoprotein conjugate using CuAAC [280, 281]. Alkyne-
incorporated SSβg was also generated using the same method. using a similar approach, Finn and colleagues intro-
duced azide- and alkyne-containing unnatural amino acids to virus-like particles, while the incorporation of the
functional handles did not affect the particles' self-assembly [282]. Accessibility of the newly introduced azido or
alkynyl groups was verified by labelling the virus-like particles using alkynyl- or azido-functionalised fluorophores, biotin,
and doTA(gd). Recently, chemical approaches to introduce azide groups to proteins using aqueous diazotransfer
reactions were also developed [283].
Introduction of Alkene Tags
A number of methods have been developed to incorporate alkene groups into proteins either
in vitro
or in bacteria or yeast. using methionyl-tRnA synthetase, Tirrell and co-workers could replace the methionine res-
idues with alkene-containing methionine analogues, such as homoanaline, in proteins expressed in a methionine auxotrophic
strain of
E. coli
[284, 285]. The Schultz group has genetically encoded o-allyltyrosine and phenylselenocysteine in
E. coli
with genetically engineered tRnA/aminoacyl-tRnA synthetase [286, 287]. The same group also introduced several alkene-
containing unnatural amino acids into proteins in yeast using tRnA/aminoacyl-tRnA synthetase pairs [288]. use of pyrroly-
syl-tRnA synthetase has been exploited to incorporate the alkene-containing nonnatural amino acid,
6-n-allyloxycarbonyl-L-lysine into proteins site-specifically [289]. Chemical methods have also been developed to intro-
duce alkene groups to proteins using reactions of o-mesitylenesulfonylhydroxylamine with cysteine thiol groups on protein,
followed by replacement with a thiol nucleophile containing alkene [207].
2.3.5.2 Introduction of Peptide Tags
Peptide tags can be introduced to a target protein via genetic approaches using a
plasmid encoding fusion of the target protein and a peptide tag, which either have high affinity to other molecular parts, or
can be a substrate for a particular enzyme for further modification [5-7].
Tetracysteine Tag
In search of substituents for large fluorescent proteins [290, 291], the Tsien group has developed an
arsenic-modified fluorescein derivative (FlAsH) system (Figure 2.6) [292]. genetic introduction of four cysteine residues
at the i, i + 1, i + 4, and i + 5 positions on a β hairpin of a protein promoted their binding with FlAsH, making the protein of
interest fluorescent. benefiting from the small size of the tetracysteine tag (CCXXCC) and small molecule fluorophores,
xx-
A
SS
As
S
S
S
S
S
S
-Cys-Cys-Xxx-Xxx-Cys-Cys
(genetically encoded sequence on protein
for FlAsH recognition)
As
As
HO
O
O
O
HO
O
COOH
COOH
FlAsH-EDT
2
(uorescence OFF)
FlAsH-protein complex
(uorescence ON)
fIgure 2.6
Protein fluorescent labelling via a tetracysteine tag using FlAsH.
