Puromycin technology for in vitro evolution and proteome exploration
Etsuko Miyamoto-Sato* & Hiroshi Yanagawa
Department of Biosciences and Informatics, Faculty of Science and Technology,
Keio University, Yokohama 223-8522, Japan.
*Correspondence should be addressed to E. Miyamoto-Sato.
e-mail: nekoneko@educ.cc.keio.ac.jp(Received 1 November, 2006 Accepted 9 November, 2006)
Abstract
Puromycin, an analogue of the 3’ end of
aminoacyl-tRNA, is transferred non-specificallyto growing polypeptide chains, causing prematuretermination of translation. However, we foundthat, at very low concentration, puromycin istransferred specifically to the carboxyl (C-)
terminus of the full-length protein. The term
puromycin technology refers to methods whichhave been developed based on this property,
including the in vitro virus (IVV) display
technique, in which an mRNA (genotype) and its
protein (phenotype) are linked via puromycin, andthe technique of specific in vitro C-terminal
protein labeling. Novel and convenient
applications based on puromycin technology havebeen employed not only for evolutionary protein
engineering, such as protein-directed selection,
but also in various fields of proteomics research,such as fluorescence labeling, affinity purification(pull-down assay), protein-protein interaction
analysis, and protein chips. Here, we review theproperties of puromycin that make thistechnology possible, and the already extensive
range of applications of this technology.

to develop a highly specific labeling system forproteins, in which puromycin derivatives bearing
a fluorescein moiety are used to label the
C-terminal end of a full-length protein (Figure1B) [14-15].

Kyewords: puromycine, evolution, proteome, in
vitro virus (IVV), C-terminal protein labeling
Introduction
Puromycin is a very powerful inhibitor ofprotein synthesis in both in vivo and in vitrotranslation systems [1-8]. Structurally, it
resembles the 3' end of an aminoacyl-tRNA
molecule [2], and it is able to enter the ribosomal
A site to be transferred to nascent polypeptide
chains by peptidyl transferase [9-11]. However,
we showed that, at very low concentration in acell-free translation system, puromycin istransferred specifically to the C-terminal of the
full-length protein [12]. We utilized this finding
to link mRNA and protein to form a so-called invitro virus (IVV) for evolutionary protein
engineering [13]. The IVV provides a system for
mRNA display, in which a genotype molecule(mRNA) is linked to the corresponding phenotype
molecule (protein) through puromycin in acell-free translation system (Figure 1A) [13-14].
We also utilized the same property of puromycin

Figure 1. Puromycin technology; the C-terminal
protein labeling and IVV methods. (A) IVV formationon the ribosome (1). Puromycin at the 3’-terminal endof a spacer (Fluor-PEG Puro; 14) ligated to mRNA canenter the ribosomal A-site to bind covalently to the
C-terminal end of the encoded full-length protein in theribosomal P-site. Fluor stands for fluorescein. Puro
represents puromycin. (B) C-Terminal labeling ofproteins on the ribosome (14,15). A puromycin
derivative (Fluor-dCpuro; 16) can enter the ribosomal
A-site to bind covalently to the C-terminal end of theprotein in the ribosomal P-site.

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Viva Origino 34 (2006) 148-154

The development of proteomics has led to anincreasing interest in cell-free translation systems,
because of their rapidity and ease of handling.
Several new in vitro protein production
technologies yielding large amounts of protein
have been developed [16-17], and in combination
with puromycin technology, have been utilized
for evolutionary protein engineering [13-14, 18],
as well as in proteomics. Recently, in vitro protein
selection using mRNA display methods such as
IVV [19-21] and RNA-peptide fusions [22-23]
have been employed for the analysis ofprotein-protein interactions using cDNA libraries.
This display technology for protein selection andscreening has already achieved spectacular results.
Moreover, we have reported various otherapplications, including in vitro analyses ofprotein-protein interactions using C-terminallylabeledproteinswithmicroarrays
andfluorescencecross-correlation
spectroscopyFCCS) [24-25]. Here we present an overview ofthe present status and prospects of puromycin
technology.
1. Puromycin technology
The antibiotic puromycin [1], which is ananalogue of the 3’ end of Tyr-tRNA Tyr [2], acts inboth prokaryotes and eukaryotes [3-5] as an
inhibitor of peptidyl transferase [6-7]. It has twomodes of inhibitory action. The first is by acting
as an acceptor substrate which attacks
peptidyl-tRNA (donor substrate) in the P site toform a nascent peptide [6-9]. The second is bycompeting with aminoacyl-tRNA for binding to
the A’ site, which is the binding site of the 3’ endof aminoacyl-tRNA within the peptidyltransferase active site [10-12]. It has beenreported that the polypeptides released by
puromycin are not full-length protein [6].
Similarly, it has been shown that growing peptide
chains on ribosomes are transferred to the
α-amino group of puromycin, which interrupts
the normal reaction of peptide bond formation [7].
Therefore, these conventional studies suggested
that puromycin is a non-specific inhibitor ofprotein synthesis as a result of competition withaminoacyl-tRNA. However, since most of thestudies on puromycin were performed atrelatively high concentrations, the behavior of
puromycin at lower concentrations was still an
open question at that time. It had been reported
that full-length protein which fails to be released
from ribosomes at the final stage of protein
synthesis, requires treatment with puromycin or
RFs to be released [26-27]. These results led us tohypothesize that puromycin at very low
concentrations, which would not effectively
compete with aminoacyl-tRNA [12], might act asa non-inhibitor and bond specifically to

