Recombinant Protein Expression in Mammalian Cells: Methods and Protocols (Methods in Molecular Biology, 2810)

Preface
Contents
Contributors
Chapter 1: PEI-Mediated Transient Gene Expression in CHO Cells
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Plasmids
		2.3 Transfection
		2.4 Culture Vessel
	3 Methods
		3.1 Plasmid Purification
		3.2 Routine Cell Culture
		3.3 Cell Expansion for Transfection
		3.4 Transfection
		3.5 Analysis of Antibody Production
	4 Notes
	References
Chapter 2: Transient Co-expression of Membrane Protein Complexes in Mammalian Cells
	1 Introduction
	2 Materials
		2.1 Plasmid DNA Purification
		2.2 Cell Cultivation and Transient Transfection
		2.3 Pull-Down Assays
		2.4 Large-Scale Protein Purification
	3 Methods
		3.1 Plasmid Design
		3.2 Plasmid Preparation
		3.3 General Cell Cultivation
		3.4 Small-Scale Transient Co-transfection Protocol (10 mL Cultures)
		3.5 Pull-Down Assays
		3.6 Large-Scale Co-transfection
		3.7 Large-Scale Purification
	4 Notes
	References
Chapter 3: The Use of Baculovirus-Mediated Gene Expression in Mammalian Cells for Recombinant Protein Production
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Transposition and Bacmid Preparation
		2.3 Bacmid PCR
		2.4 Transfection into Sf9 Cells and BacMam Virus Amplification
		2.5 BacMam Virus Concentration
		2.6 Transduction of Mammalian Cells for Small-Scale Expression Testing
		2.7 Small-Scale Purifications and Analysis of the Small-Scale Expression Tests
	3 Methods
		3.1 Routine Cell Culture Maintenance of Sf9 Insect Cells in Sf-900 III SFM Medium
		3.2 Routine Cell Culture Maintenance of Expi293F Mammalian Cells in FreeStyle 293 Medium
		3.3 Transposition and Bacmid Preparation
		3.4 Bacmid PCR
		3.5 Transfection into Sf9 Cells and BacMam Virus Amplification
		3.6 BacMam Virus Concentration
		3.7 Transduction of Mammalian Cells for Small-Scale Expression Testing
		3.8 Small-Scale Purifications and Analysis of the Small-Scale Expression Tests
	4 Notes
	References
Chapter 4: Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Transfection
		2.3 Harvest, Cell Lysis, and Supernatant Preparation
		2.4 Immunoaffinity Chromatography
		2.5 Buffer exchange Step Before Anion-Exchange Chromatography
		2.6 Anion-Exchange Chromatography
		2.7 Ultrafiltration
		2.8 Digital Polymerase Chain Reaction (dPCR)
	3 Methods
		3.1 Cell Culture
		3.2 Transient Transfection
		3.3 Harvest
		3.4 Cell Pellet Lysis and Supernatant Preparation
			3.4.1 Freeze-Thaw Lysis (See Note 14)
			3.4.2 Supernatant Preparation
		3.5 Immunoaffinity
		3.6 Buffer Exchange and Anion-Exchange Chromatography (AEX)
			3.6.1 Buffer Exchange
			3.6.2 Anion-Exchange Chromatography (AEX)
		3.7 Buffer exchange and Concentration by Ultrafiltration
		3.8 Determination of the Amount of Genome-Containing AAV Particles
	4 Notes
	References
Chapter 5: Protein Expression via Transient Transfection of Mammalian Cells in a WAVE Bioreactor
	1 Introduction
	2 Materials
		2.1 Transient Transfection
		2.2 SDS-PAGE Analysis
	3 Methods
		3.1 WAVE Bioreactor Preparation
		3.2 Transient Transfection of Expi293F Cells
		3.3 SDS-PAGE Analysis
	4 Notes
	References
Chapter 6: Small-Scale Cultivation and Transfection of ExpiCHO and HEK293E Cells in Single-Use Orbitally Shaken Bioreactors
