Journal of Insect Biotechnology and Sericology 82, 00-00 (2013)

Residue-specific incorporation of phenylalanine analogues
into protein biosynthesis in silkworm cultured cells
Hidetoshi Teramoto＊ and Katsura Kojima
Silk Materials Research Unit, Genetically Modified Organism Research Center,
National Institute of Agrobiological Sciences (NIAS), 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan(Received August 1, 2013; Accepted October 21, 2013)

Here we describe the residue-specific incorporation of para-substituted L-phenylalanine (Phe) analogues into
protein biosynthesis in Bombyx mori cultured cells, BmN, which express B. mori phenylalanyl-tRNA synthetase(BmPheRS) mutants with relaxed amino acid specificity. Aminoacylation capabilities of BmPheRS mutants(Ala450 to Gly and Thr407 to Ala or Gly mutants in the α-subunit) were first investigated in vitro and then the incorporation of Phe analogues into a reporter protein, EGFP, was investigated using BmN cells expressing one of
the above mutants. We also evaluated the effects of lowering the Phe concentration in culture media, which competes with Phe analogues in aminoacylation reactions inside cells, and found that lowering Phe concentration
was effective in increasing the substitution rate from Phe to its analogues in EGFP. We conducted electrophoretic
and mass spectrometric analyses of synthesized EGFP to clarify the incorporation of Phe analogues. We obtained direct mass spectrometric data demonstrating the incorporation of p-bromo- and p-cyano-L-phenylalanine
and indirect electrophoretic data, implying the incorporation of p-azido- and p-iodo-L-phenylalanine into protein
biosynthesis in BmN cells.
Key words: unnatural amino acids, phenylalanine analogues, aminoacyl-tRNA synsthetase, BmN cells, silkworm, Bombyx mori

INTRODUCTION
In vivo incorporation of unnatural amino acids (UAAs)
into proteins has become a powerful tool, especially inprobing protein functions in living systems and in creatingnovel proteins with novel biological functions (Davis andChin, 2012; Neumann, 2012; Zheng and Kwon, 2012). In
vivo UAAs incorporation has been achieved with two major methods: site-specific (Neumann, 2012) and residuespecific (Singh-Blom et al., 2013). The site-specific method
employs orthogonal tRNA/aminoacyl-tRNA synthetase(aaRS) pairs specific for target UAAs, which are incorporated into proteins at specific sites in response to uniquecodons such as UAG. In contrast, the residue-specificmethod can be achieved in a relatively simple way: it requires only the engineering of aaRSs to expand their recognition capabilities. To date, various organisms fromunicellular (Chin et al., 2003; Link and Tirrell, 2005;
Wang et al., 2001; Wiltschi et al., 2008; Ye et al., 2013)
to multicellular (Bianco et al., 2012; Greiss and Chin,
2011; Parrish et al., 2012) have been engineered to incorporate UAAs bearing a wide variety of functional groups
into proteins in vivo by using one of the two methods
above. We recently reported on the residue-specific incorporation of p-chloro-L-phenylalanine (ClPhe) into protein
biosynthesis in BmN cells, the ovary-derived cultured cellsof the domesticated silkworm, Bombyx mori (Teramoto et*To whom correspondence should be addressed.
Fax: +81-29-838-6028. Tel: +81-29-838-6213.
Email: teramoto@affrc.go.jp

al., 2012). Here we report further expansion of the repertoire of para-substituted L-phenylalanine (Phe) analogues
which can be residue-specifically incorporated as building
blocks in protein biosynthesis in BmN cells.
B. mori has been one of the most important animals inindustrial applications because of its incredible ability inprotein biosynthesis: they secrete hundreds of milligramsof silk proteins per larva to make cocoons before pupation. Since ancient times, silk fiber has been attractive forits brilliant texture, toughness, and many other excellent
characteristics. Silk has also been used as a surgical suture and can be easily transformed into various shapes, including transparent film and three-dimensional porous
materials (Rockwood et al., 2011; Tamada, 2005). These
properties of silk have prompted many researchers to develop silk-based materials equipped with excellent biocompatibility and mechanical strength for use in biomedical
fields (Kawakami et al., 2011; Leal-Egaña and Scheibel,2010). In addition, B. mori is useful as a bio-factory to
produce recombinant proteins by baculovirus infection
and transgenic techniques (Hong et al., 2010; Lee et al.,2012; Tomita, 2011). To expand the repertoire of aminoacid building blocks in B. mori protein biosynthesis would
therefore largely increase the utility of B. mori as an industrial animal.
We have previously cloned two genes encoding two
subunits (α and β) of B. mori phenylalanyl-tRNA synthetase (BmPheRS), where its amino acid recognition site is
located in the α-subunit, and have succeeded in relaxing theamino acid specificity of BmPheRS by amino acid substitution from Ala450 to Gly in its α-subunit to recognize

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ClPhe and p-bromo-L-phenylalanine (BrPhe) (Teramoto
and Kojima, 2010). We have subsequently demonstrated
that ClPhe can be incorporated into protein biosynthesisin BmN cells expressing the αA450G BmPheRS mutant(Teramoto et al., 2012). However, incorporation of BrPhe
was not observed, possibly due to its relatively slow recognition by the mutant under competitive conditions with
the natural substrate, Phe.
On the other hand, the αT415A Saccharomyces cerevisiae PheRS (ScPheRS) mutant was previously reported tobe effective for the incorporation of BrPhe into proteinsin Escherichia coli (Kwon et al., 2006). A sequence alignment shows that the αT415 residue of ScPheRS corresponds to the αT407 residue of BmPheRS (Teramoto andKojima, 2010). Therefore, this amino acid residue could
be another target for introducing novel mutations to relax
the amino acid specificity of BmPheRS. In addition, removal of natural substrates which compete against UAAs
from culture media is a quite effective method to maximize the incorporation efficiency of UAAs (Singh-Blomet al., 2013). Although protein biosynthesis in BmN cells
slows down extremely in culture media completely lacking Phe, which is one of the essential amino acids of B.
mori, just lowering Phe concentration would be feasible.
Such a decrease of Phe concentration would accelerate theaminoacylation reaction of tRNAPhe with Phe analogues
bearing larger functionalities than Cl and might accomplish the incorporation of BrPhe and other Phe analogues
into protein biosynthesis in BmN cells.
In this study, we aimed to further expand the aminoacid repertoire of B. mori protein biosynthesis by meansof (1) employment of novel BmPheRS mutants bearing
different amino acid mutations (αT407A and αT407G) and(2) lowering Phe concentration in culture media. We investigated the incorporation of six para-substituted Phe
analogues, ClPhe, BrPhe, p-iodo-L-phenylalanine (IPhe),p-amino-L-phenylalanine (AmPhe), p-cyano-L-phenylalanine (CyPhe), and p-azido-L-phenylalanine (AzPhe) (Fig. 1),
and revealed that BrPhe, CyPhe, and presumably AzPheand IPhe were incorporated into protein biosynthesis in
BmN cells in place of Phe.

MATERIALS AND METHODS
Materials
All chemicals used in this study were of reagent grade
and used as received. Phe analogues, ClPhe, BrPhe, IPhe,and AzPhe, were from Bachem AG (Bubendorf, Switzerland), and AmPhe • HCl and CyPhe were from Watanabe
Chemical Industries (Hiroshima). An ovary-derived cellline of B. mori, BmN, was provided by the RIKEN BioResource Center and adapted to Grace’s insect medium(Life Technologies; Carlsbad, CA, USA) supplemented