full-length protein at the stop codon.
Indeed, we confirmed that puromycin and its
derivatives bond only to full-length protein atvery low concentrations (such as 0.04 µM), where
they are “non-inhibitors” of protein synthesis, by
32
using
P-labeledrCpPuro(2’-ribocytidylyl-(3'→5')-puromycin) in an E. coli
S30 extract cell-free translation system [12]. Our
results provided the first evidence that specificbonding of puromycin to the full-length protein
occurs at the stop codon during the process oftermination of protein synthesis at very low
concentration. In other words, puromycin at
sufficiently low concentrations only has theopportunity to be bonded to proteins at a stopcodon, where it does not need to compete withaminoacyl-tRNA. Since termination is a relatively
slow step involving a translational pause in
eukaryotes [28-29] and E. coli [30], it is possiblethat puromycin even at very low concentrations
can bind to the A' site, and compete with RFs torelease the full-length protein from ribosomes.
Accordingly, under these conditions, puromycin
can be incorporated specifically at the C-terminusof the full-length protein. This concept is the basisof puromycin technology [12,14]. The
combination of this puromycin technology with invitro translation has yielded novel methods, suchas IVV and C-terminal labeling techniques, forprotein selection and screening [19-21],
fluorescence labeling [19-21], and affinity
purification (pull-down assay), as well as protein
chips for proteomics [24,25], andsuperior
methods for evolutionary protein engineering.
2. In vitro virus; IVV
For Darwinian evolution, genotype assignment
to the phenotype is essential. Evolutionary
molecular engineering through in vitro RNA
selection using ribozyme-type assignment
molecules was proposed [31-33] and shown to beeffective [34-35]. However, greater progress has
been achieved with in vitro protein selection using
fusion-type assignment molecules, in which
genotype molecules are bound to phenotype
molecules, such as ribosome display [36], mRNA
display, including IVV [12-14,18], and DNA
display [37-38], based on cell-free translation
systems. Since these display technologies are in
vitro versions of phage display [39], permitting
global search of a large sequence space, such as
the whole protein sequence space.
We developed IVV, the first application of
puromycin technology, for mRNA display in1997 [13]. Shortly thereafter, RNA-peptide
fusions were independently reported for mRNA
display [18]. The principle of puromycin-149-

Viva Origino 34 (2006) 148-154

technology has been applied in both methods,
which are based on the use of puromycin as aphysical linkage between mRNA and its encoded
full-length protein to obtain assignment molecules.
These methods have already achieved substantial
results, though early work uncovered someproblems in obtaining large libraries and highly
efficient enrichment, as well as involving rather
tedious processes [13,18]. The efficiency of theligation between the mRNA and the puromycin
spacer was initially very low (about 20-30%), but
this was improved (to about 70%) by alternative
ligation methods such as splint ligation [40] andphoto-linked ligation [41]. Further, we havereported a highly efficient single-strand ligation(over 90%) using a fluorescein-conjugatedpolyethylene glycol puromycin spacer (PEG-Puro
spacer; Figure 2A) [14]. The efficiency of fusion
formation was initially less than 10% [13] or 1%[18], but this has been improved to 40% [39,42-43]. In our experiments, we have obtained
more than 70% fusion formation [14] throughsingle-strand ligation using a flexible PEG-Puro
spacer and an optimized mRNA template in acell-free wheat germ translation system or rabbit
reticulocyte lysate translation system.
We also demonstrated that single-strand
ligation using the flexible fluorescein-conjugated