	1 Introduction
	2 Materials
		2.1 Cell Culture and Transfection
	3 Methods
		3.1 Media Preparation
		3.2 Thawing Vials of Frozen Cells
		3.3 Routine Cultivation of Cells in an OSR50
		3.4 Routine Cultivation of Cells in an OSR600
		3.5 Transfection of ExpiCHO Cells in an OSR50
		3.6 Transfection of ExpiCHO Cells in an OSR600
		3.7 Transfection of HEK293E Cells in an OSR50
		3.8 Transfection of HEK293E Cells in an OSR600
	4 Notes
	References
Chapter 7: Recombinant Protein Production from Stable CHO Cell Pools
	1 Introduction
	2 Materials
		2.1 Parental Cell Thawing
		2.2 Parental Cell Freezing
		2.3 Plasmid DNA Preparation (MAXIPREP)
		2.4 Polyethylenimine (PEI) MAX Solution
		2.5 Transfection of Parental CHO Cells with PEI MAX
		2.6 Stable Pool Generation
		2.7 Stable Pool Freezing
		2.8 Stable Pool Fed-Batch Production
			2.8.1 Volumetric Titer Estimation of SmT1 Protein by SDS-PAGE
		2.9 Purification of Secreted His-tagged Proteins
	3 Methods
		3.1 Parental Cell Thawing and Maintenance
		3.2 Parental Cell Freezing
		3.3 Plasmid DNA Preparation
		3.4 Linear PEI MAX (1 mg/mL Solution)
		3.5 Transfection of Parental Cells (20 mL Scale)
		3.6 Selection with MSX for Stable Pool Generation
		3.7 Stable Pool Freezing
			3.7.1 Research Cell Bank 1 (RCB1, See Table 1)
			3.7.2 Research Cell Bank 2 (RCB2, See Table 1)
		3.8 CHO Stable Pool Fed-Batch Production
			3.8.1 Stable Pool Thawing and Maintenance
			3.8.2 Fed-Batch Production Seeding (-3 dpTs (Days Post-temperature Shift))
			3.8.3 Fed-Batch Production Temperature Shift (0 dpTs)
			3.8.4 Fed-Batch Production Monitoring
			3.8.5 Fed-Batch Production Harvesting (10 dpTs)
			3.8.6 Volumetric Titer Estimation of SmT1 Protein by SDS-PAGE
		3.9 Purification of Secreted His-tagged Proteins
	4 Notes
	References
Chapter 8: A Versatile Method for Inducible Protein Production in 293 Cells Using the PiggyBac Transposon System
	1 Introduction
	2 Materials
		2.1 DNA Cloning
		2.2 Establishment of Stable Cell Lines
		2.3 Protein Production
	3 Methods
		3.1 DNA Cloning
			3.1.1 cDNA Cloning in the pDONR221 Vector
				Primer Design
				PCR Amplification
				Gel Purification
				BP Cloning
			3.1.2 Subcloning in PB-T-RfA Vectors: LR Cloning
		3.2 Establishment of Stable Cell Lines
			3.2.1 Thawing 293-F Cells
			3.2.2 Transfection
			3.2.3 Selection for Stable Transfectants
			3.2.4 Cell Freezing
		3.3 Scale-Up and Protein Production
			3.3.1 Scale-Up (~Days 8-15)
			3.3.2 Induction (~Days 15-22)
		3.4 Assessment of Protein Production Levels
	4 Notes
	References
Chapter 9: Recombinant CHO Cell Pool Generation Using PiggyBac Transposon System
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Plasmids
		2.3 Transfection and Genetic Selection
	3 Methods
		3.1 Plasmid Purification
		3.2 Routine Cell Culture
		3.3 Transfection
		3.4 Genetic Selection
		3.5 Fed-Batch Culture
	4 Notes
	References
Chapter 10: Stable Expression by Lentiviral Transduction of Cells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Virus Production in Helper Cells (See Note 4)
		3.2 Concentration of Virus by Centrifugation
		3.3 Determination of the Virus Titer by Flow Cytometry (for Lentivirus Preparations That Transfer a Fluorophore Gene)
