Dixon and Gottfried Otting Research School of Chemistry, Australian National University, Canberra, Australia Cell-free protein synthesis offers rapid access to proteins that are selective
Trang 1Optimization of an Escherichia coli system for cell-free synthesis
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 with [15N]amino acids and suitable for
analysis by NMR spectroscopy without chromatographic
purification A system based on an Escherichia coli cell
ex-tract was optimized with regard to protein yield and minimal
usage of 15N-labelled amino acid, and examined for the
presence of metabolic by-products which could interfere
with the NMR analysis Yields of up to 1.8 mg of human
cyclophilin A per mL of reaction medium were obtained by
expression of a synthetic gene Equivalent yields were
obtained using transcription directed by either T7 or tandem
phage k pR and pL promoters, when the reactions were
supplemented with purified phage T7 or E coli RNA
polymerase Nineteen samples, each selectively labelled with
a different 15N-enriched amino acid, were produced and
analysed directly by NMR spectroscopy after
ultracentri-fugation Cross-peaks from metabolic by-products were evident in the15N-HSQC spectra of 13 of the samples All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis No significant transamination activity was observed except for [15N]Asp, where an enzyme in the cell extract efficiently converted Aspfi Asn This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate
In addition, the activity of peptide deformylase appeared to
be generally reduced in the cell-free expression system Keywords: cell-free protein synthesis; human cyclophilin A;
15N-HSQC spectrum; NMR; selective stable isotope labe-ling
High-yield, cell-free protein synthesis systems are now
available that allow efficient high-throughput production
of protein samples for structural genomics and other
applications [1–7] They are also useful for the production
of toxic proteins that interfere with cell replication An
attractive application for this method is in production of
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
Protein yields of up to 6 mgÆmL)1have been reported
using expression systems based on Escherichia coli extracts
[2,10,15,16] At these yields, protein concentrations are sufficiently high to allow the recording of NMR spectra without further concentration of the reaction medium In particular, 15N-HSQC spectra of selectively 15N-labelled proteins can be recorded without purification of the protein, because only signals from15N-labelled amide groups are detected [16] 15N-HSQC spectra present well-resolved, highly sensitive fingerprint information that is uniquely suited to assess the NMR properties of a protein prior to structure determination [17,18]
The present study focussed on optimization of a cell-free
E coli expression system with regard to residue-selective
15N-labelling of proteins The expression system has been shown previously to be suitable for direct NMR analysis of the reaction mixture, resulting in minimal sample handling [16] However, the NMR spectroscopic analysis can be complicated by the formation of undesired by-products which originate from metabolic reactions of the15N-labelled amino acids due to the presence of a broad range of enzymes
in the E coli cell extract used in these reactions [16] While the amino groups from unincorporated [15N]amino acids do not give rise to cross-peaks in 15N-HSQC spectra due to rapid proton exchange with the water, some of the by-products seem to engage the labelled amino groups in amide bonds
Here we present a spectral catalogue of metabolic by-products obtained for amino acids with [15N]amino[aN] groups to provide a reference for the NMR analysis of
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: gottfried.otting@anu.edu.au
Abbreviations: aaRS, amino-acyl tRNA synthetase; hCypA, human
cyclophilin A; RNAP, RNA polymerase; s-CYPA, synthetic gene
encoding hCypA.
(Received 24 June 2004, revised 9 August 2004,
accepted 27 August 2004)
Trang 215N-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 compare, when transcription is performed by T7
RNA polymerase from a T7 promoter or by E coli RNA
polymerase from tandem phage k pR and pL promoters;
(b) does cross-labelling among different amino acids occur
due to amino- or amido-transferase activity and, if so, can it
be suppressed; (c) which amino acids are prone to the
formation of amide-containing products; (d) are all
by-products sufficiently small to be separated from the protein
product by dialysis or ultrafiltration; (e) what are the
minimum concentrations of labelled amino acids required
for good protein yields and (f) which buffer can be used to
replace the large amount of glutamate present in the original
medium described by Yokoyama and coworkers [2,15] and
also in the buffer of a commercial rapid translation system
[11], to enable selective labelling with [15N]Glu?