PEG-Puro spacer (Figure 2B) instead of DNA[13,18] or triethylene glycol (TEG) [39] spacer
provides an improvement of mRNA stability and
IVV formation [14]. Moreover, we found thatmRNA having a 5’-untranslated region including
SP6 promoter and Ȑ 29 enhancer (a part oftobacco mosaic virus Ȑ), and an A8 sequence(eight consecutive adenylate residues) without astop codon at the 3’ end is suitable for fusion
formation [14]. The use of mRNA without a stopcodon is preferable to establish fusions of mRNAand its encoded peptide or protein, because thisavoids competition of puromycin with RFs[12,13]. Optimization of the 5’- and 3’-terminal
sequences of mRNA templates offers theadvantage of easy handling because of highefficiency of IVV formation, simplifying formerly
tedious processes.
3. In vitro evolution and proteome exploration
In vitro display technology has many
advantages [44]. It offers a greater library
diversity (1012-13/ml) compared with phage display[39]. In fact, various aptamers have beensuccessfully evolved from random libraries using
this approach [45-48]. Evolution of antibody
mimics by using mRNA display [49] or ribosome
display [50] and identification of epitope-like
consensus motifs [51] have also been reported.

Figure 2. PEG puromycin spacer for the IVV method and puromycin analogues for the C-terminal protein labelingmethod.
(A)
The
structureofPEG
puromycin
spacer.
Fluor-PEG
Puro
spacer[p(dCp)2-T(Fluor)p-PEGp-(dCp)2-puromycin] was synthesized from Puro(Fmoc)-CPG, Polyethylene glycol (PEG,
average mol. wt 2,000), deoxycytidine phosphoramidite (dC-amidite), thymidine(fluorescein) phosphoramidite[T(Fluor)-amidite]. (B) The structure of fluorescence-labeled puromycin. A fluorophore (herein, fluorescein) was
chemically joined to puromycin through a dC linker.

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Screening for binding streptavidin aptamer
provided 10,000-fold stronger binding than thatobtained with the in vivo method [52], supporting
the superiority of the method. Screening for ATP
binding also gave good results [53]. In manycases, desired functional peptides or proteins havebeen obtained by the application of display
technology.
mRNA display methods, including IVV, havebeen successfully used to analyze protein-protein
interactions (PPI) [19-22], and drug-protein
interactions [54], starting from a cDNA library. In
order to adapt the technology to protein function
research, such as PPI analysis, in vivo preparationof bait proteins used to be required before in
vitro-based manipulation [22-23]. Our developed
cell-free cotranslation technique with IVV (Figure3) has provided a totally in vitro manipulation thatis suitable for high-throughput, genome-wide
analysis with multiple bait proteins as a result ofin vitro bait translation instead of in vivo bait
preparation [19]. Cotranslation of bait and prey
proteins should also be advantageous for theformation of protein complexes [55-56]. Thisapproach currently appears to offer the best
opportunity to obtain a comprehensive data set
including not only direct, but also indirectinteractions in a single experiment.
The display technology can also be applied toprotein chip systems. A microarray in which
mRNA-peptide fusions are immobilized byhybridization to a surface-bound DNA captureprobe has been developed, resulting in an
addressable mRNA-peptide fusion microarray[55]. This provides a protein chip in which theprotein is linked to its genetic information. Thissystem can be used to analyze genetic information
associated with interactions of a large-diversity
protein library versus a large-diversity library of
nucleotides, sugars, etc. This is potentially ahigh-throughput method, which should contribute
greatly to proteomics. Epitope-mRNA fusions
have been bound to the surface and the interaction
with fluorescence-labeled antibody has been
analyzed [57].

