Intein-mediated synthesis of proteins containing carbohydrates and other molecular probes

Article abstract of DOI:10.1021/ja0000192

We report here the use of an intein-mediated protein ligation strategy for the synthesis of glycoproteins and proteins containing various molecular probes. Several simple cysteine derivatives are synthesized and used as nucleophiles for specific incorpora

Full text of DOI:10.1021/ja0000192

VOLUME 122, NUMBER 23
J UNE 14, 2000
© Copyright 2000 by the
American Chemical Society
Intein-Mediated Synthesis of Proteins Containing Carbohydrates and
Other Molecular Probes
Thomas J. Tolbert and Chi-Huey Wong*
Contribution from the Department of Chemistry and Skaggs Institute of Chemical Biology, The Scripps
Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
ReceiVed January 3, 2000
Abstract: We report here the use of an intein-mediated protein ligation strategy for the synthesis of glycoproteins
and proteins containing various molecular probes. Several simple cysteine derivatives are synthesized and
used as nucleophiles for specific incorporation onto the C-terminus of proteins expressed as N-terminal intein
fusions. Examples of C-terminal carbohydrate labeling, fluorescence labeling, incorporation of a metal chelator,
and incorporation of a nucleotide are presented.
Introduction
complicates the isolation of proteins with such modifications
from natural sources and makes it desirable to find other sources
for these modified proteins. Because of this, development of
simple methods that allow the incorporation of site-specific
synthetic and natural modifications is of interest for biochemical
and biophysical studies.
Studies of protein splicing mechanisms have led to the
development of self-cleaving affinity purification systems that
utilize intein-generated thioesters to cleave fusion proteins from
their attached affinity tags.10 These intein-generated thioesters
can be utilized in expressed protein ligation reactions where
synthetic peptides or bacterially expressed proteins are ligated
to the C-terminus of proteins expressed as intein fusions.11-14
Site-specific incorporation of probes and natural modifications
into proteins can be of great use in biochemical and biophysical
studies. Examples of studies that benefit from introduction of
labels at specific sites include fluorescence resonance energy
transfer studies,1 nitroxide spin labeling NMR studies,2 studies
of enzymatic catalysis and protein structure-function relation-
ships,3-5 screening of combinatorial libraries,6,7 and incorpora-
tion of heavy metals in electron microscopy and X-ray crystal-
lography.8,9 In addition, some posttranslational modifications
such as glycosylation are heterogeneous or transient, which
(1) Eis, P. S. Methods Enzymol. 1997, 278, 330-343.
(2) Hubbell, W. L.; Gross, A.; Langen, R.; Lietzow, M. A. Curr. Opin.
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Bartels, H.; Bashan, A.; Agmon, I.; Kessler, M.; Pioletti, M.; Avila, H.;
Anagnostopoulos, K.; Peretz, M.; Auerbach, T.; Franceschi, F.; Yonath,
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121, 7965-7966.
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Vence, L. M.; Perler, F. B.; Benner, J.; Ducera, R. B.; Hirvonen, C. A.;
Pelletier, J. J.; Paulus, H.; Xu, M.-Q. Gene 1997, 192, 271-281.
(11) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 6705-6710.
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1998, 120, 23-29.
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J. J.; Oprian, D.; Schreiber, S. L.; Wiley: D. C.; Harrison, S. C. Nat. Struct.
Biol. 1999, 6, 953-959.
(12) Evans, T. C. J.; Benner, J.; Xu, M.-Q. Protein Sci. 1998, 7, 2256-
2264.
(8) Ceska, T. A. J. Struct. Biol. 1999, 127, 135-140.
(13) Cotton, G. J.; Muir, T. W. Chem. Biol. 1999, 6, R247-R256.
10.1021/ja0000192 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/27/2000

5422 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Tolbert and Wong
Figure 1. Intein-based fusion protein expression system and expressed protein ligation. (a) Intein-catalyzed thioester formation with a mutated
intein fusion protein (b) Thioester exchange reaction with a thiol resulting in cleavage of the intein from the protein. (c) Expressed protein ligation
between protein thioester and a peptide containing an N-terminal cysteine resulting in a native peptide bond.
Expressed protein ligation has been utilized for the incorporation
synthetic peptides containing fluorescence and phosphoryla-
tion,11,15,16 the semisynthesis of cytotoxic proteins,12 the forma-
tion of cyclic peptides and proteins,17-20 the formation of small
proteins and peptides for thiamine and antibiotic biosynthetic
studies,21,22 and in selective isotopic labeling of protein domains
for NMR studies.23,24 Here we report some investigations
combining expressed protein ligation with simple cysteine
derivatives to develop ligation reagents that can be used to
selectively modify the C-terminus of bacterially expressed
proteins. These ligation reagents can be easily synthesized by
coupling protected forms of cysteine with desired labeling
molecules. Labeling of proteins expressed as intein fusions with
these cysteine derivatives is very simple since the ligation
reaction can be combined with affinity purification, allowing
C-terminally modified proteins to be obtained from crude
bacterial lysate in a single step. Using a variety of synthetic
cysteine derivatives we have incorporated biotin, fluorescence,
a metal chelator, a nucleotide, and glycosylation onto the
C-terminus of a 392-amino acid bacterially expressed pro-
tein.
cysteine results in a expressed protein ligation reaction where
there is a thioester exchange and then a S-N acyl shift which
results in a native peptide bond (Figure 1).13,28-30
Synthesis of Cysteine Derivatives. Our strategy for synthe-
sizing C-terminal protein-labeling reagents was to couple
labeling molecules to the carboxylic acid of protected cysteine
derivatives commonly used in protein synthesis. By coupling
labeling molecules to cysteine through its carboxylic acid, either
directly or through a linker, we synthesized cysteine derivatives
which are linked to labeling molecules but still capable of
undergoing Cys-thioester ligation28,29 reactions after deprotec-
tion. Using this approach, many of the existing reagents currently
used to randomly label protein amines can be easily adapted to
form C-terminal ligation reagents.
We chose two protected forms of cysteine as starting materials
for these labeling reagents, N-R-t-Boc-S-trityl-L-cysteine (Boc-
Cys(Trt)-OH) and N-R-Fmoc-S-tert-butylthio-L-cysteine (Fmoc-
Cys(StBu)-OH). The Boc-Cys(Trt)-OH protected form of
cysteine served as a simple, relatively inexpensive starting
(14) Noren, C. J.; Wang, J.; Perler, F. B. Angew. Chem., Int. Ed. 2000,
39, 450-466.