Materials and methods
Materials
L-[U-15N]Arginine.HCl,L-[15N]aspartic acid,L-[U-15
N]aspa-ragine.H2O, L-[15N]cysteine, L-[15N]glutamic acid,
L-[15N]glutamine[aN], [15N]glycine, L-[15N]histidine[aN]
HCl, L-[15N]methionine, L-[15N]isoleucine, L-[15
N]phenyl-alanine,L-[15N]serine,L-[U-15N]tryptophan,L-[15N]tyrosine
andL-[15N]valine were purchased from Cambridge Isotope
Laboratories (Andover, MA, USA) L-[15N]Alanine,
L-[15N]leucine and L-[15N]threonine were obtained from
Spectra Stable Isotopes (Columbia, MD, USA) and
L-[15N]lysine[aN].2HCl was from Euriso-top (Saint-Aubin,
France) Oligonucleotides were purchased from Auspep
(Parkville, Australia) Vent DNA polymerase and RNase
inhibitor were from Promega, and creatine kinase and
E colitotal tRNA were from Roche E coli RNA
poly-merase (RNAP) holoenzyme was purified as described
previously [16] Spectra/Por 2 dialysis 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 pRand pLpromoters 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 produced in vivo in strain BL21(DE3)/pLysS/pBH964
as a standard for comparison with protein production using
the cell-free system The construction 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 RNAP) under the transcriptional control of the
phage k pLpromoter in vector pMA200U [22] T7 RNAP
was produced in vivo using strain BL21(DE3)recA/
pKO1166 Construction of pKO1166, and the production
and purification of the protein are also described in the
Supplementary material
Characterization of proteins Protein purity was assessed from SDS/PAGE gels that were stained with Coomassie blue Except where specified, protein concentrations were determined by the method of Bradford [23] using bovine serum albumin as the standard The molecular masses of purified proteins were confirmed
by ESI mass spectrometry, using a VG Quattro II mass spectrometer (VG Biotech, Altrincham, UK) The proteins were extensively dialyzed into 0.1% (v/v) formic acid prior
to mass spectrometric analysis
Cell-free protein synthesis The Km values of the 20 aminoacyl-tRNA synthetases (aaRS) for each cognate amino acid, from E coli strains where possible, were compiled (using the website http:// brenda.bc.uni-koeln.de/) and the aaRSs were categorized into three major groups (Table 1) Groups I–III comprise aaRS with Km values for the appropriate amino acid
< 10 lM, between 10 and 50 lM, and between 50 and
500 lM, respectively Based on these Km values and the frequency of occurrence of each amino acid in the primary sequence of hCypA, the concentrations of [15N]amino acids chosen for hCypA labelling were 50 lMfor [15N]Trp and [15N]Tyr, 150 lM for [15N]Ile, [15N]Thr and [15N]His, 0.35 mM for remaining Group II [15N]amino acids, and
1 mM for those in Group III The S30 cell extract from
E colistrain A19 was prepared by the procedure of Pratt [24], followed by concentration with polyethylene glycol
8000 as described by Kigawa et al [2]
In vitro synthesis of PpiB, using the k-promoter vector pKE874 and E coli RNAP holoenzyme, was essentially
as described previously [16] For in vitro protein synthesis
of hCypA, the inner chamber reaction mixtures (total volume 0.7 mL) contained 55 mM HEPES/KOH (pH 7.5), 1.7 mM dithiothreitol, 1.2 mM ATP, 0.8 mM
Table 1 Classification of the 20 aminoacyl-tRNA synthetases by K m
values Data compiled from http://brenda.bc.uni-koeln.de/ All values are for E coli unless noted otherwise.
Enzyme K m (m M ) Group Enzyme K m (m M ) group Group
TrpRS b
a
Value for Saccharomyces carlsbergensis, no data available for
E coli; b value for Lupinus luteus and bovine, no data available for E coli;cvalue for Paracoccus denitrificans, no data available for E coli.dvalue for Bacillus subtilis, no data available for E coli;
e no data available, assigned to group II based on side-chain rigidity;fno data available, assigned to group III based on simi-larity with Glu and Met.