with a 10% fetal bovine serum (FBS) (Life Technologies).
Grace’s insect medium with lowered Phe concentration
was prepared by adding a requisite amount of Phe to thespecially-prepared Grace’s insect medium completely lacking Phe (Nacalai tesque; Kyoto). The temperature for the
BmN culture was set to 26°C throughout the experiments.
Plasmids construction
A commercially available expression vector pQE-30(QIAGEN; Hilden, Germany) was used for recombinant
protein expression in E. coli strain M15[pREP4] (QIAGEN). The pQE-30 plasmids encoding the wild-type
BmPheRS or one of the αA450G, αT407A, and αT407G
BmPheRS mutants were constructed, as reported previously (Teramoto and Kojima, 2010). For introducing amino acid substitutions, that is, Thr407 to Ala and Gly inthe α-subunit of BmPheRS, the following primer sets
were used: 5’-CAACCCCTACGCCGAACCCAGCATGG
AAA-3’ and 5’-ATTTCCATGCTGGGTTCGGCGTAGGG
GTTG-3’ for the T407A mutation and 5’-CAACCCCTACGGCGAACCCAGCATGGAAA-3’ and 5’-ATTTCCATGCTGGGTTCGCCGTAGGGGTTG-3’ for the T407G mutation, where the original Thr codon and the mutated nucleotides are respectively underlined and in bold. An expression
plasmid bearing an improved ie1 promoter derived fromthe B. mori nuclear polyhedrosis virus (Kojima, 2002)
was used to express the wild-type or one of the mutantsof the α-subunit of BmPheRS, the wild-type β-subunit of
BmPheRS, and an enhanced green fluorescent protein(EGFP) as a reporter protein (bearing a Strep tag on its
N-terminal) in BmN cells, as with the previous study(Teramoto et al., 2012).
In vitro aminoacylation assay
In vitro-transcribed B. mori tRNAPhe and recombinant
BmPheRSs (wild-type or one of αA450G, αT407A, and
αT407G mutants) were prepared, as reported previously(Teramoto and Kojima, 2010). The capability of each recombinant BmPheRS to aminoacylate B. mori tRNAPhewith Phe and its analogues was evaluated in vitro basedon the previous report (Teramoto and Kojima, 2010).
Briefly, aminoacylation reactions were performed for 30
min at 30°C in 10 μL of reaction mix (0.1 M Na-HEPES
pH 7.5, 30 mM KCl, 10 mM MgCl2, 2 mM Mg-ATP, and2.5 μM in vitro-transcribed B. mori tRNAPhe) with 25 μM
amino acids and 0.01 A280 units of recombinant BmPheRSs,
where one A280 unit is defined as the protein amount thatgives an A280 value of 1 when dissolved in 80 μL of 6 M
guanidine hydrochloride and is measured lengthwise in a
1-cm path. Reactions were stopped by adding 16.7 μL of
a quenching solution, which was a 4:1 mixture of acidurea PAGE sample buffer (0.1 M NaOAc pH 5.0, 8 M
urea, 0.05% xylene cyanol, and 0.05% bromophenolblue)