(15) Konstantin, S.; Muir, T. W. J. Biol. Chem. 1998, 273, 16205-16209.
(16) Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc.
1999, 121, 1100-1101.
Results and Discussion
The C-terminal labeling described in this paper takes advan-
tage of the intein fusion protein expression system developed
by Chong et al.10 (commercially available from NEB), which
is broadly applicable to the expression of proteins in bacte-
ria.11,12,15-22,25,26 The intein fusion protein system utilizes an
intein from the Saccharomyces cereVisiae VMA gene which is
mutated so that it only undergoes the first step of protein
splicing, intein-catalyzed thioester formation. In this system a
protein is expressed as an N-terminal fusion to a mutated intein
which also contains a chitin-binding domain on its C-terminus.
The fusion protein is isolated on chitin resin, and then the desired
protein is released by addition of thiol agents which cleave the
intein-generated thioester forming an intein fragment still bound
to the chitin resin and the desired protein fragment with a
C-terminal thioester. Many factors contribute to the successful
expression and cleavage of desired proteins using this protein
expression system including the structure of the desired protein,
which may interfere with intein function, and the identity of
the C-terminal amino acid, which can activate in vivo cleavage
or inactivate in vitro cleavage.27 After isolation of the desired
protein as a thioester, addition of a peptide with an N-terminal
(17) Evans, T. C. J.; Benner, J.; Xu, M.-Q. J. Biol. Chem. 1999, 274,
18359-18363.
(18) Scott, C. P.; Abel-Santos, E.; Wall, M.; Wahnon, D. C.; Benkovic,
S. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13638-13643.
(19) Camarero, J. a.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 5597-
5598.
(20) Iwai, H.; Plu¨ckthun, A. FEBS Lett. 1999, 459, 166-172.
(21) Kinsland, C.; Taylor, S. V.; Kelleher, N. L.; McLafferty, F. W.;
Begley, T. P. Protein Sci. 1998, 7, 1839-1842.
(22) Roy, R. S.; Allen, O.; Walsh, C. T. Chem. Biol. 1999, 6, 789-
799.
(23) Otomo, T.; Ito, N.; Kyogoku, Y.; Yamazaki, T. Biochemistry 1999,
38, 16040-16044.
(24) Xu, R.; Ayers, B.; Cowburn, D.; Muir, T. W. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 388-393.
(25) Zamore, P. D.; Bartel, D. P.; Lehmann, R.; Williamson, J. R.
Biochemistry 1999, 38, 596-604.
(26) Gilbert, M.; Stayton, P. S. Protein Expression Purif. 1999, 16, 243-
250.
(27) Chong, S.; Williams, K. S.; Wotkowicz, C.; Xu, M.-Q. J. Biol. Chem.
1998, 273, 10567-10577.
(28) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science
1994, 266, 776-779.
(29) Tam, J. P.; Lu, Y.-A.; Liu, C.-F.; Shao, J. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 12485-12489.
(30) Tam, J. P.; Yu, Q.; Miao, Z. Biopolymers 1999, 51, 311-332.

Synthesis of Proteins Containing Probes
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5423
Figure 2. Synthesis of cysteine derivatives from Boc-Cys(Trt)-OH. (a) HBTU, HOBt, NMM, and R-NH2. (b) Trifluoroacetic acid, triisopropylsilane,
and H2O. (c) Cysteine derivatives synthesized from Boc-Cys-(Trt)-OH.
Figure 3. Synthesis of cysteine derivative 7 from Fmoc-Cys(StBu)-OH. (a) HBTU, HOBt, NMM, ethanol amine. (b) Tetrazole, 5′-dimethoxytrityl-
2′-deoxythymidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. ( c) I2, collidine, THF, H2O. (d) Trichloroacetic acid. (e) Piperidine,
DMF.
material which could be coupled to labeling reagents and then
deprotected in a single trifluoroacetic acid treatment to yield a
C-terminal labeling reagent as shown in Figure 2. Using Boc-
Cys(Trt)-OH, the fluorescent molecule anthranilic acid, a metal-
chelating diethylenetriaminepentaacetic acid (DTPA) deriva-
tive,31 the sugar 2-acetamido-1-amino-1,2-dideoxyglucopyranose
(GlcNAc-NH2), and the glycosylated amino acid H-Asn-
(GlcNAc)-OH were attached directly to the carboxylic acid of
cysteine and deprotected to form cysteine derivatives 1-4. Boc-
Cys(Trt)-OH was also used to produce the biotin and dansyl
derivatives 5 and 6 by coupling biotin and dansyl chloride to
cysteine through ethylenediamine linkers. The Fmoc-Cys(StBu)-
OH protected form of cysteine is useful starting material for
attaching acid labile labeling reagents to cysteine, and it also
allows the sulfhydryl group of cysteine to remain protected until
the protein ligation reaction where it can be deprotected in situ
with an excess of thiol agent. Fmoc-Cys(StBu)-OH was used
to form the 3′-phospho-deoxythymidine cysteine derivative 7
(Figure 3) by first coupling ethanol amine to Fmoc-Cys(StBu)-
OH and then coupling the free hydroxyl of the resulting product
to a deoxythymidine phosphoramidite commonly used in DNA
synthesis, 5′-dimethoxytrityl-2′-deoxythymidine,3′-[(2-cyano-
ethyl)-(N,N-diisopropyl)]-phosphoramidite (dT-CE phosphor-
amidite). Once the phosphoramidite was coupled to cysteine,
oxidation with iodine gave the 3′-phosphate, and removal of
the dimethoxytrityl with trichloroacetic acid and deprotection
of the â-cyanoethyl and Fmoc-protecting groups with piperidine
resulted in the cysteine-3′-phospho-deoxythimidine derivative
7.
