Optimization of an
Escherichia coli
system for cell-free synthesis
of selectively
15
N-labelled proteins for rapid analysis by NMR
spectroscopy
Kiyoshi Ozawa, Madeleine J. Headlam, Patrick M. Schaeffer, Blair R. Henderson, Nicholas E. Dixon
and Gottfried Otting
Research School of Chemistry, Australian National University, Canberra, Australia
Cell-free protein synthesis offers rapid access to proteins that
are selectively labelled w ith [
15
N]amino acids and s uitable for
analysis by NMR spectroscopy without chromatographic
purification. A system b ased on an Escherichia c oli cell ex-
tract was optimized with regard to protein y ield and m inimal
usage of
15
N-labelled amino acid, and examined for the
presence of metabolic by-products which could interfere
with the NMR analysis. Yields of u p to 1.8 mg of human
cyclophilin A per m L of reaction m edium were obtained b y
expression of a synthetic gene. Equivalent yields were
obtained using transcription directed by either T7 or tandem
phage k p
R
and p
L
promoters, when the reactions were
supplemented with purified phage T7 or E. coli RNA

selectively isotope-labelled samples, as in vitro expression
uses much smaller volumes and therefore requires corres-
pondingly smaller quantities of expensive isotope-labelled
amino acids than conventional in vivo systems. This has
been exploited both for the production of stable-isotope
labelled proteins for NMR spectroscopy [2,4,7–11], as well
as for the incorporation of amino acid analogues [12] such
as selenomethionine [13] and 3-iodo-
L
-tyrosine [14] for
X-ray crystallography.
Proteinyieldsofupto6mgÆmL
)1
have been re ported
using expression systems based on Escheri chia coli extracts
[2,10,15,16]. At these yields, protein concentrations are
sufficiently high t o allow the recording of NMR spectra
without further concentration of the reaction medium. In
particular,
15
N-HSQC spectra of selectively
15
N-labelled
proteins can be recorded without purification of the protein,
because only signals from
15
N-labelled a mide groups are
detected [16].
15
N-HSQC spectra present well-resolved,

Correspondence to G. Otting, Australian National University,
Research School of Chemistry, Canberra, ACT 0200, Australia.
Fax: +61 2 61250750, Tel.: +61 2 61256507,
E-mail:
Abbreviations: aaRS, amino-acyl tRNA synthetase; hCypA, human
cyclophilin A; RNAP, RNA polymerase; s-CYPA, synthetic gene
encoding hCypA.
(Received 2 4 June 200 4, revised 9 August 2004,
accepted 27 August 2004)
Eur. J. Biochem. 271, 4084–4093 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04346.x
15
N-labelled protein samples produced in high-throughput
mode without further purification or concentration steps. In
addition, the following issues were addressed: (a) how do
yields c ompare, when transc ription is performed by T7
RNA polymerase from a T7 promoter or by E. coli RNA
polymerase from tandem phage k p
R
and p
L
promoters;
(b) does c ross-labelling among different amino acids occur
due to amino- or amido-transferase activity and, if so, can it
be s uppressed; (c) which amino acids are prone to the
formation of amide-containing by-products; (d) are all by-
products sufficiently small to be separated from the pr otein
product by dialysis or ultrafiltration; (e) what are the
minimum concentrations of labelled amino acids required
for good protein y ields and (f) w hich buffer c an be used to
replace the large amount of glutamate present in the original

L
-[
15
N]glut amine [aN], [
15
N]glycine,
L
-[
15
N]histidine[aN].
HCl,
L
-[
15
N]methionine,
L
-[
15
N]isoleucine,
L
-[
15
N]phenyl-
alanine,
L
-[
15
N]serine,
L
-[U-

(Parkville, Australia). Vent DNA polymerase and RNase
inhibitor were from Promega, and creatin e kinase and
E. coli total tRNA were from Roche. E. coli RNA poly-
merase (RNAP) holoenzyme was purified as described
previously [16]. Spectra/Por 2 d ialysis tubing was purchased
from Spectrum Laboratories Inc. (Rancho Dominguez,
CA, USA).
E. coli strains A19 [16], BL21(DE3)/pLysS [19] and
BL21(DE3)recA [20] were as described previously. The
plasmid pKE874 [16] was used for production of the E. coli
peptidyl-prolyl cis-trans isomerase PpiB under the control of
tandem phage k p
R
and p
L
promoters. Plasmid pBH964 was
used for cell-free expression of a synthetic gene (s-CYPA)
that encodes human cyclophilin A (hCypA) under control
of the phage T7 promoter in vector pETMCSI [21]. hCypA
was p roduced in vivo in strain BL21(DE3)/pLysS/pBH964
as a standard for comparison with protein production using
the cell-free system. The co nstruction of the s-CYPA gene
and plasmid pBH964, as well as the procedure for
purification of hCypA, are described in detail in the
Supplementary material.
Plasmid pKO1166 contains phage T7 gene 1 (which
encodes T7 R NAP) under the transcriptional control of t he
phage k p
L
promoter in vector pMA200U [22]. T7 RNAP

500 l
M
, respectively. Based on these K
m
values and the
frequency of occurrence of each amino acid in the primary
sequence of hCypA, the concentrations of [
15
N]amino acids
chosen for hCypA labelling were 50 l
M
for [
15
N]Trp and
[
15
N]Tyr, 150 l
M
for [
15
N]Ile, [
15
N]Thr and [
15
N]His,
0.35 m
M
for remaining Group II [
15
N]amino acids, and

M
) group Group
ThrRS
a
 0.002 I LeuRS  0.05 III
TrpRS
b
¼ 0.005 I GluRS  0.05 III
IleRS  0.005 I AspRS  0.06 III
TyrRS  0.008 I SerRS ¼ 0.07 III
HisRS  0.008 I MetRS  0.08 III
ArgRS ¼ 0.011 II ValRS  0.10 III
CysRS
c
¼ 0.013 II GlnRS  0.15 III
PheRS
d
 0.028 II GlyRS  0.16 III
AsnRS  0.032 II AlaRS  0.34 III
ProRS
e
– II LysRS
f
– III
a
Value for Saccharomyces carlsbergensis, no data available for
E. coli;
b
value for Lupinus luteus and bovine, no data available
for E. coli;

N]aminoacid(atthe
concentration given above), 1 m
M
each of 19 unlabelled
L
-amino ac ids, 1 5 m
M
magnesium acetate, 175 lgÆmL
)1
E. coli total t RNA, 0.05% (w/v) N aN
3
,168lLof
concentrated S30 extract (containing 5.2 mg of total
protein), 16 lgÆmL
)1
of supercoiled plasmid DNA
(pBH964, as described above), 150 U of RNase inhibitor
and 93 lgÆmL
)1
of T7 RNAP. For labelling with
[
15
N]Glu, ammonium or potassium acetate (200 m
M
)
was used instead of potassium glutamate (208 m
M
).
The inner chamber reaction mixtures were dialyzed in
Spectra/Por 2 tubing with a nominal size cutoff of

10% (v/v) D
2
O to provide a lock signal. In addition, spectra
were recorded after dialysis of the samples overnight at 4 °C
in Spectra/Por 2 tubing, against buffer comprising 1 0 m
M
sodium phosphate (pH 6.5), 100 m
M
NaNO
3
,5m
M
dithiothreitol and 5 0 l
M
NaN
3
. The dialyzed samples were
concentrated to a final v olume o f about 0.6 mL using
Millipore Ultra-4 centrifugal filters (MWCO 10 000) and
D
2
O was added to a final concentration of 10% (v/v) before
NMR measurement.
Results
Cell-free protein synthesis enhanced by T7 RNA
polymerase
Prior to the preparation of
15
N-labelled samples of hCypA
and analysis by NMR spectroscopy, the performance of the