Incorporation of Phe analogues in BmN cells

and 80% glycerol, to each 10 μL reaction solution followed by vortexing. Unreacted and aminoacylated B. moritRNAPhe were separated by electrophoresis on a 10% acidurea polyacrylamide gel in 0.1 M NaOAc (pH 5.0) at 4°C
at 9 V/cm until xylene cyanol migrated about 12 cm. Separated tRNAs were visualized with GelRed (Biotium Inc.;
Hayward, CA, USA).
Cell culture assay
Incorporation of Phe analogues in EGFP synthesized in
BmN cells in place of Phe by BmPheRS mutants was investigated based on our previous report (Teramoto et al.,2012). BmN cells (1.4-3.0 × 106) were seeded in 25 cm2
culture flasks and transfected with the plasmid vectors expressing the wild-type or one of the mutants of the
α-subunit of BmPheRS (1.5 μg/flask) and the wild-typeβ-subunit of BmPheRS (1.5 μg/flask) and EGFP (3 μg/
flask) under a serum-free condition using the HilyMax
transfection reagent (24 μL/flask) (Dojindo; Kumamoto)according to the manufacturer’s instructions. After overnight transfection, the medium was changed to a fresh one
supplemented with 10% FBS –with or without Phe analogues. Although gene expressions from transfected plasmids were generally expected to become maximal in two
to three days after transfection, the amount of EGFP accumulated in cells was not sufficient for analyzing the incorporation of Phe analogues by mass spectrometry at thattime. Hence, the transfected cells were cultured for 9 days
at 26°C with two medium changes at a three-day interval.
The cells were then harvested and lysed in Buffer W(100 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA)
supplemented with 1% (v/v) Igepal CA-630 by a 10-min
incubation on ice. The cleared lysates obtained after centrifugation (9500 × g, 4°C, 15 min) were purified under anative condition using Strep-Tactin spin columns (IBA;
Göttingen, Germany) according to the manufacturer’s instructions. Five micro liters of the affinity-purified EGFP
eluted with 50 μL of Buffer BE (100 mM Tris-HCl pH 8,
150 mM NaCl, 1 mM EDTA, 2 mM D-biotin) were subjected to SDS-PAGE. Electrophoresis was performed bystandard techniques using Any kD TGX precast gels (BioRad; Hercules, CA, USA) at a constant voltage of 200 V.
Separated proteins were stained by EzStain AQua (ATTO;
Tokyo).
In-gel digestion
The affinity-purified EGFP was separated on 12.5% ePAGEL precast gels (ATTO) at a constant current of
20 mA and visualized by EzStain Aqua (ATTO). Slices
containing EGFP were excised from the gels and cut into
small pieces. The gel pieces were then destained in 50%
acetonitrile/ 100 mM NH4HCO3 at 37°C with shaking,washed thoroughly in 50% methanol/ 10% acetic acid,3

equilibrated in 50 mM NH4HCO3, dehydrated in acetonitrile, and dried in a vacuum centrifuge. The minimum volume of the solution containing 0.02 μg/μL Trypsin Gold
(Promega; Fitchburg, WI, USA) in 40 mM NH4HCO3/
10% acetonitrile was infiltrated into the dried gel pieces
and the extra solution was removed. The hydrated gel
pieces were covered with a digestion buffer (40 mM
NH4HCO3, 10% acetonitrile) and incubated at 37°C overnight. Digested peptide fragments were extracted with water followed by 50% acetonitrile/ 5% trifluoroacetic acid
(TFA) and then dried in a vacuum centrifuge. The dried
peptide fragments were stored at −20°C until analysis.
MALDI-TOF-MS
The trypsin-digested peptide fragments were dissolvedin a small amount of TA (2:1 mixture of 0.1% TFA andacetonitrile) and mixed with α-cyano-4-hydroxycinnamic
acid solution saturated in TA. The mixtures were spottedon a target plate and dried at room temperature. MALDITOF-MS spectra were collected in the positive ion reflector mode with an autoflex III mass spectrometer (Bruker
Daltonics; Billerica, MA, USA). The mass spectrometricdata was analyzed by mMass software (Niedermeyer andStrohalm, 2012).

RESULTS AND DISCUSSION
In vitro aminoacylation assay of the recombinant
BmPheRS mutants
Either the wild-type BmPheRS or one of the αA450G,
αT407A and αT407G BmPheRS mutants were expressedin E. coli and affinity-purified. Their capabilities to aminoacylate B. mori tRNAPhe with six para-substituted Phe
analogues (Fig. 1) were assayed in vitro. Aminoacylation
was detected by acid-urea polyacrylamide gel electrophoresis (acid-urea PAGE) (Fig. 2). The wild-type BmPheRS
aminoacylated the tRNA only with Phe. As previously reported, B. mori tRNAPhe was aminoacylated with ClPheand BrPhe by the αA450G mutant (Teramoto and Kojima,
2010). These two Phe analogues were also recognized by
the αT407A and αT407G mutants. The tRNA was only
slightly aminoacylated with IPhe by the αA450G mutant,as previously reported (Teramoto and Kojima, 2010). The
αT407A mutant exhibited a similar result in aminoacylation with IPhe while the αT407G mutant showed a much
improved recognition of IPhe. It was found that AmPhe
was not a good substrate for all three mutants and that
CyPhe was weakly recognized by all three mutants.
AzPhe, which bears an azide functionality useful for chemoselective reactions (Lallana et al., 2011; van Berkel etal., 2011), was found to be well recognized by the αT407G
mutant while the other two mutants showed a weaker recognition.