Trang 3each of CTP, GTP and UTP, 0.64 mM 3¢,5¢-cyclic AMP,
68 lM folinic acid, 27.5 mM ammonium acetate, 208 mM
potassium glutamate, 80 mM creatine phosphate,
250 lgÆmL)1 creatine kinase, a [15N]amino acid (at the
concentration given above), 1 mM each of 19 unlabelled
L-amino acids, 15 mM magnesium acetate, 175 lgÆmL)1
E coli total tRNA, 0.05% (w/v) NaN3, 168 lL of
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
[15N]Glu, ammonium or potassium acetate (200 mM)
was used instead of potassium glutamate (208 mM)
The inner chamber reaction mixtures were dialyzed in
Spectra/Por 2 tubing with a nominal size cutoff of
12–14 kDa for 10 or 12 h at 37C with gentle shaking
against the outer chamber solution (14 mL) that was
changed after 3 and 6 h The inner chamber assembly and
the outer chamber buffer were housed within a 50-mL
polypropylene tube The outer chamber solution had the
same composition as the inner chamber reaction mixture,
except that S30 extract, tRNA, plasmid DNA, T7 RNAP,
creatine kinase and RNase inhibitor were omitted, and the
concentration of magnesium acetate was increased to
19.3 mM For SDS/PAGE analysis, in vitro reaction samples
were diluted two-fold with gel loading mix containing 2%
(w/v) SDS and heated for 2 min at 90C The reaction
mixtures containing hCypA were clarified by
ultracentrifu-gation (100 000 g, 4 h) and stored at 4C
NMR spectroscopy
All NMR spectra were recorded at 25C using a Varian
INOVA 600 MHz NMR spectrometer equipped with a
probe operating at room temperature.15N-HSQC spectra
were recorded with 5 mm sample tubes using t1max¼
32 ms, t2max¼ 102 ms and total recording times of
17–24 h NMR measurements were made using the
super-natant from the clarified reaction mixtures after addition of
10% (v/v) D2O to provide a lock signal In addition, spectra
were recorded after dialysis of the samples overnight at 4C
in Spectra/Por 2 tubing, against buffer comprising 10 mM
sodium phosphate (pH 6.5), 100 mM NaNO3, 5 mM
dithiothreitol and 50 lMNaN3 The dialyzed samples were
concentrated to a final volume of about 0.6 mL using
Millipore Ultra-4 centrifugal filters (MWCO 10 000) and
D2O 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 of15N-labelled samples of hCypA
and analysis by NMR spectroscopy, the performance of the
cell-free expression system was explored with respect to a
number of parameters We first examined the yields
obtained when transcription is conducted by T7 RNAP
from the T7 promoter rather than by E coli RNAP
holoenzyme from phage k promoters as in our previous
work [16]
Even with promoters recognized by E coli RNAP, supplementation of the S30 extract with RNAP is important for good protein yields [16] When using T7 promoters, addition of T7 RNAP was critically required T7 RNAP, when purchased from commercial sources, is the most expensive component of our cell-free system T7 RNAP is, however, relatively easy to produce and purify,
as it is a much smaller and simpler protein than E coli RNAP holoenzyme, which is composed of five different subunits (a2bb¢xr) [25] We adapted published methods [26] to develop a simple procedure for the isolation of T7 RNAP from a strain containing a plasmid that directed overproduction of the protein under control of the heat-inducible k pL promoter Up to 25 mg of pure T7 RNAP could be obtained within a few days from 5 g of cells This is sufficient for many hundreds of in vitro reactions
Our initial attempts to purify T7 RNAP were hindered by proteolytic cleavage This was controlled by use of an ompT– E coli host strain [BL21(DE3)recA], limitation of the time cultures were treated at 42C during induction of synthesis of T7 RNAP to 30 min, followed by treatment for
2 h at 40C [26], and use of a-toluenesulphonyl fluoride in the buffer during cell lysis
E coliPpiB [16] was produced equally well in vitro when using either T7 or k promoters (data not shown) Corre-spondingly similar yields were expected for hCypA, as PpiB and hCypA are functional homologues with similar three-dimensional structures and amino acid compositions, and the codon 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 compar-able to those obtained for PpiB with either promoter system (Fig 1, lane 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
In order to achieve maximal yields in the cell-free system, smaller mass amounts of T7 RNAP were required than