4. Full-length protein labeling at the
C-terminal end
As already mentioned, puromycin and its
derivatives at low concentrations can bind
specifically to the carboxyl terminus of full-length
proteins [12] and a fluorescein-puromycin
conjugate has been successfully used for the
C-terminal fluorescein labeling of proteins [15].
This fluorescence-labeling method is useful for
analyzing protein-protein or protein-DNA
interactions with DNA microarrays or withfluorescencecross-correlation
spectroscopy(FCCS), and for pull-down assay [24].
The efficiency of C-terminal labeling isreported to be between 50% and 95% [15].To
confirm the general utility of this labeling method,
we chemically synthesized various puromycin
analogs in which one or two (deoxy)cytidylicacid(s) (i.e., dC, dCdC, rC, or rCrC) were inserted
between the fluorophore (fluorescein, RhG,TAMRA, Cy3, or Cy5) and puromycin as a linker
to enhance the incorporation of puromycin
analogs into proteins by mimicking the CCA
sequence at the 3’ end of tRNA [24]. These
puromycin analogs were added to the wheat germin vitro translation system [17]. Among them, wefound that the Fluor-dC-puromycin conjugates(Figure 2B) gave the best yield because of the useof the dC linker. The dC linker should be inferior
as an inhibitor to the rC linker in the middle ofprotein synthesis and superior to the rC linker as a
non-inhibitor for specific bonding to thefull-length protein in the final stage of protein
synthesis. In addition, we have optimized anmRNA template having a certain sequence at the
3’ end (XA8 sequence; XhoI and eight
consecutive adenylate residues) to provide 3- toFigure 3. IVV selection based on cell-freecotranslation. The selection procedure iscomposed of (I) transcription, (II) cell-freecotranslation, coupling translation and interactionof tagged bait protein and prey IVV library toform complexes, (III) IVV two-step selection(Rigaut et al. 1999), and (IV) RT-PCR to
amplify the library. IgG beads (IgG) for theprotein A (zz domain) tag are used for the 1st stepof the purification, and calmodulin beads (Cal) forthe calmodulin binding protein (CBP) tag are used
for the 2nd step of the purification.

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5-fold higher formation efficiency of C-terminallylabeled proteins [14]. We concluded that mRNA
templates with the XA8 sequence as a 3’end andSP6 + Ȑ29 as a 5’ UTR are clearly preferable
for the C-terminal labeling of proteins to facilitate
high-throughput proteome exploration.
5. Proteome exploration using c-terminal
protein labelingThis C-terminus labeling method has many
advantages. Because the protein is labeled only at
the C-terminus, most proteins can maintain theirfunctionality. The method is suitable forquantitative analysis because there is a single
fluorophore on each protein molecule. The effectof labeling on the protein activity should besmaller than with the GFP fusion method, becauseof the much smaller size of the label. We haveshown that C-terminal fluorescence labeling isvery useful for many applications, including
detection of protein-protein or protein-DNA
interactions with a DNA microarray or with
FCCS, and pull-down assay [24-25].
Recently, the C-terminal labeling method has
been applied to observe protein expression in vivo
[58-59]. The X-dC-puromycin conjugates bearing
various fluorescent and biotin moieties (Figure2B) have been shown to be a new class ofreagents for studying protein expression not onlyin vitro, but also in vivo [58]. These compounds
are readily incorporated into expressed protein
products in cell lysates in vitro and efficiently
cross the cell membrane to function in protein
synthesis in vivo. This method was used to
examine whether dopamine receptor activation
stimulates local protein synthesis in living cells[59]. The system enables direct monitoring andimaging of protein expression with fluorescentpuromycin-labeled protein, and has the potential
to provide high spatial and temporal resolution inliving cells.
Conclusions and outlook
Puromycin technology in combination withcell-free translation systems allows the processingof huge libraries, is not limited by cellulartransformations, and is not biased by in vivo
environments. In vitro display technologies using
this approach for mRNA display, such as IVV,
have proved to be valuable tools for many
applications other than merely selecting
polypeptide binders. They have great potential for
directed evolution of protein stability and affinity,
for the selection of enzymatic activities,
antibodies, and functional peptides fromrandom-peptide libraries, and for proteomics
research, including protein-protein interactions,drug-protein
interactions,andpotentiallyDNA-protein interactions from cDNA libraries.
The C-terminal protein labeling technology isvery useful for analyzing protein-protein orprotein-DNA interactions with DNA microarrays
or with FCCS in model systems, and is expected
to have wide applicability. Thus, puromycin
technology is considered to be a next-generation
technology that will have a great impact in many
areas of biotechnology, medicine and proteomics.
Acknowledgements
We thank Dr. M. Ishizaka and S. Fujimori foruseful discussions. Also, we thank N. Hirai and K.
Masuoka for experimental assistances. This work
was supported in part by a grant of the Genome
Network Project from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
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