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Fig. 1.  Structures of the natural substrate, L-phenylalanine (Phe), and its para-substituted analogues investigated inthis study. ClPhe: p-chloro-L-phenylalanine, BrPhe: p-bromo-L-phenylalanine, IPhe: p-iodo-L-phenylalanine, AmPhe: pamino-L-phenylalanine, CyPhe: p-cyano-L-phenylalanine, AzPhe: p-azido-L-phenylalanine.

Fig. 2.  In vitro aminoacylation of B. mori tRNAPhe with Phe and six para-substituted Phe analogues by the wild-type
BmPheRS or one of the αA450G, αT407A, or αT407G BmPheRS mutants. After 30 min aminoacylation reactions at
30°C, the unreacted and aminoacylated tRNAs were separated by acid-urea PAGE where the aminoacylated tRNAs(aa-tRNAPhe) exhibited slower mobility than the unreacted tRNA.

Effects of expressing BmPheRS mutants on protein biosynthesis in BmN cells under normal and
low Phe conditions
Expression of BmPheRS mutants with relaxed aminoacid specificity might be toxic for living systems because
they might recognize inappropriate amino acids as a substrate. Such adverse effects arising from the expression in

BmPheRS mutants can be speculated to be more serious
when the concentration of the native substrate, Phe, in theculture media is lowered. We then tested the effects of expressing BmPheRS mutants in BmN cells on their protein
biosynthesis activity under normal and low Phe conditions
using EGFP as a reporter protein.
Fig. 3A shows EGFP fluorescence in BmN cells ex-

Incorporation of Phe analogues in BmN cells

5Fig. 3.  Effects of expressing the BmPheRS mutants on protein biosynthesis in BmN cells. (A) Images showing
EGFP fluorescence in BmN cells expressing the wild-type BmPheRS or one of the αA450G, αT407A, or αT407G
BmPheRS mutants. The cells were cultured for 9 days under normal (0.9 mM) and low (0.1 mM) Phe concentrations.
The dark-field images detecting fluorescence of EGFP were superimposed on the bright-field images. (B) The SDSPAGE of affinity-purified EGFP obtained from BmN cells cultured under each condition shown in Panel A. The constant volume of the affinity-purified EGFP solution was loaded on SDS-PAGE gels to compare the relative amount ofsynthesized EGFP among different culture conditions.

pressing the wild-type BmPheRS or one of the αA450G,
αT407A, and αT407G BmPheRS mutants. The cells were
cultured for 9 days under normal (0.9 mM) and low(0.1 mM) Phe conditions. The synthesized EGFP was affinity-purified from cell lysates and subjected to SDS-PAGE(Fig. 3B). Under both normal and low Phe conditions, adrastic decrease of EGFP synthesis was observed whenthe αT407G mutant was expressed, suggesting a misincorporation of inappropriate amino acids in place of Phe.
This observation is consistent with the in vitro assay results where the αT407G mutant showed mostly relaxed
substrate specificity (Fig. 2). On the other hand, the othertwo mutants exhibited little effects on EGFP synthesis under normal Phe concentration. However, EGFP synthesis
largely decreased when the αT407A mutant was expressed
under low Phe conditions. This observation shows that themisincorporation of inappropriate amino acids by the
αT407A mutant was negligible with sufficient amounts ofPhe but became noticeable when Phe was lowered. In
contrast, expression of the αA450G mutant continued tohave little effect on EGFP synthesis even under low Phe

conditions. From these experiments, it was found that the
αT407G mutant is inappropriate for assays using BmN
cells due to its excessive relaxation of its amino acidspecificity and that the αT407A mutant could be only employed under normal Phe conditions.
It was previously reported that L-lysine (Lys) and Ltryptophan (Trp) was misincorporated in place of Phe
when the αT415G ScPheRS mutant was expressed in E.
coli (Kwon et al., 2006), where the αT415 residue in
ScPheRS corresponds to the αT407 residue in BmPheRS
according to sequence alignment (Teramoto and Kojima,
2010). It is thus likely that Lys and/or Trp were misincorporated in place of Phe in protein biosynthesis in BmN
cells when EGFP synthesis decreased.
Incorporation of BrPhe in EGFP synthesized in
BmN cells expressing the αT407A BmPheRS mutant
Fig. 2 shows that the αT407A BmPheRS mutant recognizes BrPhe in vitro as well as the αA450G mutant. We
then investigated whether BrPhe can be incorporated into

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protein biosynthesis in BmN cells expressing the αT407A
mutant under normal Phe conditions. The synthesized
EGFP was affinity-purified and subjected to SDS-PAGE(Fig. 4A). The SDS-PAGE data exhibited that the amountof EGFP gradually decreased with the increase in BrPhe
concentration. Such a decrease of EGFP was not observedin the cells expressing the wild-type BmPheRS even in
the presence of 1 mM BrPhe. When BmPheRS mutants
successfully aminoacylate B. mori tRNAPhe with Phe analogues in BmN cells, the analogues will be equally incorporated into all newly-synthesized proteins, including
essential enzymes for protein biosynthesis machinery,
which would slow down the biosynthesis of proteins, including EGFP. In fact, a marked decrease of EGFP waspreviously observed for the incorporation of ClPhe withthe αA450G mutant (Teramoto et al., 2012). Hence, a decrease of EGFP synthesis could be indicative of the incorporation of Phe analogues into protein biosynthesis.
MALDI-TOF-MS analysis of the trypsin-digested fragments of EGFP showed that BrPhe was partially incorporated in place of Phe (Fig. 4B). This result implies thatthe αT407A mutant is more appropriate for incorporating
BrPhe into proteins in BmN cells than the αA450G mutant which did not show detectable incorporation of BrPhe
under the same assay conditions (Teramoto et al., 2012).
As with the previous results observed for ClPhe in­corporation (Teramoto et al., 2012), the mobility of EGFP
in SDS-PAGE did not change by BrPhe incorporation(Fig. 4A). From a rough estimation of relative peak intensities in MALDI-TOF-MS spectra (Fig. 4B) by assumingthat ionization efficiencies of the analyzed peptides and its
counterparts containing BrPhe are identical, the replacement ratio from Phe to BrPhe was estimated to be at mostca. 5%. Since EGFP contains 14 Phe residues, the 5% replacement denotes that, at most, one Phe residue was replaced by BrPhe in EGFP. This replacement leads toa ~ 79 Da increase of the molecular weight of EGFP
which is ca. 30 kDa including purification tags. Such afaint increase of molecular weight will have practically noeffects on the mobility of EGFP. For this reason, we did
not observe any changes in the EGFP mobility in SDSPAGE concerning the incorporation of different Phe analogues, as described later.
Increased substitution rate by reducing Phe concentration in culture medium
As previously reported, ClPhe can be incorporated into
protein biosynthesis in BmN cells expressing the αA450G
BmPheRS mutant by simply adding it to the culture
medium which contain standard concentrations of Phe(0.9 mM) (Teramoto et al., 2012). It will be important toknow whether the replacement ratio from Phe to ClPhe
increases when the Phe concentration in the medium islowered. Fig. 5A shows the SDS-PAGE pattern of the affinity-purified EGFP, expressed in normal (0.9 mM) andlow (0.1 mM) Phe conditions in the presence of ClPhe. A
drastic decrease in the EGFP amount was observed when
the cells expressing the αA450G mutant were cultured under low Phe conditions. The observed, relatively larger
decrease of EGFP synthesis compared to those under normal Phe conditions could be attributable to an increased
incorporation of ClPhe into protein biosynthesis. As expected, MALDI-TOF-MS analysis of the trypsin-digested
peptide fragments demonstrated that lowering the Pheconcentration in the culture medium led to a relatively increased replacement ratio from Phe to ClPhe (Fig. 5B).
Incorporation of BrPhe into EGFP synthesized in
BmN cells expressing the αA450G BmPheRS mutant under low Phe conditions
An increased replacement ratio from Phe to ClPhe under low Phe conditions prompted us to investigate the incorporation of BrPhe into EGFP synthesized in BmN cells
expressing the αA450G BmPheRS mutant under low Phe
conditions (0.1 mM). SDS-PAGE of the affinity-purified
EGFP showed that its amount gradually decreased with
the increase of BrPhe concentration when the αA450G
mutant was expressed (Fig. 6A). This behavior is similar
to the case of the αT407A mutant shown in Fig. 4A, suggesting that BrPhe was incorporated into protein biosynthesis. MALDI-TOF-MS analysis of the trypsin-digested
fragments of EGFP confirmed that a small portion of Phe
was replaced with BrPhe (Fig. 6B). This observation demonstrated that lowering Phe concentration in the culture
medium is effective for incorporating Phe analogues
which are not normally incorporated into protein biosynthesis in BmN cells.
Incorporation of CyPhe, AzPhe, and IPhe in EGFP
synthesized in BmN cells expressing the αA450G
BmPheRS mutant under low Phe conditions
Incorporation of CyPhe, AzPhe, and IPhe, which wererelatively weakly recognized with the αA450G BmPheRS
mutant (Fig. 2), was investigated. It was previously reported that these three Phe analogues were all incorporated
into protein biosynthesis in E. coli expressing the αA294G
E. coli PheRS mutant (Kirshenbaum et al., 2002). Thismutant has a homologous amino acid substitution with the
αA450G BmPheRS mutant.
Incorporation of CyPhe was first investigated under low
Phe conditions. EGFP synthesis in BmN cells cultured under a 0.1 mM Phe condition was not impeded (Fig. 6A),
implying no remarkable incorporation of CyPhe. This observation might reflect the in vitro assay results (Fig. 2),that is, the recognition of CyPhe by the αA450G mutant
was weaker than that of BrPhe, which was incorporated inIncorporation of Phe analogues in BmN cells