Ligation of Cysteine Derivatives to a Model Protein. To
test the ability of the cysteine derivatives to modify the
C-terminus of proteins expressed as N-terminal intein fusions,
we attempted to ligate them to the Escherichia coli maltose
binding protein (MBP) as a model system. The MBP served as
a convenient model system because expression of a MBP-intein
fusion protein has been reported previously and it is a well-
defined system in terms of expression and purification charac-
teristics.10 Each cysteine derivative was tested by cleaving the
MBP-intein fusion protein under conditions in which Cys-
thioester ligation28,29 should take place, then analyzing whether
the cysteine derivative modified the protein or not. Briefly, the
MBP-intein fusion protein was isolated on a chitin column,
and then cleavage of the MBP-intein fusion protein and ligation
of the cysteine derivatives to the MBP was initiated by adding
a solution containing 30 mM 2-mercaptoethanesulfonic acid (2-
MESA)12 and 1 mM cysteine derivative at pH ) 7.5. Cleavage
and ligation can be conducted with the cysteine derivatives alone
at higher concentration (15 mM) without any extra thiol agent
to induce cleavage, but the additional thiol agent was added to
reduce the amount of cysteine derivative required. Cleaved MBP
(31) Anelli, P. L.; Fedeli, F.; Gazzotti, O.; Lattuada, L.; Lux, G.; Rebasti,
F. Bioconjugate Chem. 1999, 10, 137-140.

5424 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Tolbert and Wong
Figure 4. (a) HPLC profile of cleavage reaction of MPB-intein fusion protein with 1 mM derivative 3 and 30 mM 2-MESA. (b) HPLC profile of
cleavage reaction of MBP-intein fusion protein cleaved with 30 mM DTT. (c) Coelution of the differently modified forms of MBP in a 50:50
mixture of the cleavage reactions shown in a and b.
Figure 5. Electrospray mass spectra of MBP ligated to cysteine derivative 3. (a) Mass-to-charge ratio (m/z) spectra of MBP 14. (b) Mass spectra
of MBP 14.
Table 1. Test Ligation of Various Cysteine Derivatives to the
MBP
was eluted off of the chitin resin with H2O, reduced with
dithiothreitol, and then the protein sample was desalted, and
small molecule contaminants were removed using reverse phase
HPLC. Due to the large size of the MBP, approximately 43
KDa, and the relatively small size of the modifications that were
ligated to the MBP, the largest of which caused a mass increase
cysteine
modified
MBPb
observed
massc
calculated
massd
of 0.55 KDa, reverse phase HPLC did not separate modified
and unmodified forms of the MBP (Figure 4). Ligation of the
cysteine derivatives to the MBP was detected by collecting the
entire MBP HPLC peak and analyzing the sample by electro-
spray mass spectrometry. Most of the cysteine derivatives gave
very clean mass spectra indicating nearly quantitative ligation
(greater than 95%) of the cysteine derivatives to the MBP.
Figure 5 illustrates a representative mass spectra for MBP ligated
to derivative 3, and Table 1 shows the masses of the modified
proteins observed for different test ligation reactions.
Of the cysteine derivatives synthesized, only derivative 6
failed to ligate cleanly to the MBP using the procedure described
above. Difficulty was encountered with the dansyl cysteine
derivative 6 because of its low water solubility at neutral pH.
The dansyl cysteine derivative 6 was soluble in water at or below
pH 5.0 but quickly precipitated out of solution when the pH
was neutralized to 7.0. To overcome this obstacle, ligation of
the MBP to the dansyl cysteine derivative was accomplished
by a two-step cleavage and ligation procedure where the MBP
thioester was first formed at pH 7.0, the the pH was then
adjusted to 5.0, and the dansyl cysteine derivative 6 was added
to the protein solution. Using this procedure, a small amount
of the MBP protein precipitated irreversibly at the acid pH, but
the MBP which remained in solution was ligated cleanly to 6
as detected by electrospray mass spectrometry.
derivativea
cysteine
11
12
13
14
15
16
17
18
43033
43150
43480
43236
43361
43305
43315
43382
43042
43162
43489
43245
43360
43311
43318
43390
1
2
3
4
5
6
7
a Cysteine derivative used in ligation reaction. b Modified form of
MBP resulting from the ligation reaction. c Mass of the modified MBP
observed by electrospray mass spectrometry. d Calculated mass for the
ligation of the respective cysteine derivatives.
Application of C-terminal Modification to Protein Phos-
phorylation and Glycosylation. Part of the utility of this
C-terminal labeling technique is the ability to easily add
synthetic derivatives with unique chemical activities onto the
C-terminus of bacterially expressed proteins. One type of unique
chemical activity that is advantageous to incorporate into
proteins is the addition of chemical groups which serve as
substrates for subsequent enzymatic modification. To demon-
strate this, two of the cysteine derivatives synthesized above,
compounds 4 and 7, were chosen specifically for their ability
to serve as substrates for enzyme-catalyzed transferase reactions.
The deoxythymidine-3′-phosphate cysteine derivative 7 was

Synthesis of Proteins Containing Probes
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5425
Figure 6. Enzymatic transfer of phosphate and galactose to two differently modified forms of MBP. (a) Transfer of phosphate to MBP modified
with 7 using T4-polynucleotide kinase (b) Transfer of galactose to MBP modified with 4 using â-1,4-galactosyltransferase.
prepared in hopes that it would serve as a substrate for T4-
polynucleotide kinase when attached to the C-terminus of
proteins. T4-polynucleotide kinase has been used to label peptide
and peptide nucleic acid (PNA) amines with 32P using 2′-
deoxycytidine 3′-phosphoroimidazolide,32 and we reasoned that
coupling cysteine to a deoxynucleotide 3′-phosphate would
allow specific phosphorylation of the C-terminus of proteins
expressed as intein fusions. The modified form of MBP 18, MBP
modified with deoxythymidine-3′-phosphate cysteine derivative
7, was first prepared as described above and then dialyzed to
remove 2-MESA and the excess of derivative 7. Then MBP 18
was placed under standard T4-polynucleotide kinase phospho-
rylation conditions33 (Figure 6a), and the resulting modified
protein was analyzed by electrospray mass spectrometry. An
increase in mass of 80 was observed for the T4-polynucletide
kinase reacted MBP (19) relative to the starting 18, indicating
successful phosphorylation. A useful application of this C-
terminal protein phosphorylation would be to incorporate a
single 32P onto the C-terminus of proteins expressed as intein
fusions in a manner analogous to nucleic acid 5′-32P labeling
by substituting γ-32P ATP for ATP in the T4-polynucleotide
kinase reaction.
analyzed by electrospray mass spectrometry, and an increase
in mass corresponding to transfer of galactose was observed
(155 observed, 162 calculated). Although not undertaken in this
study, further extension of the resulting glycoprotein to sialyl
Lewis X-34,35 or R-galactosyl epitope-type36 structures should
be possible since the N-acetyllactosamine disaccharide can serve
as a substrate for other glycosyltransferases. Also, since aspar-
agine â-linked to N-acetylglucosamine is a substrate for the
endo-â-N-acetylglucosaminidase from Mucor hiemalis, it may
be possible to transfer high mannose-type sugars in transgly-
cosidase reactions similar to the types reported by Mizuno et
al.37 By using this approach oligosacharrides of medium to large
size might be incorporated onto the C-terminus of bacterially
expressed proteins by starting with glycopeptides having a
single N-linked N-acetylglucosamine, and this may offer a route
to the synthesis of glycoproteins with homogeneous glyco-
forms.