E coli RNAP, when the same proteins were produced under control of the T7 and k promoters, respectively (data not shown) A further advantage of the T7 system lies in its better tolerance with respect to temperature changes: lowering the reaction temperature from 37 to 30C decreased protein yields insignificantly, whereas this tem-perature change resulted in decreased yields when the expression was under control of the k promoters This may
be because pKE874 also directs low-level synthesis of a thermolabile version of the k cI repressor, which may repress transcription by E coli RNAP at 30C The optimized concentrations of T7 or E coli RNAP used in the present work were found to be suitable for production of many different proteins
Trang 4Optimization of other conditions forin vitro protein synthesis
Several other parameters were assessed individually to maximize protein yields In particular, the optimal quan-tity of concentrated S30 extract in the reaction mixture was determined for each new preparation, but results from several batches nevertheless gave similar results In addition, the optimal concentrations of MgCl2 and template DNA were found to vary with different batches
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 be stable with respect to proteolysis After two months of storage at 4C, hCypA was not significantly degraded, as evaluated by NMR measurements
Cell-free synthesis of PpiB with increasing concentra-tions of amino acids showed that almost no protein was synthesized when each amino acid was present at 10 lM
(Fig 1, lane 2) This result confirmed that the extract is depleted in natural amino acids Absence of free amino acids from the cell extract is a prerequisite for efficient incorporation of labelled amino acids into target proteins Furthermore, the protein yields increased with increasing amino acid concentration (Fig 1, lanes 1–8), indicating that the concentrations of amino acids limit protein synthesis at < 1 mM After ultracentrifugation to remove ribosomes and ribosome-associated translation factors, the target proteins were found to be the most abundant protein in the reaction mixtures, provided the amino acids had been supplied at high concentrations (Fig 1) Remarkably, reduction of the concentration of amino acids to 30 lM lowered the yield only by about 50% (Fig 1, lanes 4 and 8)
In vitro synthesis of h CypA selectively labelled with [15N]amino acids
The achievable yields of proteins would be expected to depend on the concentrations of available amino-acylated tRNAs As the loading efficiency of the amino-acyl tRNA synthetases depends on their Km values, amino acids processed by synthetases with low Kmvalues are expected
to be more readily available for protein synthesis than others To limit the expense of use of labelled amino acids, their concentrations in the inner and outer chambers were adjusted according to their frequency in the primary structure of the protein and according to the Kmvalues of their respective tRNA synthetases (Table 1) Our results confirmed that labelled amino acids from Groups I and II (Table 1) could be used at reduced concentrations (still several-fold above the respective Kmvalues) without 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 synthesis of PpiB under control of phagek promoters.
Identical volumes of reaction products were loaded into lanes of a 15%
SDS/polyacrylamide gel, which were stained with Coomassie blue.
Lanes 1, 3, 5 and 7: reaction mixtures before the start of in vitro
synthesis of PpiB, with transcription by E coli RNAP from tandem
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 was present
at a concentration of 10 l M (lanes 1 and 2), 30 l M (lanes 3 and 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.
Fig 2 Cell-free synthesis of hCypA under control of the T7 promoter
with minimized concentrations of labelled amino acids from the three
different groups defined in Table 1 The samples were centrifuged for
4 h at 100 000 g before analysis by SDS/PAGE (15%) The gel was
loaded with the soluble fractions (supernatants) of hCypA synthesized
in vitro during 10 h at 37 C with different concentrations of
15
N-labelled amino acids (all other amino acids were at 1 m M ), and
stained with Coomassie blue Lane 1: 1 m M [ 15 N]Glu; lane 2: 50 l M
[ 15 N]Tyr; lane 3: 150 l M [ 15 N]Thr; lane 4; 350 l M [ 15 N]Asn; lane 5;
1 m M [15N]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 potassium glutamate.
