Table Of ContentMethods in
Molecular Biology 1118
Kirill Alexandrov
Wayne A. Johnston Editors
Cell-Free
Protein
Synthesis
Methods and Protocols
M E T H O D S I N M O L E C U L A R B I O LO G Y ™
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfi e ld, Hertfordshire, AL10 9AB, UK
F or further volumes:
h ttp://www.springer.com/series/7651
C ell-Free Protein Synthesis
M ethods and Protocols
E dited by
K irill A lexandrov and Wayne A. Johnston
Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia
E ditors
K irill Alexandrov W ayne A. J ohnston
I nstitute for Molecular Bioscience I nstitute for Molecular Bioscience
The University of Queensland The University of Queensland
St. Lucia, QLD, Australia St. Lucia, QLD, Australia
I SSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-781-5 ISBN 978-1-62703-782-2 (eBook)
D OI 10.1007/978-1-62703-782-2
S pringer New York Heidelberg Dordrecht London
L ibrary of Congress Control Number: 2013957131
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P refa ce
A dvances in Life Sciences and Biotechnology have historically relied on the ability to replicate
the building blocks of life in vitro, in order to elucidate their mode of action. Much bio-
technological progress in the last 40 years has been focused on developing more effi cient
analysis and synthesis technologies for both DNA and proteins. However, while orders of
magnitude reduction in costs for DNA sequencing and synthesis was achieved during the
last decade, the throughput and cost of technologies for protein production and engineer-
ing have changed comparatively little.
C ell-free protein expression is a rapid and high-throughput methodology for conver-
sion of DNA-encoded genetic information into protein-mediated biochemical activities. It
holds the promise to narrow the technological gap between DNA and protein technologies
and provide a platform for broad application of synthetic biology principles in the Life
Sciences.
C ell-free technologies have developed in two opposite but complementary directions:
scale-up and miniaturization. Scale-up aims to produce preparative amounts of high-value
recombinant proteins rapidly and without involvement of a recombinant host.
Miniaturization aims to extract the most information out of the smallest amount of the
largest possible number of proteins or protein variants at the lowest possible cost.
Combination of both directions is expected to provide us with a powerful platform for
protein analysis, engineering, and manufacturing.
T his book is aimed to bring together the key opinion leaders of cell-free technology
development and provide case studies and detailed protocols for application of cell-free
methodology. The book aims to cover the main directions in the development of cell-free
technologies including several recently developed cell-free systems. The book also presents
a number of applications of cell-free systems that range from discovery of biofuel enzymes
to in vitro assembly of viruses.
T arget groups: Biochemists, bioengineers, biotechnologists, cell biologists, and chemical
and synthetic biologists.
S t. Lucia, Q LD , Australia K irill A lexandrov
W ayne A . J ohnston
v
C ontents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Production of Eukaryotic Cell-Free Lysate from Leishmania tarentolae . . . . . . 1
Wayne A. Johnston and Kirill Alexandrov
2 Bioinformatics Analysis and Optimization of Cell-Free Protein Synthesis . . . . . 17
Alexander A. Tokmakov, Atsushi Kurotani, Mikako Shirouzu,
Yasuo Fukami, and Shigeyuki Yokoyama
3 A Cell-Free Expression Screen to Identify Fusion Tags
for Improved Protein Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Andrew Kralicek
4 One-Pot, Microscale Cell-Free Enzyme Expression and Screening. . . . . . . . . . 55
Aarthi Chandrasekaran and Anup K. Singh
5 Cell-Free Translation of Biofuel Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Taichi E. Takasuka, Johnnie A. Walker, Lai F. Bergeman,
Kirk A. Vander Meulen, Shin-ichi Makino, Nathaniel L. Elsen,
and Brian G. Fox
6 Cloning-Independent Expression and Screening
of Enzymes Using Cell-Free Protein Synthesis Systems . . . . . . . . . . . . . . . . . . 97
Yong-Chan Kwon, Jae-Kwang Song, and Dong-Myung Kim
7 High-Level Cell-Free Production of Membrane
Proteins with Nanodiscs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Christian Roos, Lei Kai, Stefan Haberstock, Davide Proverbio,
Umesh Ghoshdastider, Yi Ma, Slawomir Filipek, Xiaoning Wang,
Volker Dötsch, and Frank Bernhard
8 Cell-Free Protein-Based Enzyme Discovery
and Protein–Ligand Interaction Study. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Sabrina Guillemer, Cécile Persillon, Jean-Michel Masson,
and Gilles Ravot
9 Human Cell Extract-Derived Cell-Free Systems for Virus Synthesis . . . . . . . . . 149