–
E. coli host strain [BL21(DE3)recA], limitation of
thetimeculturesweretreatedat42°C during i nduction of
synthesis of T7 RNAP to 30 min, followed by treatment for
2 h at 40 °C [ 26], and use of a-toluenesulphonyl fluoride in
the buffer during cell lysis.
E. coli PpiB [16] was p roduced equally well in vitro when
using either T7 or k promoters (data not shown). Corre-
spondingly similar y ields were expected f or hCypA, as PpiB
and hCypA are f unctional homologues with similar three-
dimensional structures and amino acid compositions, and
the c odon usage of the CYPA gene had been adjusted for
the E. coli expression system by construction of an artificial
gene. The protein yields obtained in vitro for hCypA with
the T7 promoter system were about 1.5–1.8 mgÆmL
)1
of
cell-free reaction medium, and were indeed closely c ompar-
able to those obtained for PpiB with either promoter system
(Fig. 1 , l ane 8 and Fig. 2, lane 1 ). With transcription under
the control of the k promoters, however, cell-free synthesis
of hCypA was below the detection limit of SDS/PAGE with
Coomassie blue staining, even though the same plasmid
produced excellent yields in vivo (data not shown). More-
over, PpiB was always produced in vitro as a fully soluble
protein, whereas a portion (15–20%) of hCypA was
invariably found in the insoluble fraction. This is probably
due to the pI of hCypA being close to the pH of the reaction
mixture (pH 7.5); the pI value of PpiB is about one unit
lower.

of S30 extract. Concentration of the S30 extract by
dialysis against a solution of PEG 8000 [2] reduced the
volume of the in vitro reaction mixture, but had little
effect on protein yields.
The concentration of tRNA was found to affect the yields
of proteins. For production of aspartyl-tRNA synthetase
[16], for example, the optimal tRNA concentration was
about 45 lgÆmL
)1
, whereas tRNA concentrations of 87 and
175 lgÆmL
)1
worked equally well for PpiB and hCypA.
Proteins produced and stored in the reaction mixture
appeared to b e stable with respect to proteolysis. After t wo
months of storage at 4 °C, hCypA was not significantly
degraded, as evaluated by NMR measurements.
Cell-free synthesis of PpiB with i ncreasing concentra-
tions of amino acids showed that almost n o protein was
synthesized wh en e ach amino acid was present at 10 l
M
(Fig. 1 , lane 2). This result confirmed that t he extract is
depleted in natural amino acids. Absence of free amino
acids from t he cell extract is a prerequisite for efficient
incorporation of labelled amino acids in to target proteins.
Furthermore, the protein yields increased with increasing
amino acid c oncentration ( Fig. 1, lanes 1 –8), indicating
that the concentrations of amino acids limit protein
synthesis at < 1 m
M

structure of the protein and according to the K
m
values of
their respective tRNA synthetases (Table 1). Our results
confirmed that labelled amino acids from Groups I a nd II
(Table 1) could b e used a t r educed concentrations (still
several-fold above the respective K
m
values) witho ut signi-
ficantly affecting protein yields. Figure 2 shows a compar-
ison of yields obtained for hCypA with substantially
reduced concentrations of Tyr, Thr and Asn, compared to
Fig. 1. Cell-free sy nthesis of PpiB under control o f phage k promoters.
Identical vo lumes of re action products were lo ade d in to lanes o f a 15%
SDS/polyacrylamide gel, which were stained with Coomassie blue.
Lanes1,3,5and7:reactionmixturesbeforethestartofin vitro
synthesis of PpiB, with transcription by E. coli RNAP from tande m
phage k promoters (0 h reactions). Lanes 2, 4, 6 and 8: corresponding
mixtures after synthesis for 12 h at 37 °C. Each amino acid w as p resen t
at a concentration of 10 l
M
(lanes 1 and 2), 30 l
M
(lanes 3 a nd 4),
300 l
M
(lanes 5 and 6) or 1 m
M
(lanes 7 and 8). Mobilities of molecular
mass markers (kDa) were as indicated.

15
N]His. These fractions were subjected to NMR measurements
without further purification (Fig. 3). Mobilities of molecular mass
markers (kDa) were as indicated. The [
15
N]Glu-labelled hCYPA
sample in lane 1 was produced with 200 m
M
ammonium acetate in the
reaction buffer, whereas the samples in the other lanes were produced
with 208 m
M
potassium glutam ate.
Ó FEBS 2004 Cell-free synthesis of
15
N-labelled proteins (Eur. J. Biochem. 271) 4087
those obtained when all amino acids were at 1 m
M
.The
similarity in protein production levels is corroborated by the
similarity in cross-peak intensities observed in
15
N-HSQC
NMR spectra recorded of the reaction mixtures (Fig. 3).
The standard reaction mixture [2,15,16] contains a high
concentration of potassium glutamate, which makes it
unsuitable for selective labelling of Glu residues in target
proteins. A buffer with 208 m
M
potassium