		3.4 Determination of the Virus Titer by Colony-Forming Assay (For Lentivirus Preparations that Confer Antibiotic Selection)
		3.5 Transduction of Cells by Lentiviral Vectors
	4 Notes
	References
Chapter 11: Expression of Multispecific Antibodies
	1 Introduction
	2 Materials
		2.1 Basic Cell Culture Techniques
		2.2 Plasmid Preparation and Linearization
		2.3 Upstream Fed-Batch Materials
		2.4 Purification by Differential Binding to Protein  A
	3 Methods
		3.1 Generation of the Three-Plasmid DNA Constructs
		3.2 Plasmid Preparation and Linearization
		3.3 Basic Cell Culture Techniques
		3.4 Ratio Optimization with Three Plasmids by Transient Transfection Using PEI
		3.5 Stable Transfection
		3.6 Selection of Bulk Pool Populations and Freezing of Cryovials
		3.7 Fed-Batch Process
		3.8 Titer Measurement of Fully Assembled Product
		3.9 Harvest
		3.10 Purification by Differential Binding to Protein A
	4 Notes
	References
Chapter 12: Expression of Bispecific Antibodies Using Cellular Display
	1 Introduction
	2 Materials
		2.1 Design of Alternate Splicing Constructs
		2.2 Cloning of the Genes of Interest
		2.3 Enrichment of Cells Based on Cellular Display
		2.4 Enrichment of Cells Based on Quality Attributes
	3 Methods
		3.1 Design of Alternate Splicing Constructs
			3.1.1 Design of Basic Construct (With Weak Splice Donor in the Fc)
			3.1.2 Optimization of Cellular Display and Secretion
			3.1.3 Cloning of the Genes of Interest
		3.2 Enrichment of Cells Based on Cellular Display
		3.3 Enrichment of Cell Populations Based on Product Quality
	4 Notes
	References
Chapter 13: Generation of Monoclonal Chinese Hamster Ovary Cell Lines Using DISPENCELL-S3
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Equipment
		2.3 Cells
	3 Methods
		3.1 Culture of CHO Cells
		3.2 DISPENCELL-S3 Setup
		3.3 Media Preparation for DISPENCELL-S3 Seeding into One 96-Well Plate
		3.4 Cell Sample Filtration
		3.5 Cell Sample Characterization and Dilution
		3.6 Single-Cell Isolation with DISPENCELL-S3
		3.7 DISPENCELL-S3 Multiwell Plate Analysis
		3.8 Monoclonal Outgrowth Assessment
		3.9 Clone Expansion
	4 Notes
	References
Chapter 14: Enhancing Cell Line Stability by CRISPR/Cas9-Mediated Site-Specific Integration Based on Histone Modifications
	1 Introduction
	2 Materials
		2.1 Reagents and Kits
		2.2 Cells, Buffer, and Media
		2.3 Equipment
		2.4 Bioinformatic Tools and References
	3 Methods
		3.1 ChIP Sequencing
		3.2 Data Analysis ChIP-Seq and Identification of Integration Sites
		3.3 Cloning of CRISPR Plasmids
			3.3.1 Designing Guide RNAs (gRNA)
			3.3.2 Annealing and Phosphorylation of gRNAs
			3.3.3 Preparation of the Plasmid Backbone
			3.3.4 Digestion and Dephosphorylation of the Plasmid Backbone
			3.3.5 Ligation of gRNA and Backbone Plasmid
			3.3.6 Transformation
		3.4 Cloning of the Donor Plasmid
			3.4.1 Planning and Preparation of the Homology Arms
			3.4.2 Cloning of the Donor Plasmid
		3.5 Transfection and Selection
			3.5.1 Transfection
			3.5.2 Selection
		3.6 Examining of Site-Specific Integration
			3.6.1 Determination of Fluorescence Intensities
			3.6.2 Cas9-Targeted Sequencing of Integration Sites
	4 Notes
	References
Chapter 15: Bench-Scale Stirred-Tank Bioreactor for Recombinant Protein Production in Chinese Hamster Ovary (CHO) Cells in Sus...