Fig. 4. Analysis of EGFP produced in BmN cells expressing
the αT407A BmPheRS mutant cultured in the presence of
BrPhe. (A) The SDS-PAGE of the affinity-purified EGFP produced under varied conditions. The constant volume of theaffinity-purified EGFP solution was loaded on SDS-PAGE gelsto compare the relative amount of synthesized EGFP among
different culture conditions. (B) MALDI-TOF-MS spectra of thetrypsin-digested peptide fragments, TIFFKDDGNYK ([M + H]+= 1347.66 Da) and AEVKFEGDTLVNR ([M + H]+ = 1477.76 Da),
from the affinity-purified EGFP. The numbering at the left ofthe spectra corresponds to Panel A. Replacement of one Phe
residue in the fragment peptides by BrPhe leads to a theoretical mass increase of 77.91 Da (79Br-1H). The sample from
Condition 5 did not give good quality spectra due to a low
quantity of EGFP.

EGFP under the 0.1 mM Phe condition. When the concentration of Phe was further lowered to 0.02 mM, a significant decrease of EGFP synthesis was observed (Fig. 6A),
indicating the incorporation of CyPhe into protein biosynthesis. MALDI-TOF-MS analysis confirmed that a part ofPhe was substituted with CyPhe in EGFP (Fig. 6B).
We next investigated the incorporation of AzPhe, which
was moderately recognized with the αA450G mutant invitro (Fig. 2). Similar to CyPhe, EGFP synthesis was not
disrupted under the 0.1 mM Phe condition (Fig. 6A), im-

7

Fig. 5.  Analysis of EGFP produced in BmN cells expressing
the αA450G BmPheRS mutant cultured in the presence of
ClPhe under different Phe concentrations. (A) The SDS-PAGEof the affinity-purified EGFP expressed under normal (0.9 mM)
and low (0.1 mM) Phe conditions. The constant volume of theaffinity-purified EGFP solution was loaded on SDS-PAGE gelsto compare the relative amount of synthesized EGFP among
different culture conditions. (B) MALDI-TOF-MS spectra of thetrypsin-digested peptide fragment, TIFFKDDGNYK ([M + H]+= 1347.66 Da), from the affinity-purified EGFP. The numberingon the left of the spectra corresponds to Panel A. The replacement of one Phe residue in the fragment peptide to ClPhe
leads to a theoretical mass increase of 33.96 Da (35Cl-1H).