CypA
15
N-HSQC spectra were recorded of hCypA samples
produced in vitro with 19 different
15
N-labelled amino
acids. Spectra w ith acceptable s ensitivity cou ld be r ecorded
at 25 °C using the reaction mixtures at pH 7.5 (Fig. 3).
Sample handling was kept to a minimum to explore the
potential of this methodology for high-throughput protein
analysis. Although spectra recorded before and after
ultracentrifugation were not significantly different, all data
presented in F ig. 3 were recorded after the ribosomes and
other m acromole cular assemblies h ad been removed by
ultracentrifugation to avoid the formation of a precipitate
during data acquisition. The NMR chemical shifts of
purified hCypA are k nown [27], allowing the identification
of individual c ross-peaks f rom t he protein a nd detection of
any additional cross-peaks due to metabolic by-products.
The spectra recorded for hCypA produced with
15
N-labelled
Asn, Gln, Ile, Leu, Phe and Tyr c ontained only cross-peaks
from th e p rotein. Samples with
15
N-labelled A rg, A sp, His,
Lys, Met, Thr and Val contained a few additional cross-
peaks, due to limited metabolic conversion of the labelled
amino acids. The additional peaks were, however, less
intense than the average of the protein cross-peaks. Samples

(fourth panel of Fig. 3). The intensities of the undesired
[
15
N]Asn cross-peaks were about two thirds of those of the
[
15
N]Asp cross-peaks, indicating highly efficient transami-
nation/transamidation. This suggests that the labelled
amino group is not released in the form o f ammonia
because i t would h ave been diluted by the presence of
27.5 m
M
ammonium present in the reaction buffer. The
E. coli asparagine synthetases A and B (asnA and asnB gene
products) s ynthesize A sn from Asp and may be r esponsible
for this activity in the S30 extract. Remarkably, no evidence
of transamination was observed i n the [
15
N]Asn s ample.
(The additional cross-peaks in Fig. 3 are due to the side-
chain amides which were labelled in the [U-
15
N]Asn
substrate). We further noted that the amido/aminotrans-
ferase activity could be suppressed by replacing potassium
glutamate in the buffer b y ammonium or potassium acetate
(last p anel of Fig. 3). This new buffer did not affect the
production levels of the proteins tested (hCypA, PpiB and
ubiquitin) to a significant degree (e.g. see l ane 1 in Fig. 2).
We also compared the buffers with 200 m

15
N-amino acids. The spectra were recorded at 25 °C and pH 7.5 using the in vitr o
reaction mixture after centrifugation (100 000 g, 4 h) and addition of 10% D
2
O. The assignments of the backbone a mide cross- peaks are indicated
by the one-letter amino acid symbols and the sequence numbers. Sq uares i dentify the c ross peaks which could b e assigned acc ording to the
previously publ ished assignment [27]. Circles identify the cross-peaks from metabolites. Dotted squares mark the p osit ions of cross-peaks w hic h
were assigned at pH 6.5 [27] but are absent from the present spectra or observable only at lower plot levels. The spectrum recorded with [
l5
N]Asn
also contains the cross-peaks from t he side-chain amide gr oups (backbone and side-chain NH groups were labelled in the amino acid used in the
reaction). Th e cross-peak from the side-chain N H of W121 i s labelled W121e. Question marks in the spectra of [
15
N]Met and [
15
N]Val hCypA
identify tentative n ew assignments of cross-peaks which had n ot been assigned previously [27].
4088 K. Ozawa et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Ó FEBS 2004 Cell-free synthesis of
15
N-labelled proteins (Eur. J. Biochem. 271) 4089
Fig. 3. (Continued).
4090 K. Ozawa et al.(Eur. J. Biochem. 271) Ó FEBS 2004
The cell-free expression system lends itself to t he addition
of specific enzyme inhibitors, such as the alanine racemase
inhibitor, b-chloro-
L
-alanine [28]. When we t ested the effect
of adding b-chloro-
L