Trang 5those obtained when all amino acids were at 1 mM The
similarity in protein production levels is corroborated by the
similarity in cross-peak intensities observed in15N-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 mM potassium D-glutamate
performed equally well as one withL-glutamate, suggesting
that glutamate served as an osmolyte rather than playing a
more specific role However, this buffer was still unsuitable
for selective labelling with Glu, presumably because of the
presence of glutamate racemase in the S30 extract The
glutamate could not be substituted by 200 mM betaine,
which had an inhibitory effect on the synthesis of PpiB and
hCypA In contrast, reaction mixtures with ammonium or
potassium acetate instead of potassium glutamate were
found to perform well Maximal yields of PpiB and hCypA
were obtained with either acetate salt at concentrations in
the range of 200–230 mM (e.g Figure 2, lane 1)
Further-more, this alternate medium suppressed amino- or
amido-transferase activities which otherwise incorporated the
a-amino-nitrogen of Asp into the a- and side-chain NH2
groups of Asn (see below)
15
N-HSQC NMR spectra of selectively15N-labelledhCypA
15N-HSQC spectra were recorded of hCypA samples
produced in vitro with 19 different 15N-labelled amino
acids Spectra with acceptable sensitivity could be recorded
at 25C 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 Fig 3 were recorded after the ribosomes and
other macromolecular assemblies had been removed by
ultracentrifugation to avoid the formation of a precipitate
during data acquisition The NMR chemical shifts of
purified hCypA are known [27], allowing the identification
of individual cross-peaks from the protein and detection of
any additional cross-peaks due to metabolic by-products
The spectra recorded for hCypA produced with15N-labelled
Asn, Gln, Ile, Leu, Phe and Tyr contained only cross-peaks
from the protein Samples with15N-labelled Arg, Asp, 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
with 15N-labelled Ala, Asp, Cys, Glu, Gly, Ser and Trp
contained additional cross-peaks that were more intense
than the average protein peaks In all cases, the cross-peaks from metabolic by-products were removed easily by dialysis of the sample prior to NMR analysis (data not shown) For proteins with a molecular mass similar to hCypA, dialysis can be achieved simply by transfer of the tubing containing the reaction mixture to a buffer suitable for NMR measurements The Spectra/Por 2 dialysis tubing (12–14 kDa cut-off) was found to retain a 9.5-kDa globular protein under these conditions Comparison of spectra obtained with selectively 15N-labelled PpiB and hCypA showed closely related sets of metabolite HSQC cross-peaks (data not shown)
There was no evidence for transaminase activity except for the [15N]Asp-labelled sample produced in the conven-tional way in the presence of potassium glutamate Under those conditions, cross-peaks of the backbone and side-chain amides of the [15N]Asn-labelled sample were also observed in the NMR spectrum of[15N]Asp-labelled hCypA (fourth panel of Fig 3) The intensities of the undesired [15N]Asn cross-peaks were about two thirds of those of the [15N]Asp cross-peaks, indicating highly efficient transami-nation/transamidation This suggests that the labelled amino group is not released in the form of ammonia because it would have been diluted by the presence of 27.5 mM ammonium present in the reaction buffer The
E coliasparagine synthetases A and B (asnA and asnB gene products) synthesize Asn from Asp and may be responsible for this activity in the S30 extract Remarkably, no evidence
of transamination was observed in the [15N]Asn sample (The additional cross-peaks in Fig 3 are due to the side-chain amides which were labelled in the [U-15N]Asn substrate) We further noted that the amido/aminotrans-ferase activity could be suppressed by replacing potassium glutamate in the buffer by ammonium or potassium acetate (last panel 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 lane 1 in Fig 2)
We also compared the buffers with 200 mM potassium acetate or 200 mMammonium acetate for the synthesis of [15N]Asp- and [15N]Glu-hCypA with regard to the number and positions of HSQC cross-peaks from metabolites No significant differences were observed (data not shown) Quite generally, the cell-free expression system allows the use of high concentrations of unlabelled amino acids in the reaction mixtures to dilute the effects of incorporation of amino acids that are15N-labelled by transamination In this situation, the main effect of transamination is a reduction of the degree of15N-labelling in the amino acid of interest In the case of [15N]Gly, much of the label appeared to have been trapped in an amide group of a low molecular mass metabolite (Fig 3)
Fig 3 15 N-HSQC spectra of hCypA selectively labelled with 15 N-amino acids The spectra were recorded at 25 C and pH 7.5 using the in vitro reaction mixture after centrifugation (100 000 g, 4 h) and addition of 10% D 2 O The assignments of the backbone amide cross-peaks are indicated
by the one-letter amino acid symbols and the sequence numbers Squares identify the cross peaks which could be assigned according to the previously published assignment [27] Circles identify the cross-peaks from metabolites Dotted squares mark the positions of cross-peaks which 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 [l5N]Asn also contains the cross-peaks from the side-chain amide groups (backbone and side-chain NH groups were labelled in the amino acid used in the reaction) The cross-peak from the side-chain NH of W121 is labelled W121e Question marks in the spectra of [ 15 N]Met and [ 15 N]Val hCypA identify tentative new assignments of cross-peaks which had not been assigned previously [27].