plying no remarkable incorporation of AzPhe. When Phe
is lowered to 0.02 mM, EGFP synthesis significantly decreased, as with the case of CyPhe (Fig. 6A). This result
strongly implies that AzPhe was incorporated into protein
biosynthesis. However, MALDI-TOF-MS analysis of theaffinity-purified EGFP did not give any detectable peaks
demonstrating the incorporation of AzPhe (data notshown). Trials to detect the incorporation of AzPhe by achemoselective reaction to azide groups (Lallana et al.,2011) also failed (data not shown). We supposed that thesubstitution rate from Phe to AzPhe was too small to be
detected regardless of the observed adverse effects on
EGFP synthesis. Alternatively, unwanted photolysis of aromatic azide groups during cell culture, cell lysis, and/or
EGFP purification might have made AzPhe undetectable.
We finally investigated the incorporation of IPhe, which

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Teramoto and Kojima

Fig. 6. Analysis of EGFP produced in BmN cells expressing the αA450G BmPheRS mutant cultured in the presenceof BrPhe, CyPhe, AzPhe, or IPhe under varied Phe concentrations. (A) SDS-PAGE of the affinity-purified EGFP. A
constant volume of the affinity-purified EGFP solution was loaded on SDS-PAGE gels to compare the relative amountof synthesized EGFP among different culture conditions. (B) MALDI-TOF-MS spectra of the trypsin-digested peptide
fragments, EFHHHHHH ([M + H]+ = 1117.48 Da) and/or TIFFKDDGNYK ([M + H]+ = 1347.66 Da), from the affinity-purified EGFP. The numbering on the left of the spectra corresponds to Panel A. The replacement of one Phe residue inthe fragment peptides to BrPhe and CyPhe leads to theoretical mass increases of 77.91 Da (79Br-1H) and 25.00 Da(12C14N-1H), respectively.

was only slightly recognized with the αA450G mutant invitro (Fig. 2). EGFP synthesis in BmN cells cultured under the 0.02 mM Phe condition seemed to be slightly decreased (Fig. 6A). A more apparent decrease of EGFP was
observed when the Phe concentration was further lowered
to 0.01 mM. This observation strongly suggests the incorporation of IPhe into protein biosynthesis in BmN cells
under severely restricted Phe concentration. However,similar to the case of AzPhe, a direct observation of thereplacement from Phe to IPhe by MALDI-TOF-MS analysis failed (data not shown). We speculated that the incorporation of IPhe strongly impaired protein biosynthesis
activity in BmN cells, most likely due to the presence ofa bulky iodo group even when the replacement ratio from
Phe to IPhe was too low to be detected.
In this study, we succeeded in expanding the repertoireof amino acids used in protein biosynthesis in BmN, theovary-derived B. mori cultured cells. We found that the
αT407A BmPheRS mutant is effective for the incorpora-tion of BrPhe in protein biosynthesis in BmN cells. Moreover, we found that BrPhe, CyPhe, and most likely AzPheand IPhe can be incorporated into protein biosynthesis in
BmN cells expressing the αA450G BmPheRS mutant
when concentration of the competing amino acid, Phe, inthe culture media was adequately lowered. These findings
would contribute to create novel proteins containing
UAAs in B. mori cultured cells and even in B. mori larvae in the near future.

ACKNOWLEDGEMENTS
The authors thank Dr. Jun Ishibashi (NIAS) for his support in MALDI-TOF-MS analysis and Dr. Qiang Zhang
for her technical assistance. This work was supported by
JSPS KAKENHI Grant Numbers 20780039, 22658019,
and 24688008.

Incorporation of Phe analogues in BmN cells

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