the activity of p eptide deformylase ( D. Mouradov and
T. Huber, unpublished data). I n the present case of hCypA,
retention of the N-formyl group enables the ob servation o f
the cross-peaks from the amino-terminal amide protons.
Interestingly, ESI mass spectrometric analysis of hCypA
produced in vivo in E. coli also showed N-terminal hetero-
geneity. Although  70% of the protein s tarted with a
deformylated Met (18 012 Da),  20% had N-terminal
N-formyl-Met (18 040 Da) and  10% had l ost the
N-terminal Met (17 881 Da ).
In our experiments, weak cross-peaks with broad line
shapes were difficult to detect. Hence not all signals assigned
previously [27] could be observed. As the original assign-
ments h ad been reported f or pH 6.5, we measured the
15
N-HSQCspectraafterdialysisatthispH.ThelowerpH
value enhanced some of the signals as expected. For
example, the cross-peak of Asp9 was observed at pH 6.5
(data not shown), whereas it was missing from the spectrum
recorded at pH 7.5 (Fig. 3). In contrast to the previously
reported assignment [27], there was no evidence for more
than a s ingle conformation of His70, neither at pH 7.5 nor
at pH 6.5.
Discussion
In this study, several aspects of a cell-free protein
synthesis system b ased on an E. coli cell extract were
investigated and optimized. O ne po int is the observation
that T7 RNA polymerase performs as well as the E. coli
holoenzyme in the in vitro coupled transcription/transla-
tion system. As many modern plasmid vectors used for

latter condition is easily fulfilled. For most amino acids,
purification of the prod uced
15
N-labelled p rote in is not
required for identification of the HSQC cross-peaks from
the protein. In the few cases where cross-peaks from
metabolites and protein could overlap, a simple dialysis step
is sufficient to r emove the metabolites. The easy removal of
the i nterfering signals from metabolites is a clear advantage
of the cell-free expression system over in-cell NMR analyses
[29,30]. Interestingly, only low-mass metabolites appear to
be produced also when [U-
15
N]protein is s ynthesized in vivo
using ammonium chloride [31].
Most importantly, the transf er of the
15
N-label t o other
amino acids is insignificant for 1 8 of the amino acids and
can b e suppressed for [
15
N]Asp b y use of a modified
medium in which glutamate is replaced by acetate. Notably,
these r esults were achieved without preparation of
extracts from auxotrophic E. coli strains. Replacement of
glutamate by acetate has recently also been described by
Klammt et al.[7].
Cell-free expression kits for large-scale protein produc-
tion have become commercially available, making cell-free
expression a r eadily accessible technology [7,11,32]. Our

15
N-labelled proteins (Eur. J. Biochem. 271) 4091
Acknowledgements
We thank Dr S imon Bennett for m easurements o f ESI mass spectra.
K.O. and G.O. thank the Australian Research Coun cil for a CSIRO-
Australian Postdoctoral and Federa tion Fellowships, respectively.
Financial support by the Australian Research Council is gratefully
acknowledged.
References
1. Spirin, A.S., Baranov, V.I., Ryab ova, L.A., Ovodov, S.Y. &
Alakhov, Y.B. (1988) A continuous cell-free translation system
capable of producing polypeptides in high yield. Science 242,
1162–1164.
2. Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M ., Ito, Y., S hi-
bata,T.&Yokoyama,S.(1999)Cell-freeproductionandstable-
isotope l abeling of m illigram quantities of proteins. FEBS Lett.
442, 15–19.
3. Yokoyama, S. (2003) Protein expression systems for structural
genomics and proteomics. Curr. Opin. Chem. Biol. 7, 39–43.
4. Shi,J.,Pelton,J.G.,Cho,H.S.&Wemmer,D.E.(2004)Protein
signal assign ments using sp ecific labeling a nd cell-free synthesis.
J. Biomol. N MR 28, 2 35–247.
5. Swartz, J.R., Jewett, M.C. & Woodrow, K.A. (2004) Cell-free
protein synthesis with prokaryotic combined t ranscription-trans-
lation. Methods Mol. Biol. 267, 169–182.
6. Jewett, M.C. & Swartz, J.R. (2004) Mimicking the Escherichia coli
cytoplasmic environment activates long-lived and efficient cell-free
protein synthesis. Biotech. Bioeng. 86, 1 9–26.
7. Klammt, C., Lo
¨