Trang 7Fig 3 (Continued).
Trang 8The cell-free expression system lends itself to the addition
of specific enzyme inhibitors, such as the alanine racemase
inhibitor, b-chloro-L-alanine [28] When we tested the effect
of adding b-chloro-L-alanine (0.5 mM) to the reaction
mixture for the synthesis of [15N]Ala labelled PpiB,
however, only a single cross-peak belonging to a minor
metabolite was removed from the set of metabolite signals
(data not shown) The metabolic enzymes and pathways in
E coliare not sufficiently well understood to suppress all
side reactions in this way
Single additional peaks present in the15N-HSQC
spec-trum of [15N]Met and [15N]Val labelled hCypA were still
present after dialysis and were attributed to the amide
protons of Met1 and Val2 These had not been reported in
the original assignment of hCypA [27], in agreement with
the frequent observation that the HNresonances of
amino-terminal residues are broadened beyond detection by fast
base-catalyzed exchange with the water In the case of our
cell-free expression system, the amino-terminal Met of PpiB
has been observed (by proteolysis and mass spectrometry)
to remain predominantly N-formylated due to saturation of
the activity of peptide deformylase (D Mouradov and
T Huber, unpublished data) In the present case of hCypA,
retention of the N-formyl group enables the observation of
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 started with a
deformylated Met (18 012 Da), 20% had N-terminal
N-formyl-Met (18 040 Da) and 10% had lost 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 had been reported for pH 6.5, we measured the
15N-HSQC spectra after dialysis at this pH The lower pH
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 single 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 based on an E coli cell extract were
investigated and optimized One point 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
protein overproduction are based on transcription from
the T7 promoter, it is convenient if the same vectors can
be used in the cell-free system In the case of hCypA,
good protein yields were obtained with transcription by
T7 RNAP from the T7 promoter, whereas no production
could be detected in vitro with a phage k promoter and
transcription by the E coli RNAP holoenzyme However,
no difference between the two systems was observed for
the closely related protein PpiB This phenomenon may
be explained in different ways Possibly, hCypA has less
favourable folding characteristics than PpiB Alternat-ively, the relative complexity of the E coli RNAP holoenzyme may offer more targets for inhibitory protein–protein interactions
Whereas the present study required significant NMR measurement time to record each15N-HSQC spectrum, this
is no longer prohibitive with the increased sensitivity available on high-field NMR spectrometers equipped with cryogenic probeheads [16] We thus envisage that parallel production of a large number of selectively labelled protein samples followed by the recording of 15N- or 13C-HSQC spectra will provide a practical approach to support resonance assignments, provided that sample handling can
be kept to a minimum The present study shows that this latter condition is easily fulfilled For most amino acids, purification of the produced 15N-labelled protein 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 remove the metabolites The easy removal of the interfering 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-15N]protein is synthesized in vivo using ammonium chloride [31]
Most importantly, the transfer of the15N-label to other amino acids is insignificant for 18 of the amino acids and can be suppressed for [15N]Asp by use of a modified medium in which glutamate is replaced by acetate Notably, these results 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 readily accessible technology [7,11,32] Our strategy extends the applicability of the system with regard
to protein analysis by NMR spectroscopy The catalogue of
15N-HSQC spectra in Fig 3 provides the basis for the straightforward identification of metabolite cross-peaks by visual inspection, as their generation is insensitive to the identity of the target protein
Ultimately, the formation of metabolites could be avoided by the use of a completely reconstituted cell-free system comprising only the minimum set of enzymes necessary for translation [33] Currently achievable yields, however, do not justify the effort associated with the purification of the large number of enzymes required If all metabolites generated by the E coli S30 extract could be identified, it might be possible to suppress their production
by the use of appropriate enzyme inhibitors This would provide the benefit of less isotopic dilution and thereby improved labelling efficiency and enhanced sensitivity of the NMR analysis In the absence of enzyme inhibitors, increased yields may be obtained by providing the rapidly metabolized amino acids in excess, beyond the quantities suggested by the Km values of the aminoacyl-tRNA synthetases (Table 1) and the number of amino acids present in the protein The data of Fig 3 provide a guideline for a corresponding rebalancing of amino acid concentra-tions They apply equally to the in-cell NMR analysis [30] of selectively labelled proteins
Trang 9We thank Dr Simon Bennett for measurements of ESI mass spectra.