Tominari Kobayashi, Kodai Machida, and Hiroaki Imataka
10 Cell-Free Protein Synthesis in Microfluidic 96-Well Plates . . . . . . . . . . . . . . . . 157
Kirsten Jackson, Ruba Khnouf, and Z. Hugh Fan
11 Preparation of Multiple Site-Specific Mutant Proteins
for NMR Studies by PCR-Directed Cell-Free Protein Synthesis . . . . . . . . . . . . 169
Kiyoshi Ozawa and Ruhu Qi
12 Site-Specific Incorporation of Unnatural Amino Acids
into Proteins by Cell-Free Protein Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Kiyoshi Ozawa and Choy Theng Loh
vii
viii Contents
13 In Vitro Translation of Papillomavirus Authentic and Codon-Modified
L1 Capsid Gene mRNAs in Mouse Keratinocyte Cell-Free Lysate . . . . . . . . . . 205
Kong-Nan Zhao
14 An Optimized Yeast Cell-Free Lysate System
for In Vitro Translation of Human Virus mRNA . . . . . . . . . . . . . . . . . . . . . . . 219
Xiao Wang, Liang Zhao, and Kong-Nan Zhao
15 In Vitro Translation-Based Protein Kinase Substrate Identification . . . . . . . . . 231
Szilvia K. Nagy and Tamás Mészáros
16 Preparation of Protein Arrays Using Cell-Free Protein Expression . . . . . . . . . . 245
Elizabeth A. Cook and Mingyue He
17 Posttranscriptional Control of Protein Synthesis
in Drosophila S2 Cell-Free System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Motoaki Wakiyama and Shigeyuki Yokoyama
18 Cell-Free Membrane Protein Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Tomomi Kimura-Soyema, Mikako Shirouzu, and Shigeyuki Yokoyama
19 The PURE System for Protein Production . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Yoshihiro Shimizu, Yutetsu Kuruma, Takashi Kanamori,
and Takuya Ueda
20 A Cell-Free Protein Synthesis System from Insect Cells . . . . . . . . . . . . . . . . . . 285
Toru Ezure, Takashi Suzuki, and Eiji Ando
21 A Cell-Free Expression Platform for Production
of Protein Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Xristo Zárate and David W. Galbraith
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
C ontributors
K IRILL A LEXANDROV • Institute for Molecular Bioscience, The University of Queensland,
S t. Lucia, Q LD , A ustralia
E IJI A NDO • Analytical & Measuring Instruments Division, Clinical & Biotechnology
Business Unit, S himadzu Corporation, K yoto, J apan
L AI F . B ERGEMAN • Department of Biochemistry, U niversity of Wisconsin, M adison , W I , U SA
F RANK B ERNHARD • Centre for Biomolecular Magnetic Resonance, I nstitute for Biophysical
Chemistry, Goethe-University of Frankfurt/Main, F rankfurt/Main, G ermany
A ARTHI C HANDRASEKARAN • Joint BioEnergy Institute (JBEI), E meryville, C A, U SA;
S andia National Laboratories, L ivermore, C A, U SA
E LIZABETH A . C OOK • Protein Technology Group, Babraham Research Campus, B abraham
Bioscience Technologies Ltd , C ambridge, U K
V OLKER D ÖTSCH • Centre for Biomolecular Magnetic Resonance, I nstitute for Biophysical
Chemistry, Goethe-University of Frankfurt/Main, F rankfurt/Main, G ermany
N ATHANIEL L . E LSEN • Department of Biochemistry , U niversity of Wisconsin, M adison,
W I , U SA
T ORU E ZURE • Analytical & Measuring Instruments Division, Clinical & Biotechnology
Business Unit, S himadzu Corporation, K yoto, J apan
S LAWOMIR F ILIPEK • Faculty of Chemistry , U niversity of Warsaw, W arsaw, P oland
B RIAN G . F OX • Department of Biochemistry , U niversity of Wisconsin, M adison, W I , U SA
Y ASUO F UKAMI • Graduate School of Science, K obe University, N ada , J apan; R esearch
Center for Environmental Genomics, K obe University, N ada , J apan
D AVID W . G ALBRAITH • School of Plant Sciences and BIO5 Institute, University of Arizona,
T ucson, A Z , U SA
U MESH G HOSHDASTIDER • Centre for Biomolecular Magnetic Resonance, I nstitute for
Biophysical Chemistry, Goethe-University of Frankfurt/Main, F rankfurt/Main,
G ermany; F aculty of Chemistry , U niversity of Warsaw, W arsaw, P oland
S ABRINA G UILLEMER • Protéus, Parc Georges Besse, N îmes , F rance
S TEFAN H ABERSTOCK • Centre for Biomolecular Magnetic Resonance, Institute for Biophysical
Chemistry , G oethe-University of Frankfurt/Main, F rankfurt/Main, G ermany
M INGYUE H E • The Inositide Laboratory , B abraham Institute, Babraham Research
Campus , C ambridge, U K
Z . H UGH FAN • Department of Mechanical and Aerospace Engineering , U niversity of
Florida, G ainesville, F L, U SA; D epartment of Biomedical Engineering, U niversity of
Florida, G ainesville, F L, U SA
H IROAKI I MATAKA • Department of Materials Science and Chemistry , G raduate School of
Engineering, University of Hyogo, H imeji, J apan; M olecular Nanotechnology Research
Center , G raduate School of Engineering, University of Hyogo, H imeji, J apan; R IKEN
Systems and Structural Biology Center , T surumi-ku, Yokohama, J apan
K IRSTEN J ACKSON • Department of Biomedical Engineering, U niversity of Florida,
G ainesville, F L, U SA
ix