Conclusions
The approach presented here for C-terminally incorporating
molecular probes and natural modifications into bacterially
expressed proteins should be useful for biochemical studies
which require site-specific labeling or stoichiometric labeling
of any protein which can be expressed as an intein fusion
protein. By utilizing the intein fusion protein expression system
developed by Chong et al.,10 the ligation of our labeling
molecules to a protein is combined with affinity purification,
allowing isolation and labeling of a bacterially expressed protein
to occur in a single step. The synthesis of the cysteine derivatives
used in this paper to C-terminally modify proteins was ac-
complished in a few synthetic steps, and many of the labeling
reagents which are commonly used to randomly label protein
amines can be easily adapted to this C-terminal labeling strategy
by reacting them with protected forms of cysteine. Using this
approach to attach molecules that can serve as substrates for
subsequent enzymatic transformations should allow relatively
simple molecules to be extended to more complex structures
The glycosylated dipeptide H-Cys-Asn(GlcNAc)-OH (4) was
prepared in order to demonstrate the incorporation of a C-
terminal glycosylation tag into a bacterially expressed protein
which could be subsequently extended to a more complex
oligosaccharide using glycotransferases. Bovine â-1,4-galacto-
syltransferase accepts a wide range of substrates which contain
N-acetylglucosamine (GlcNAc) as a terminal non-reducing sugar
residue, including GlcNAc both N-linked and O-linked to
peptides and proteins.34,35 The modified form of MBP 15, MBP
modified with the glycopeptide 4, was first prepared as described
above, and then dialyzed to remove 2-MESA and the excess of
derivative 4. Galactose was then transferred to 15 by incubating
the modified MBP with galactosyltransferase in the presence
of UDP-galactose (Figure 6b). The resulting protein, 20, was
(32) Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E. Bioconjugate Chem. 1998,
9, 415-417.
(33) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, 1989; Vol. 2, pp
10.59-10.61.
(36) Fang, J.; Li, J.; Chen, X.; Zhang, Y.; Wang, J.; Guo, Z.; Zhang,
W.; Yu, L.; Brew, K.; Wang, P. G. J. Am. Chem. Soc. 1998, 120, 6635-
6638.
(37) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.; Kawakami, T.;
Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem. Soc. 1999, 121, 284-
290.
(34) Seitz, O.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 8766-8776.
(35) Witte, K.; Sears, P.; Martin, R.; Wong, C.-H. J. Am. Chem. Soc
1997, 119, 2114-2118.

5426 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Tolbert and Wong
Compound 3. N-R-t-Boc-S-trityl-L-cysteine (0.107 g, 0.230 mmol),
1,3-diisopropyl carbodiimide (0.144 g, 1.15 mmol), and 1-hydroxy-
benzotriazole (0.177 g, 1.15 mmol) were combined and dissolved in
10 mL of DMF. The resulting mixture was stirred for 30 min, and
then diisopropylethylamine (0.088 g, 0.690 mmol) and 2-acetamido-
1-amino-1,2-dideoxy-glucopyranose (0.12 g, 0.543 mmol) were added
to the reaction. This mixture was stirred for 12 h, and then the solvent
was removed by rotovap. The residue was dissolved in EtOAc, washed
with H2O three times, and then dried over MgSO4 and evaporated. The
residue was crudely purified by flash chromatography (0-6% MeOH
in CH2Cl2), and the resulting crude product was dissolved in a solution
containing trifluoroacetic acid (10 mL), triisopropylsilane (0.109 g, 0.69
mmol), and H2O (0.1 mL). The resulting solution was stirred for 25
min, and then the solvent was removed by rotovap. The residue was
dissolved in a mixture of H2O (15 mL) and CH2Cl2 (10 mL), the organic
layer was separated from the aqueous layer, and the aqueous layer was
extracted four times with 15 mL of CH2Cl2. The aqueous layer was
concentrated by rotovap, and the resulting residue was purified by C18
reverse phase HPLC (5% acetonitrile in H2O containing 0.1% TFA)
affording 0.054 g of 3 (72% yield). 1H NMR (400 MHz, D2O) δ 5.06
(d, 1H, J ) 9.7), 4.13 (t, 1H, J ) 5.5), 3.87-3.82 (m, 2H), 3.70 (dd,
1H, J ) 12.5, 4.7), 3.57 (t, 1H, J ) 9.0), 3.50-3.41 (m, 2H), 3.07 (dd,
1H, J ) 15.1, 5.5), 3.01 (dd, 1H, J ) 15.1, 5.5) 1.97 (s, 3H); 13C
NMR δ 177.31, 170.99, 81.03, 80.18, 76.64, 71.80, 62.85, 56.84, 56.53,
27.10, 24.48; HRMS (FAB) 346.1044 (M + Na+), calcd 346.1049.
after being attached to proteins. In addition to incorporating
molecular probes, expressed protein ligation shows promise for
incorporating glycosylation into proteins, as illustrated by the
addition of the GlcNAc glycosylation tag, which could be further
extended to more complicated oligosacharrides using glyco-
transferases.
Experimental Section
Materials and Methods. Chemicals were purchased from Aldrich
and Sigma. Protected amino acids were obtained from Novabiochem
and Bachem. Plasmid pMYB5, which expresses an MBP-intein fusion
protein, was obtained as part of the Impact T7 protein purification
system with self-cleavable affinity tag, sold by New England Biolabs,
Inc. Recombinant T4-polynucleotide kinase was purchased from New
England Biolabs, and galactosyltransferase from bovine milk was
purchased from Sigma. NMR spectra were recorded on a Brucker AMX
400 or a Brucker DRX 500 NMR spectrometer. Electrospray mass
spectrometry of protein samples were conducted with a PE SCIEX API-
III biomolecular mass analyzer.
Compound 1. N-R-t-Boc-S-trityl-L-cysteine (0.100 g, 0.216 mmol)
and N,N′-carbonyldiimidazole (0.035 g, 0.216 mmol) were combined
in a round-bottom flask covered with aluminum foil and dissolved in
10 mL of DMF. After stirring this mixture for 30 min at room
temperature, anthranilic acid (0.030 g, 0.216 mmol) and triethylamine
(0.044 g, 0.432 mmol) were added. The reaction was stirred for an
additional 24 h, and then the solvent was removed under vacuum. The
residue was crudely purified by flash chromatography (0-2% MeOH
in CH2Cl2), and the resulting crude product was dissolved in a solution
containing trifluoroacetic acid (5 mL), H2O (130 µL), and triisopro-
pylsilane (0.10 g, 0.65 mmol). This mixture was stirred for 30 min in
a flask covered with aluminum foil and then evaporated under vacuum.
The resulting residue was dissolved in a mixture of 25 mL of H2O and
25 mL of CH2Cl2, and the pH of the aqueous layer was adjusted to 7.0
with 1 M NaOH. The aqueous layer was separated from the organic
layer, extracted three times with 20 mL of CH2Cl2, and then
concentrated under vacuum. C18 reverse phase HPLC purification of
the residue (0-80% acetonitrile in H2O, 0.1% TFA) afforded 0.037 g
of a white solid (71% yield). 1H NMR (400 MHz, MeOD) δ 8.48 (dd,
1H, J ) 8.4, 1.1), 8.11 (dd, 1H, J ) 7.8, 1.4), 7.59 (ddd, 1H, J ) 7.8,
7.8, 1.4), 7.22 (ddd, 1H, J ) 8.4, 7.8, 1.1), 4,34 (dd, 1H, J ) 6.2, 5.1),
3.21 (dd, 1H, J ) 14.8, 5.1), 3.12 (dd, 1H, J ) 14.8, 6.2); 13C NMR:
δ 171.44, 166.75, 141.13, 135.35, 132.75, 125.25, 122.28, 118.90,
57.48, 26.18; HRMS (MALDI-FTMS) 241.0646 (MH+), calcd 241.0647.
Compound 2. N-R-t-Boc-S-trityl-L-cysteine (0.84 g, 0.181 mmol),
HBTU (0.825 g, 0.217 mmol), and HOBt (0.042 g, 0.272 mmol) were
combined and dissolved in CH2Cl2 (20 mL) and stirred for 30 min,
and then 4-methylmorpholine (0.055 g, 0.544 mmol) and N2,N2-Bis-
[2-[bis[2-(1,1-dimethyethoxy)-2-oxoethyl]-amino]ethyl]-L-lysine 1,1-
dimethylethyl ester (0.142 g, 0.181 mmol) prepared by the method of
Anellin et al.31 were added. After the reaction mixture was stirred for
3 h at room temperature, the solvents were removed by rotovap. The
residue was crudely purified by flash chromatography (15-25% EtOAc
in hexanes), and then the crude product was dissolved in a mixture
containing TFA (12 mL), triisopropylsilane (0.288 g, 0.181 mmol),
and H2O (0.37 mL). This solution was stirred for 2 h, and then the
solvents were removed by rotovap. The residue was redissolved in a
mixture of TFA (10 mL) and CH2Cl2 (10 mL) and stirred for 16 h at
room temperature. The solvents were removed by rotovap, and then
the residue was dissolved in a mixture of H2O (15 mL) and CH2Cl2
(15 mL). The organic layer was removed, and the aqueous layer was
extracted three times with 15 mL of CH2Cl2. The aqueous layer was
concentrated by high vacuum rotoevaporation, and then the residue
was purified by C18 reverse phase HPLC (5% acetonitrile in H2O, 0.1%
Compound 4. N-R-t-Boc-S-trityl-L-cysteine (0.088 g, 0.191 mmol),
HBTU (0.078 g, 0.205 mmol), and HOBt (0.568 g, 0.373 mmol) were
dissolved in 10 mL of DMF and stirred for 30 min under argon. After
30 min 4-methyl morpholine (0.0573 g, 0.568 mmol) and 9 H2N-Asn-
(GlcNAc)-OtBu (0.745 g, 0.191 mmol) were added to the reaction,
and it was stirred for an additional 4 h at which time the solvent was
then removed by rotovap. The residue was crudely purified by flash
chromatography (4-25% MeOH in CH2Cl2), and the resulting crude
product was dissolved in a solution containing TFA (7 mL), triisopro-
pylsilane (0.91 g, 0.57 mmol), and H2O (0.12 mL). This mixture was
stirred for 30 min under argon, and then the solvents were removed by
rotovap. The residue was dissolved in a mixture of H2O (15 mL) and
CH2Cl2 and the organic layer was separated and discarded. The aqueous
layer was extracted with 15 mL of CH2Cl2 four times and then
concentrated. The residue was purified by C18 reverse phase HPLC
(5% acetonitrile in H2O, 0.1% TFA). A white solid (0.0672 g, 80%
1
yield) was obtained. H NMR (400 MHz, D2O) δ 4.97 (d, 1H, J )
9.7), 4.73 (t, 1H, J ) 5.5), 4,19 (t, 1H, J ) 5.4), 3.80 (dd, 1H, J )
12.4, 1.9), 3.74 (t, 1H, J ) 9.9), 3.67 (dd, 1H, J ) 12.1, 4.9), 3.53 (t,
1H, J ) 9.7), 3.47-3.36 (m, 2H), 3.06 (dd, 1H, J ) 15.0, 5.4), 3.00
(dd, 1H, J ) 15.0, 5.4), 2.89 (dd, 1H, J ) 16.6, 5.5), 2.79 (dd, 1H, J
) 16.6, 5.5), 1.92 (s, 3H); 13C NMR δ 177.21, 175.78, 174.87, 170.16,
80.80, 79.99, 76.49, 71.84, 62.87, 56.68, 56.53, 51.48, 38.70, 27.39,
24.42; HRMS (FAB) 461.1312 (M + Na+), calcd 461.1318.
Compound 5. N-R-t-Boc-S-trityl-L-cysteine (0.24 g, 0.52 mmol),
HBTU (0.24 g, 0.62 mmol), and HOBt (0.12 g, 0.78 mmol) were
combined in a round-bottom flask and dissolved in 10 mL of DMF.
This mixture was stirred under argon for 20 min at room temperature,
and then 4-methyl morpholine (0.15 g, 1.56 mmol) and biotinylethyl-
enediamine (obtained from the method of Garlick and Giese38) (0.15
g, 0.52 mmol) were added to the mixture. After stirring for 3 h, the
solvents were removed under vacuum, and the residue was dissolved
in 40 mL of CH2Cl2 and extracted three times with 40 mL of H2O.
The organic layer was separated and dried over MgSO4, and the solvents
were evaporated by rotovap. The residue was crudely purified by flash
chromatography (4-8% MeOH in CH2Cl2), and then the resulting crude
product was dissolved in a solution containing trifluoroacetic acid (10
mL), H2O (320 µL), and triisopropylsilane (0.24 g, 1.56 mmol). This
mixture was stirred for 30 min and then evaporated under vacuum.