K.O and G.O thank the Australian Research Council for a
CSIRO-Australian Postdoctoral and Federation Fellowships, respectively.
Financial support by the Australian Research Council is gratefully
acknowledged.
References
1 Spirin, A.S., Baranov, V.I., Ryabova, 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.,
Shi-bata, T & Yokoyama, S (1999) Cell-free production and
stable-isotope labeling of milligram 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 assignments using specific labeling and cell-free synthesis.
J Biomol NMR 28, 235–247.
5 Swartz, J.R., Jewett, M.C & Woodrow, K.A (2004) Cell-free
protein synthesis with prokaryotic combined
transcription-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, 19–26.
7 Klammt, C., Lo¨hr, F., Scha¨fer, B., Haase, W., Do¨tsch, V.,
Ru¨terjans, H., Glaubitz, C & Bernhard, F (2004) High-level
cell-free expression and specific labeling of integral membrane
pro-teins Eur J Biochem 271, 568–580.
8 Kigawa, T., Muto, Y & Yokoyama, S (1995) Cell-free synthesis
and amino acid-selective stable isotope labeling of proteins for
NMR analysis J Biomol NMR 6, 129–134.
9 Yabuki, T., Kigawa, T., Dohmae, N., Takio, K., Terada, T., Ito,
Y., Laue, E.D., Cooper, J.A., Kainosho, M & Yokoyama, S.
(1998) Dual amino acid-selective and site-directed stable-isotope
labeling of the human c-Ha-Ras protein by cell-free synthesis.
J Biomol NMR 11, 295–306.
10 Kariya, E., Ohki, S.-Y., Hayano, T & Kainosho, M (2000)
Backbone 1 H, 13 C, and 15 N resonance assignments of an 18.2 kDa
protein, E coli peptidyl-prolyl cis-trans isomerase b (EPPIb).
J Biomol NMR 18, 75–76.
11 Parker, M.J., Aulton-Jones, M., Hounslow, A.M & Craven, C.J.
(2004) A combinatorial selective labeling method for the
assign-ment of backbone amide NMR resonances J Am Chem Soc.
126, 5020–5021.
12 Hendrickson, T.L de Cre´cy-Lagard V & Schimmel, P (2004)
Incorporation of nonnatural amino acids into proteins Annu Rev.
Biochem 73, 147–176.
13 Kigawa, T., Yamaguchi-Nunokawa, E., Kodama, K., Matsuda,
T., Yabuki, T., Matsuda, N., Ishitani, R., Nureki, O &
Yokoyama, S (2002) Selenomethionine incorporation into a
protein by cell-free synthesis J Struct Funct Genomics 2, 29–35.
14 Kiga, D., Sakamoto, K., Kodama, K., Kigawa, T., Matsuda, T.,
Yabuki, T., Shirouzu, M., Harada, Y., Nakayama, H., Takio, K.,
Hasegawa, Y., Endo, Y., Hirao, I & Yokoyama, S (2002) An
engineered Escherichia coli tyrosyl-tRNA synthetase for
site-specific incorporation of an unnatural amino acid into proteins in
eukaryotic translation and its application in a wheat germ cell-free
system Proc Natl Acad Sci USA 99, 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 proteins without purification: a high-throughput approach FEBS Lett 524, 159–162.
