Advanced Methods in Structural Biology, 1st ed. 2023
Methods in Molecular Biology Series, Vol. 2652

This volume explores the latest integrated bioprocesses and technologies used to study the production of the target recombinant protein of therapeutic or diagnostic interest; its isolation, purification, and stabilization; and the bio-interaction and structural analyses. The chapters in this book are organized into four parts. Part One covers production methods of soluble and membrane proteins in prokaryotic and eukaryotic expression systems, such as Lactococcus lactis and Escherichia coli. Part Two describes traditional and novel approaches for recombinant protein purification and stabilization, and buffers and additives. Part Three discusses automated methods in structural biology based on in silico approaches; and Part Four provides examples of advanced protein investigation methodologies to assess structural analysis such as high-throughput protein crystallization and time-resolved serial crystallography. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and comprehensive, Advanced Methods in Structural Biology is a valuable resource to those in academia (i.e., graduate students and postdoctoral researchers) and researchers in the pharmaceutical industry who wish to learn more about this developing field.  

Chapter 5 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

Preface…
Table of Contents…
Contributing Authors…

PART I VECTOR DESIGN AND PROTEIN EXPRESSION

1 Cloning and Recombinant Protein Expression in Lactococcus lactis
Susheel K. Singh, Mohammad Naghizadeh, Jordan Plieskatt, Subhash Singh, and Michael Theisen

2 E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate
Diana Gomes, Luís A. Passarinha, and Ângela Sousa

3 Mini-Bioreactor Platform for Membrane Proteins Production in Komagataella pastoris
Jorge Barroca-Ferreira, Claudio J. Maia, and Luís A. Passarinha

4 Recombinant Protein Production Using the Baculovirus Expression Vector System (BEVS)
Heather McDuffie Sullivan, Ewa Krupinska, Anna Andersson Rasmussen, Juan Manuel Orozco Rodriguez, and Wolfgang Knecht

5 High-Throughput Production and Optimization of Membrane Proteins after Expression in Mammalian Cells
Nadisha Gamage, Harish Cheruvara, Peter J. Harrison, James Birch, Charlie J. Hitchman, Monika Olejnik, Raymond J. Owens, and Andrew Quigley

6 Fluorescent Fusion Proteins Expression in Plant Cells
Shuxian Huang and Yonglun Zeng 

7 Functional Expression of the Ectodomain of Plant Receptor Kinases in Plant Suspension Culture
Hidefumi Shinohara 

PART II RECOMBINANT PROTEIN PURIFICATION AND STABILIZATION

8 A Simple Purification Method for Heat Stable Recombinant Low Molecular Weight Proteins and Peptides Via GST-Fusion Products
Patience Salavalina Okoto, Shivakumar Sonniala, Beatrice Sakhel, Djamali Muhoza, Paul Adams, and Thallapuranam Krishnaswamy Suresh Kumar  

9 eGFP as an All-in-One Tag for Purification of Membrane Proteins
Tomáš Heger, Charlott Stock, Michelle Juknaviciute Laursen, Michael Habeck, Thibaud Dieudonné, and Poul Nissen 

10 Effects of Ionic Liquids as Additives on Protein Crystallization
Crissy L. Tarvera and Marc L. Pusey

11 Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay: A Practical Approach
Filipa S. S. Engrola, João Paquete-Ferreira, Teresa Santos-Silva, Márcia A. S. Correia, Francisco Leisico, and Marino F. A. Santos

12 Biophysical Characterization of Membrane Proteins
Stephan Niebling, Osvaldo Burastero, and María García-Alai

13 Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase
Thibaud Dieudonné, Christine Jaxel, Maylis Lejeune, Guillaume Lenoir, and Cédric Montigny 

14 Preparation of Protein-Enriched Outer Membrane Vesicles from Escherichia Coli for In Situ Structural Biology of Outer Membrane Proteins
Filippo Castegnaro, Björn M. Burmann, and Johannes Thoma 

PART III PROTEIN BIO-INTERACTION ANALYSIS 

15 Receptor-Based Virtual Screening of Large Libraries in a Multi-Level in Silico Approach
Tatiana F. Vieira and Sérgio F. Sousa

16 Protein-Protein Interaction Studies Using Molecular Dynamics Simulation
Veerendra Kumar and Shivani Yaduvanshi 

17 Isolation of Bacterial Extracellular Vesicles and Identification of their Protein Cargo
Roberto Alvarez-Fernandez Garcia, Guillermo Aragoneses-Cazorla, Laura Lerma, Rafael C. Prados-Rosales, and Jose L. Luque-Garcia

18 Study of Protein Conformational Dynamics Using Hydrogen/Deuterium Exchange Mass Spectrometry
Jorge Lukas Uhrik, Tomas Henek, Joan Planas-Iglesias, Josef Kucera, Jiri Damborsky, Martin Marek, and Lenka Hernychova

19 Protein Interaction Analysis by Surface Plasmon Resonance
Dennis G. Drescher and Marian J. Drescher

PART IV PROTEIN STRUCTURAL ANALYSIS

20 High-Throughput Protein Crystallization in an Integrated Droplet-Based Microfluidic Platform
Joana Ferreira and Filipa Castro

21 Sample Preparation for Time-Resolved Serial Crystallography: Practical Considerations
Pedram Mehrabi and Eike C. Schulz

22 Using Small-Angle X-Ray Scattering to Characterize Biological Systems: A General Overview and Practical Tips
João Paquete-Ferreira, Francisco Leisico, Márcia A. S. Correia, Filipa S. S. Engrola, Teresa Santos-Silva, and Marino F. A. Santos

23 Nuclear Magnetic Resonance-Guided Structural Analysis of Moderate-Affinity Protein Complexes with Intrinsically Disordered Polypeptides
Dmitri Tolkatchev, Garry E. Smith Jr., and Alla S. Kostyukova 

24 Protein Structural Analysis by Cryogenic Electron Microscopy
Michael Hall, Erin Schexnaydre, Camilla Holmlund, and Marta Carroni 

Subject Index List…

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts