Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
PART I RECOMBINANT EXPRESSION AND ISOLATION FROM ENDOGENOUS SOURCE
1 Production of Multi-subunit Membrane Protein Complexes . . . . . . . . . . . . . . . . .
3
Burak V. Kabasakal, Qiyang Jiang,
and Christiane Schaffitzel
2 Production of Multiprotein Complexes Using the Baculovirus
Expression System: Homology-Based and Restriction-Free
Cloning Strategies for Construct Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Paola Rossolillo, Olga Kolesnikova, Karim Essabri,
Gala Ramon Zamorano, and Arnaud Poterszman
3 Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9
System and Double-Stranded DNA Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Sylvain Geny, Simon Pichard, Alice Brion,
Jean-Baptiste Renaud, Sophie Jacquemin,
Jean-Paul Concordet, and Arnaud Poterszman
4 Validation of the Production of Antibodies in Different Formats
in the HEK 293 Transient Gene Expression System . . . . . . . . . . . . . . . . . . . . . . . . . 59
Jens Ko ¨nig, Michael Hust, and Joop van den Heuvel
5 The Intervening Removable Affinity Tag (iRAT) System
for the Production of Recombinant Antibody Fragments . . . . . . . . . . . . . . . . . . . . 77
Norimichi Nomura, Yayoi Nomura,
Yumi Sato, and So Iwata
6 Isolation of Artificial Binding Proteins (Affimer Reagents)
for Use in Molecular and Cellular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Anna A. S. Tang, Christian Tiede,
Michael J. McPherson, and Darren C. Tomlinson
PART II BIOPHYSICAL CHARACTERIZATION AND SAMPLE OPTIMIZATION
7 Measurements of Protein–DNA Complexes Interactions
by Isothermal Titration Calorimetry (ITC)
and Microscale Thermophoresis (MST). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Amandine Gontier, Paloma F. Varela, Cle ´ment Nemoz,
Virginie Ropars, Magali Aumont-Nicaise,
Michel Desmadril, and Jean-Baptiste Charbonnier
8 switchSENSE Technology for Analysis of DNA Polymerase Kinetics . . . . . . . . . . 145
Guillaume Bec and Eric Ennifar
9 Sedimentation Velocity Methods for the Characterization
of Protein Heterogeneity and Protein Affinity Interactions . . . . . . . . . . . . . . . . . . . 155
Christine Ebel and Catherine Birck
vii
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ix
PART I RECOMBINANT EXPRESSION AND ISOLATION FROM ENDOGENOUS SOURCE
1 Production of Multi-subunit Membrane Protein Complexes . . . . . . . . . . . . . . . . .
3
Burak V. Kabasakal, Qiyang Jiang,
and Christiane Schaffitzel
2 Production of Multiprotein Complexes Using the Baculovirus
Expression System: Homology-Based and Restriction-Free
Cloning Strategies for Construct Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Paola Rossolillo, Olga Kolesnikova, Karim Essabri,
Gala Ramon Zamorano, and Arnaud Poterszman
3 Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9
System and Double-Stranded DNA Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Sylvain Geny, Simon Pichard, Alice Brion,
Jean-Baptiste Renaud, Sophie Jacquemin,
Jean-Paul Concordet, and Arnaud Poterszman
4 Validation of the Production of Antibodies in Different Formats
in the HEK 293 Transient Gene Expression System . . . . . . . . . . . . . . . . . . . . . . . . . 59
Jens Ko ¨nig, Michael Hust, and Joop van den Heuvel
5 The Intervening Removable Affinity Tag (iRAT) System
for the Production of Recombinant Antibody Fragments . . . . . . . . . . . . . . . . . . . . 77
Norimichi Nomura, Yayoi Nomura,
Yumi Sato, and So Iwata
6 Isolation of Artificial Binding Proteins (Affimer Reagents)
for Use in Molecular and Cellular Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Anna A. S. Tang, Christian Tiede,
Michael J. McPherson, and Darren C. Tomlinson
PART II BIOPHYSICAL CHARACTERIZATION AND SAMPLE OPTIMIZATION
7 Measurements of Protein–DNA Complexes Interactions
by Isothermal Titration Calorimetry (ITC)
and Microscale Thermophoresis (MST). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Amandine Gontier, Paloma F. Varela, Cle ´ment Nemoz,
Virginie Ropars, Magali Aumont-Nicaise,
Michel Desmadril, and Jean-Baptiste Charbonnier
8 switchSENSE Technology for Analysis of DNA Polymerase Kinetics . . . . . . . . . . 145
Guillaume Bec and Eric Ennifar
9 Sedimentation Velocity Methods for the Characterization
of Protein Heterogeneity and Protein Affinity Interactions . . . . . . . . . . . . . . . . . . . 155
Christine Ebel and Catherine Birck
vii