17 Yamazaki, T., Yoshida, M., Kanaya, S., Nakamura, H & Nagayama, K (1991) Assignments of backbone 1 H, 13 C, and 15 N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spec-troscopy Biochemistry 30, 6036–6047.
18 Yee, A., Chang, X., Pineda-Lucena, A., Wu, B., Semesi, A., Le, B., Ramelot, T., Lee, G.M., Bhattacharyya, S., Gutierrez, P., Deni-sov, A., Lee, C.-H., Cort, J.R., Kozlov, G., Liao, J., Finak, G., Chen, L., Wishart, D., Lee, W., McIntosh, L.P., Gehring, K., Kennedy, M.A., Edwards, A.M & Arrowsmith, C.H (2002) An NMR approach to structural genomics Proc Natl Acad Sci USA 99, 1825–1830.
19 Studier, F.W., Rosenberg, A.H., Dunn, J.J & Dubendorff, J.W (1990) Use of T7 RNA polymerase to direct expression of cloned genes Methods Enzymol 185, 60–89.
20 Williams, N.K., Prosselkov, P., Liepinsh, E., Line, I., Sharipo, A., Littler, D.R., Curmi, P.M.G., Otting, G & Dixon, N.E (2002)
In vivo protein cyclization promoted by a circularly permuted Synechocystis sp PCC6803 DnaB mini-intein J Biol Chem 277, 7790–7798.
21 Neylon, C., Brown, S.E., Kralicek, A.V., Miles, C.S., Love, C.A.
& Dixon, N.E (2000) Interaction of the Escherichia coli replica-tion terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance Biochemistry 39, 11989–11999.
22 Elvin, C.M., Thompson, P.R., Argall, M.E., Hendry, P., Stam-ford, N.P.J., Lilley, P.E & Dixon, N.E (1990) Modified bacter-iophage lambda promoter vectors for overproduction of proteins
in Escherichia coli Gene 87, 123–126.
23 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 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 (2002) 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 specific genes Proc Natl Acad Sci USA 82, 1074–1078.
27 Ottiger, M., Zerbe, O., Gu¨ntert, P & Wu¨thrich, K (1997) The NMR solution conformation of 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 resonances in switch-variant anti-dansyl antibodies labeled with [1–13C]methionine Biochem-istry 30, 270–278.
29 Ou, H.D., Lai, H.C., Serber, Z & Do¨tsch, V (2001) Efficient identification of amino acid types for fast protein backbone assignments J Biomol NMR 21, 269–273.
30 Serber, Z., Ledwidge, R., Miller, S.M & Do¨tsch, V (2001) Eva-luation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy J Am Chem Soc.
123, 8895–8901.
31 Gronenborn, A.M & Clore, G.M (1996) Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy Protein Sci 5, 174–177.
Trang 1032 Elbaz, Y., Steiner-Mordoch, S., Danieli, T & Schuldiner, S (2004)
In vitro synthesis of fully-functional EmrE, a multidrug
trans-porter, and study of its oligomeric state Proc Natl Acad Sci USA
101, 1519–1524.
33 Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T.,
Nishikawa, K & Ueda, T (2001) Cell-free translation
recon-stituted with purified components Nat Biotechnol 19, 751–755.
Supplementary material
The following material is available from http://www
blackwellpublishing.com/products/journals/suppmat/EJB/
EJB4346/EJB4346sm.htm
Details of the construction of the synthetic s-CYPA gene,
purification of cyclophilin A and expression and purification
of T7 RNA polymerase are given and reference to the
following supplementary figures is made:
Fig S1 Construction of the synthetic CYPA gene (s-CYPA) The gene was constructed following manipula-tion of the nucleotide sequence to increase its identity with 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 identified by arrows above the complete gene sequence The NdeI, ApaI, FokI, XhoI, EcoRI and NcoI restriction endonuclease sites are boxed The start codon (ATG) is within the NdeI site and the stop codon (TAA) is identified by a black box in the linker Fig S2 Plasmid pKO1166 This 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 pL promoter into vector pMA200U [5]