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 3-L Glass Bioreactor and Controller
		2.3 Cell Analysis
	3 Methods
		3.1 Cell Culture and Seed Train Expansion
		3.2 Bioreactor Preparation
			3.2.1 Assembly and Sterilization
			3.2.2 Medium Batching
		3.3 Operation of Bioreactor
			3.3.1 Inoculation
			3.3.2 Daily Routine Sampling
		3.4 Harvest of Bioreactor
	4 Notes
	References
Chapter 16: Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells
	1 Introduction
	2 Materials
		2.1 Reagents and Kits
		2.2 Formulas
		2.3 Equipment
	3 Methods
		3.1 Design sgRNAs Against Targeted Gene
		3.2 Construction of px458 Plasmid with Designed sgRNAs
			3.2.1 Preparation of px458 Vector
			3.2.2 Digestion of px458 Plasmid
			3.2.3 Phosphorylate and Anneal sgRNA Primers for the sgRNA Constructs (see Note 5)
			3.2.4 The sgRNA Plasmid Construction (Ligation)
			3.2.5 Transformation of sgRNA Plasmid in E. coli
			3.2.6 Analyze Transformation Colony
		3.3 Transfection of CRISPR-Cas9 Components into an IgG-Expressing CHO Cell Line
			3.3.1 Day 0: Seed Cells for Transfection
			3.3.2 Day 1: Transfection of sgRNA Plasmids with Lipofectamine 3000
			3.3.3 (Alternative to Subheading 3.3.2) Day 1: Electroporation of CRISPR Ribonucleoproteins (RNPs)
		3.4 LCA Lectin Selection of the Transfected Cells
		3.5 (Optional) Fluorescence-Activated Cell Sorting to Enrich for sgRNA Plasmid Expression (see Note 9)
		3.6 Surveyor Mutation Assay to Check the Knockout Efficiency
		3.7 Limited Dilution Cloning (Clone Isolation) (see Note 13)
		3.8 Clonal Cell Line Selection
			3.8.1 Sanger Sequencing to Analyze Gene Mutation
			3.8.2 LCA Lectin Blot to Check FUT8 Knockout Efficiency
			3.8.3 Antibody Titer Analysis
	4 Notes
	References
Chapter 17: Development of Responsive Promoters and their Utilization for Stable CHO Sensor Cell Lines
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Plasmids
		2.3 Transfection and Selection
		2.4 Fed-Batch Fermentation
		2.5 Fluorescence Assessment
	3 Methods
		3.1 Plasmid Purification
		3.2 Plasmid Cloning
		3.3 Stable Cell Line Generation
		3.4 Induction of Responsive Promoters
		3.5 Fed-Batch Cultivation and Automated Fluorescence Measurement
	4 Notes
	References
Chapter 18: CRISPR Deletion of miR-27 Impacts Recombinant Protein Production in CHO Cells
	1 Introduction
	2 Materials
		2.1 Expression Plasmid Construction
		2.2 Transfection of CHO Cell Lines
		2.3 Indel Analysis
		2.4 Analysis of Mature miRNA Expression
		2.5 Analysis of Phenotype in Batch and Fed-Batch Cultures
	3 Methods
		3.1 Guide RNA Design Considerations for Noncoding RNAs
		3.2 Generation and Preparation of sgRNA Expression Plasmid
			3.2.1 Annealing, Ligation, and Amplification of sgRNA Expression Vector
			3.2.2 Transformation
		3.3 Transfection and Generation of Stable Cell Lines
		3.4 Analysis of Indels Generated by CRISPR/Cas9 Using TOPOTA Cloning and Surveyor Assay