The resulting residue was dissolved in a mixture of 20 mL of H2O and
20 mL of CH2Cl2, and the aqueous layer was separated from the organic
layer. The aqueous layer was extracted three times with 20 mL of CH2-
Cl2 and then concentrated under vacuum. C18 reverse phase HPLC
purification of the residue (5% ACN in H2O, 0.1% TFA) afforded 0.152
1
TFA), affording 0.0606 g of a clear solid (59% yield). H NMR (400
MHz, D2O) δ 4.08 (t, 1H, J ) 5.7), 3.54 (dd, 1H, J ) 8.4, 5.7), 3.46
(t, 4H, J ) 6.5), 3.27 (ddd, 1H, J ) 13.4, 6.7, 6.7), 3.01 (dd, 1H, J )
14.9, 5.7), 2.96 (dd, 1H, J ) 14.9, 5.7), 1.85-1.76 (m, 1H), 1.64-
1.34 (m, 5H);); 13C NMR: δ 177.51, 171.83, 170.10, 65.62, 57.98,
56.92, 55.39, 48.69, 41.61, 30.43, 30.10, 27.30, 25.78; HRMS (MALDI-
FTMS) 590.2100 (MNa+), calcd 590.2108.
(38) Garlick, R. K.; Giese, R. W. J. Biol. Chem. 1988, 263, 210-215.

Synthesis of Proteins Containing Probes
J. Am. Chem. Soc., Vol. 122, No. 23, 2000 5427
g of a white solid (75% yield). 1H NMR (400 MHz, D2O) δ 4.57 (dd,
1H, J ) 7.8, 5.0), 4.39 (dd, 1H, J ) 7.8, 5.0), 4.12 (t, 1H, J ) 5.4),
3.45 (m, 1H), 3.33-3.24 (m, 4H), 3.03 (dd, 1H, J ) 14.9, 5.4), 3.00-
2.93 (m, 2H), 2.74 (d, 1H, J ) 13.2), 2.22 (t, 2H, J ) 7.3), 1.72-1.50
(m, 4H), 1.48-1.31 (m, 2H); 13C NMR δ 179.62, 170.46, 64.53, 62.70,
57.79, 56.01, 42.16, 42.12, 41.45, 37.96, 30.39, 30.12, 27.50, 27.30;
HRMS (MALDI-FTMS) 390.1646 (MH+), calcd 390.1633.
the reaction stirred for an additional 2 h. The reaction was concentrated
by under high vacuum and then dissolved in 50 mL of CH2Cl2. The
organic layer was extracted with 50 mL of H2O three times, dried over
MgSO4, and then concentrated under vacuum. Purification of the
resulting residue by flash chromatography (0-30% MeOH in CH2Cl2)
afforded 0.90 g (82% yield) of a light yellow oil. 1H NMR (400 MHz,
D2O) δ 7.41, (d, 6H, J ) 8.1), 7.28 (t, 6H, J ) 8.1), 7.21 (t, 3H, J )
8.1), 6.64 (t, 1H, J ) 5.5), 5.06 (d, 1H, J ) 5.9), 3.88 (m, 1H), 3.25-
3.22 (m, 2H), 2.76-2.73 (m, 2H), 2.68 (dd, 1H, J ) 12.5, 5.5), 2.54
(dd, 1H, J ) 12.5, 5.5); 13C NMR δ 171.28, 155.86, 144.81, 130.00,
128.50, 127.32, 80.67, 67.54, 42.42, 41.53, 34.41, 31.38, 28.72; HRMS
(MALDI-FTMS) 528.2323 (MNa+), calcd 528.2297.
Compound 9. Fmoc-Asn(GlcNAc)-OtBu39 (0.100 g, 0.256 mmol)
was dissolved in a mixture of DMF (5 mL) and piperidine (5 mL) and
stirred for 20 min. The solution was then evaporated under high vacuum
and the residue was purified by flash chromatography (60:30:10 EtOAc:
MeOH:H2O) affording 0.585 g of a light yellow solid (92% yield). 1H
NMR (500 MHz, D2O) δ 5.09 (d, 1H, J ) 9.9), 3.93 (dd, 1H, J )
12.5, 1.8), 3.87 (t, 1H, J ) 9.9), 3.80 (dd, 1H, J ) 12.5, 4.8), 3.75 (t,
1H, J ) 6.1), 3.67 (t, 1H, J ) 9.9), 3.59-3.51 (m, 2H), 2.75 (dd, 1H,
J ) 15.8, 5.9), 2.70 (dd, 1H, J ) 15.8, 6.2), 2.07 (s, 3H), 1.51 (s,
9H);); 13C NMR δ 175.20, 174.88, 173.93, 83.80, 78.83, 77.93, 74.62,
69.92, 60.92, 54.74, 51.66, 39.81, 27.56, 22.56; HRMS (MALDI-
FTMS) 414.1852 (MNa+), calcd 414.1847.
Compound 6. Cysteine-ethylenediamine (8) (0.123 g, 0.244 mmol),
dansyl chloride (0.327 g, 1.21 mmol), and triethylamine (0.074 g, 0.728
mmol) were combined in a round-bottom flask and dissolved in 20
mL of CH2Cl2. After stirring for 20 h the reaction was evaporated under
vacuum, and the residue was crudely purified by flash chromatography
(0-1% MeOH in CH2Cl2). The crude product was dissolved in a
solution containing trifluoroacetic acid (7 mL), H2O (150 µL), and
triisopropylsilane (0.116 g, 0.732 mmol). This mixture was stirred for
30 min and then evaporated under vacuum. The resulting residue was
dissolved in a mixture of 20 mL of H2O and 20 mL of CH2Cl2, the
aqueous layer was separated from the organic layer, extracted three
times with 20 mL of CH2Cl2, and then concentrated under vacuum.
C18 reverse phase HPLC purification of the residue (gradient of 0-20%
acetonitrile in H2O, 0.1% TFA) afforded 0.053 g of a clear yellow
solid (55% yield). 1H NMR (400 MHz, D2O) δ 8.61 (d, 1H, J ) 8.6),
8.37 (d, 1H, J ) 8.6), 8.21 (d, 1H, J ) 7.6), 8.00 (d, 1H, J ) 7.8),
7.80 (dd, 1H, J ) 8.6, 7.8), 7.79 (dd, 1H, J ) 8.6, 7.6), 4.10 (t, 1H,
5.7), 3.44 (s, 6H), 3.37 (ddd, 1H, J ) 14.3, 5.7, 5.7), 3.17 (ddd, 1H, J
) 14.3, 5.7, 5.7), 2.99 (t, 2H, J ) 5.7), 2.93 (dd, 1H, J ) 14.8, 5.7),
2.87 (dd, 1H, J ) 14.8, 5.7); 13C NMR δ 170.45, 141.48, 137.37,
132.62, 130.93, 130.53, 129.26, 128.45, 128.26, 128.19, 121.73, 56.82,
49.08, 44.04, 41.50, 27.30; HRMS (MALDI-FTMS) 419.1172 (MNa+),
calcd 419.1188.
Compound 7. dT-CE phosphoramidite (0.235 g, 0.315 mmol),
Fmoc-Cys(StBu)-ethanol amine 10 (0.164 g, 0.346 mmol) and tetrazole
(0.111 g, 1.58 mmol) were dried under high vacuum for 3 h prior to
the reaction. Into a 250 mL round-bottom flask which had been dried
in an oven prior to use were combined and dissolved in freshly distilled
acetonitrile (15 mL) the dT-CE phosphoramidite and Fmoc-Cys(StBu)-
ethanol amine . The reaction was initiated by addition of the dried
tetrazole in 5 mL of freshly distilled acetonitrile. The reaction was
stirred under argon at room temperature for 1 h, and then 3.8 mL of an
iodine solution (0.1 M iodine in THF:collidine:H2O, 2:2:1) were added
to the reaction. This mixture was stirred for 10 min, and then 1 M
aqueous thiosulfate solution (10 mL) was added. After the solution
stirred for 5 min, 100 mL of CH2Cl2 was added to the reaction, and
the aqueous layer was separated from the organic layer. The aqueous
layer was extracted with CH2Cl2, and the organic layers were combined
and dried over Na2SO4, and then the solvents were removed by rotovap.
The residue was dissolved in a solution containing CH2Cl2 (50 mL),
MeOH (50 mL), and trichloroacetic acid (2.5 mL of a 6.1 N solution).
This mixture was stirred for 15 min, and then the solvents were removed
by rotoevaporation. The residue was crudely purified by flash chro-
matography (0-8% MeOH in CH2Cl2), and the crude product was
dissolved in a mixture of CH2Cl2 (10 mL) and piperidine (1 mL) and
stirred for 12 h at room temperature. The reaction was rotoevaporated
and dissolved in H2O (30 mL) and EtOAc (15 mL). The organic and
aqueous layers were separated, and the aqueous layer was extracted
with EtOAc three times and then concentrated and purified by C18
reverse phase HPLC (0-80% acetonitrile in H2O (no TFA)), affording
0.96 g of a white solid (55% yield). 1H NMR (400 MHz, D2O) δ 7.65
(s, 1H), 6.27 (dd, 1H, J ) 7.3, 6.2), 4.76 (m, 1H), 4.17 (dd, 1H, J )
7.8, 3.5), 3.98-3.89 (m, 2H), 3.82 (dd, 1H, J ) 12.7, 3.5), 3.76 (dd,
1H, J ) 12.7, 4.6), 3.65 (t, 1H, J ) 6.4), 3.55-3.37 (m, 2H), 2.96 (d,
2H, J ) 6.5), 2.52 (ddd, 1H, J ) 14.1, 6.2, 3.2), 2.34 (ddd, 1H, J )
14.1, 7.3, 7.3), 1.86 (s, 3H), 1.26 (s, 9H); 13C NMR δ 177.74, 169.15,
154.19, 139.94, 114.00, 88.14, 87.49, 77.51, 66.49, 63.51, 56.59, 50.60,
46.35, 42.61, 40.24, 31.40, 14.10; HRMS (MALDI-FTMS) 557.1518
(MH+), calcd 557.1505.
Compound 10. Fmoc-Cys(StBu)-OH (0.30 g, 0.695 mmol), HBTU
(0.316 g, 0.834 mmol), and HOBt (0.159 g, 1.04 mmol) were combined
and dissolved in 15 mL of DMF. After stirring for 30 min at room
temperature 4-methylmorpholine (0.210 g, 2.08 mmol) and ethanol
amine (0.052 g, 0.851 mmol) were added. The reaction was stirred for
3 h and then concentrated by rotoevaporation. The resulting residue
was dissolved in 20 mL of EtOAc, extracted with H2O (15 mL) three
times, extracted with saturated aqueous NaCl solution (15 mL) once,
dried over MgSO4, and then rotoevaporated. The residue was purified
by flash chromatography (0-2% MeOH in CH2Cl2) affording 0.314 g
1
of a white powder (95% yield). H NMR (400 MHz, CDCl3) δ 7.76
(d, 2H, J ) 7.6), 7.59 (d, 2H, J ) 7.6), 7.40 (t, 2H, J ) 7.6), 7.31 (t,
2H, J ) 7.6), 6.75 (s, 1H), 5.80 (d, 1H, J ) 7.8), 4.51-4.36 (m, 3H),
4.21 (t, 1H, J ) 6.8), 3.71 (t, 2H, J ) 4.7), 3.60-3.39 (m, 2H), 3.11
(dd, 1H, J ) 13.5, 6.8), 3.03 (dd, 1H, J ) 13.5, 6.8), 1.34 (s, 9H); 13
C
NMR δ 170.80, 143.58, 141.26, 127.75, 127.08, 125.04, 120.01, 67.28,
61.66, 54.77, 48.59, 47.02, 42.49, 41.99, 29.78; HRMS (MALDI-
FTMS) 475.1710 (MH+), calcd 475.1725.
Expression and Purification of the Maltose Binding Protein-
Intein Fusion Protein. Each cysteine derivative was tested for
C-terminal ligation by cleaving/modifying the MBP-intein fusion
protein10 obtained from 1 L of cell growth of E. coli strain ER2566/
pMYB5 in LB media (approximately 15-30 mg of protein). The
general growth and protein purification procedures are given below.
One liter of LB media containing 50 µL/mL of ampicillin and 0.25
mM IPTG was inoculated with a 5 mL culture of ER2566/pMYB5
and incubated in a 30 °C shaker for 12-16 h. Cell pellets were obtained
by spinning the culture at 5000 g for 15 min. Cells were resuspended
in 20 mL of buffer A (20 mM Na-HEPES pH ) 7.6, 500 mM NaCl,
0.03% Triton X-100, 1 mM EDTA), and lysed by French press. The
lysate was centrifuged for 30 min at 27000g, and the clarified lysate
was decanted. Clarified lysate was loaded directly onto chitin columns
(containing 15 mL of chitin resin) which had been equilibrated with
buffer A. Chitin columns were washed with 3-5 column volumes of
buffer A after loading of the lysate and then washed with three volumes
of H2O. Once this had been done, the MBP-intein fusion was bound
to the chitin column and ready to be cleaved by the addition of cleavage
reagents.
General Cleavage Conditions for the MBP-VMA Intein Fusion
Protein. Cleavage was initiated on the chitin column by addition of 1
mM cysteine derivative dissolved in a solution containing 30 mM
2-mercaptoethanesulfonic acid, pH ) 7.5.12 The chitin column was then
flushed with argon and incubated for 24 h at 4 °C. After incubation,
the column was eluted with 2-3 column volumes of H2O. The eluent
Compound 8. N-R-t-Boc-S-trityl-L-cysteine (1.00 g, 2.16 mmol) and
N,N′-carbonyldiimidazole (0.42 g, 2.60 mmol) were combined in a 100
mL round-bottom flask and dissolved in 10 mL of DMF. The mixture
was stirred for 30 min at room temperature, until the bubbling had
ceased, and then ethylenediamine (0.65 g, 10.8 mmol) was added and
(39) Urge, L.; Kollat, E.; Hollosi, M.; Laczko, I.; Wroblewski, K.; Thurin,
J.; Otvos, L. J. Tetrahedron Lett. 1991, 32, 3445-3448.

5428 J. Am. Chem. Soc., Vol. 122, No. 23, 2000
Tolbert and Wong
was concentrated to about 1 mL using centriprep 10,000 protein
concentrators (Millipore), and then DTT was added to this solution to
a concentration of 20 mM in order to reduce any unwanted disulfide
bonds. The different modified forms of the MBP were isolated by C18
reverse phase HPLC (using a gradient of 0-80% ACN in H2O, 0.1%
TFA over 30 min, flow rate 1 mL/min.). The MBP obtained from HPLC
purification was analyzed directly by electrospray mass spectrometry.
Modified MBP 11. Cleavage and ligation were conducted as
described in the general procedure using 1 mM cysteine as a ligation
reagent. After elution of the protein and HPLC purification, electrospray
mass spectrometry gave an observed mass of 43033 (calculated mass
for the MBP-cysteine adduct is 43042).
Modified MBP 12. Cleavage and ligation were conducted as
described in the general procedure using 1 mM 1 as a ligation reagent.
After elution of the protein and HPLC purification, electrospray mass
spectrometry gave an observed mass of 43150, calcd 43162.
Modified MBP 13. Cleavage and ligation were conducted as
described in the general procedure using 1 mM 2 as a ligation reagent.
After elution of the protein and HPLC purification, electrospray mass
spectrometry gave an observed mass of 43480, calcd 43489.
Modified MBP 14. Cleavage and ligation were conducted as
described in the general procedure using 1 mM 3 as a ligation reagent.
After elution of the protein and HPLC purification, electrospray mass
spectrometry gave an observed mass of 43236, calcd 43245.
Modified MBP 15. Cleavage and ligation were conducted as
described in the general procedure using 1 mM 4 as a ligation reagent.
After elution of the protein and HPLC purification, electrospray mass
spectrometry gave an observed mass of 43361, calcd 43360.
Modified MBP 16. Cleavage and ligation were conducted as
described in the general procedure using 1 mM 5 as a ligation reagent.
After elution of the protein and HPLC purification, electrospray mass
spectrometry gave an observed mass of 43305, calculated mass 43311.
Modified MBP 17. Cleavage was initiated on the chitin column by
addition of a solution containing 30 mM 2-mercaptoethanesulfonic acid,
pH ) 7.5. The chitin column was then flushed with argon and incubated
for 12 h at 4 °C. After incubation, the column was eluted with 2-3
column volumes of H2O. The eluent, containing MBP-thioester was
concentrated to about 2 mL using centriprep 10,000 protein concentra-
tors (Millipore), and then the pH was adjusted to approximately 5.5-
6.0 as determined by pH paper. To this solution was added 2 mL of a
mixture containing 30 mM 2-mercaptoethanesulfonic acid and 2 mM
cysteine derivative 6 at pH ) 5.5-6.0. A small amount of protein
precipitated at this point, but the majority remained in solution. This
solution was incubated for 24 h at 4 °C and then purified by HPLC as
described in the general procedure. Observed 43315, calcd 43318.
Modified MBP 18. Cleavage and ligation were conducted as
described in the general procedure using 1 mM 7 as a ligation reagent.
After elution of the protein and HPLC purification, electrospray mass
spectrometry gave an observed mass of 43382, calcd 43390.
Modified MBP 19. 18 was prepared from MBP-intein as described
above, except that no HPLC purification was conducted. The Cen-
triprep-concentrated 18 solution was dialyzed against 10 mM Tris-HCl
pH 7.0 for 12 h to remove 2-MESA and 7. To this solution were added
70 mM Tris-HCl, 10 mM MgCl2, 5 mM dithiothreitol (DTT), and 5
mM ATP, and then T4-polynucleotide kinase (20 units) was added to
start the reaction (1 unit of T4-polynucleotide kinase is the amount of
enzyme required to catalyze the incorporation of 1 nmol of acid
insoluble 32P in 30 min at 37 °C.). The reaction was incubated at room
temperature for 20 h and then purified by HPLC as described in the
standard procedure. Observed 43460, calcd 43470.
Modified MBP 20. 15 was prepared from MBP-intein as described
above, except that no HPLC purification was conducted. The Cen-
triprep-concentrated MBP-GlcNAc solution was dialyzed against 50
mM HEPES pH 7.0 for 12 h to remove 2-MESA and 4. To this solution
were then added UDP-galactose (5 mM), Mn2+ (5 mM), and HEPES
pH 7.0 (50 mM), and then 0.2 units of galactosyl transferase was added
to start the reaction (1 unit of bovine â-1,4-galactosyltransferase (GalT)
will transfer 1.0 µmol of galactose from UDP-galactose to D-glucose
at pH 8.4 at 30 °C with 0.2 mg of R-lactalbumin/mL). The reaction
was incubated at room temperature for 20 h and then purified by HPLC
as described in the standard procedure. Observed 43516, calcd 43522.
Acknowledgment. The authors thank Professor Philip Daw-
son for advice and the use of the Dawson lab electrospray mass
spectrometer. In addition the authors to thank Dr. Sun Fong,
Dr. Igor Kozlov, and Dr. John Offer for helpful discussions.
JA0000192