ment of b ackbone amide NMR re sonanc es. J. Am. Che m. Soc.
126, 5020–5021.
12. Hendrickson, T.L. de Cre
´
cy-Lagard V. & Schimmel, P. (2004)
Incorporation of nonnatural amino acids into p roteins. Annu. Rev.
Biochem. 73 , 147–176.
13. Kigawa, T., Yamaguchi-Nunokawa, E., K odama, K ., Matsuda,
T., Yabuki, T., Matsuda, N., Ishitani, R., N ureki, O. &
Yokoyama, S. (2002) Selenomethionine incorporation into a
protein by cell-free synthesis. J. Struct. Funct. Genomics 2, 29–35.
14. Kiga, D., S akamoto, K., Kodama, K ., Kigawa, T., M atsuda, T.,
Yabuki, T., Shirouzu, M., Harada, Y., Nakayama, H., Takio, K.,
Hasegawa, Y., Endo, Y., Hirao, I. & Yokoyama, S. (2002) An
engineered Escherichi a coli tyrosyl-tRNA synthetase fo r site-
specific incorporation of an unnatural amino acid into proteins in
eukaryotic translation and its application in a wheat germ cell-free
system. Proc.NatlAcad.Sci.USA99, 9715–9720.
15. Kigawa, T., Yabuki, T., Matsuda, N., Matsuda, T., Nakajima, R.,
Tanaka, A. & Yokoyama, S. (2004) Preparation of Escherichia coli
extract for highly productive cell-free protein expression. J. Struct.
Funct. Genomics 5, 63–68.
16. Guignard, L., Ozawa, K., Pursglove, S.E., Otting, G. & Dixon,
N.E. (2002) NMR analysis of in vitro-synthesized prote ins without
purification: a h igh-throughp ut approach . FEB S Lett. 524,
159–162.
17. Yamazaki, T ., Yoshida , M., Kanaya, S., Nakamura, H. &
Nagayama, K. (1991) Assignments of backbone
1
H,

in Escherichia c oli. Gene 87, 123–126.
23. Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities o f protein utilizing the
principle of protein-dye binding. Anal. B iochem. 72, 248–254.
24. Pratt, J.M. (1984) Coupled transcription-translation in prokary-
otic cell-free systems. In Transcription and Translation
(Hames, B.D. & Higgins, S.J., eds), pp. 179–209. IRL Press,
Oxford, UK.
25. Young, B.A., Gruber, T.M. & Gross, C.A. (200 2) Views of tran-
scription initiation. Cell 109, 417–420.
26. Tabor, S. & Richardson, C.C. (1985) A bacteriophage T7 RNA
polymerase/promoter system for controlled exclusive expres-
sion of specifi c genes. Proc.NatlAcad.Sci.USA82, 1 074–1078.
27. Ottiger,M.,Zerbe,O.,Gu
¨
ntert, P. & Wu
¨
thrich, K . (1997) The
NMR solution c onform ation o f unligated human cyclophilin A.
J. Mol. Biol. 272, 64–81.
28. Kato, K., Matsunaga, C., Igarashi, T., Kim, H H., Odaka, A.,
Shimada, I. & Arata, Y. (1991) Complete assignment of
the methionyl carbonyl carbon r esonances in switch-variant
anti-dansyl a ntibodies labeled with [1–
13
C]methionine. Biochem-
istry 30, 270–278.
29. Ou, H.D., Lai, H.C., Serber, Z. & Do
¨
tsch, V. (2001) Efficient

the E. coli ppiB gene, using a combination of ligation and
recursive overlap extension of complementary synthetic
oligonucleotides as described above. Oligonucleotides used
for the construction of the s-CYPA are i dentified by arrows
above the complete gene sequence. T he Nd eI, Apa I, FokI,
XhoI, EcoRI and NcoI restriction endonuclease sites are
boxed. The start codon (ATG) is within t he NdeIsiteandthe
stop codon (TAA) is i dentified by a black box in the linker.
Fig. S 2. Plasmid pKO1166. T his plasmid, which directs
overproduction of T7 RNA polymerase, was constructed by
insertion of a DNA fragment bearing T7 gene 1 under
control of the bacteriophage k p
L
promoter into vector
pMA200U [5].
Ó FEBS 2004 Cell-free synthesis of
15
N-labelled proteins (Eur. J. Biochem. 271) 4093