			3.4.1 TOPOTA Cloning
			3.4.2 Surveyor Assay for Detection of Indels
			3.4.3 Next-Generation Sequencing (NGS) for Analysis of Indels
		3.5 Analysis of Mature miR-27 Expression After Targeting Using CRISPR/Cas9
		3.6 Analysis of Phenotype in Batch and Fed-Batch Cultures After Knockdown of miR-27b Using CRISPR/Cas9
	4 Notes
	References
Chapter 19: High-Throughput Protein Expression Screening of Cell-Surface Protein Ectodomains
	1 Introduction
	2 Materials
		2.1 High-Throughput Expression of Recombinant Proteins in Mammalian Cells
		2.2 Biotinylation of Expressed Proteins in the Supernatant
		2.3 ELISA Fluorescence
	3 Methods
		3.1 High-Throughput Expression of Recombinant Proteins in Mammalian Cells
			3.1.1 Day 0-Cell Seeding and DNA Preparation
			3.1.2 Day 1-Transfection
			3.1.3 Day 2-Feeding
			3.1.4 Day 6-Harvesting Expressed Proteins
		3.2 Biotinylation of Expressed Proteins in the Supernatant
		3.3 ELISA Fluorescence
			3.3.1 Plate Coating to Bind the Expressed Proteins
			3.3.2 Washing and Blocking the ELISA Plate
			3.3.3 Prepare Standards and Capture Biotinylated Proteins on the ELISA Plate
			3.3.4 Incubate Bound Proteins with Streptavidin, Alexa Fluor 488 Conjugate
			3.3.5 Imaging and Analysis
	4 Notes
	References
Chapter 20: Genome-Wide High-Throughput RNAi Screening for Identification of Genes Involved in Protein Production
	1 Introduction
	2 Materials
		2.1 Cells and Media
		2.2 Transfection Reagents and Instrumentation
		2.3 Luciferase Assay
		2.4 Data Visualization
	3 Methods
		3.1 Assay Optimization and Development
			3.1.1 Cell Number Optimization and Plate Selection
			3.1.2 Transfection Efficiency Assessment
		3.2 Primary Screen
		3.3 Secondary and Orthogonal Validation Screens
	4 Notes
	References
Chapter 21: Purification and Quality Control of Recombinant Proteins Expressed in Mammalian Cells: A Practical Review
	1 Introduction
	2 Purification Differences in Intracellular and Extracellular/Secreted Proteins
	3 Affinity Chromatography
	4 Genetically Engineered Tags
	5 Fusion Proteins with Solubility-Enhancing or Fluorescence Tags and Cleavage with Specific Proteases
	6 Parameters for Purity Optimization for Tagged Proteins
	7 Other Conventional Chromatography Techniques
		7.1 Size Exclusion Chromatography (SEC)
		7.2 Ion Exchange Chromatography (IEC)
		7.3 Mixed Mode Chromatography (MMC)
		7.4 Hydrophobic Interaction Chromatography (HIC)
		7.5 Reverse Phase Chromatography (RPC)
		7.6 Ultrafiltration (UF)
	8 Purification Strategies
		8.1 Strategy 1
		8.2 Strategy 2
		8.3 Strategy 3
		8.4 Strategy 4
		8.5 Strategy 5
		8.6 Strategy 6
		8.7 Strategy 7
		8.8 Strategy 8
		8.9 Strategy 9
		8.10 Strategy 10
		8.11 Strategy 11
		8.12 Strategy for the Purification of Large Molecules or Complexes
	9 Prone to Aggregate Proteins and Buffer Optimization
	10 Final Quality Control: Purity, Homogeneity, Identity, and Integrity
	References
Index