Mammalian Expression Technologies
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Mammalian Expression System
The production of proteins in mammalian cells is an important tool in numerous scientific and commercial areas. For example, the proteins expressed in and purified from mammalian cell system are routinely needed for life science research and development. In the field of biomedicine, proteins for human therapy, vaccination or diagnostic applications are typically produced in mammalian cells. Gene cloning, protein engineering, biochemical and biophysical charact erization of proteins also require the use of gene expression in mammalian cells. Other applications in widespread use involve screening of libraries of chemical compounds in drug discovery, and the development of cell-based biosensors.
Usage of Protein Produced in Mammalian Expression System
The proteins produced in mammalian system have the best structural and functional features that are usually most close to their cognate native form and can satisfy the following application needs or utility:
- Transgene expression
- Biochemical analyses
- Assay standards
- Functional studies of the protein (in vitro and ex vivo)
- Structural studies, including protein crystallization, protein structure and NMR
- Protein-protein interaction experiments
- Enzyme kinetics
- Immunogen for antibodies development
- Proteomic and phenomics study
- Drug target discovery and validation
- Cell line development, drug screening, and in vitro model system
- Animal studies, including in vivo functional and ADME, PK/TK and safety studies
- Physiology and pathology studies
- Diagnostic application
- Therapeutic application
- Prophylactic (vaccine) development
- Protein engineering and mutagenesis studies)
Overview of Mammalian Expression Technology
Within this technology area, many aspects contribute a successful protein production in mammalian cell system. Generally, the following lines of consideration are helpful in making a given protein expression project productive:
- Transgene bioinformatics analysis and genetic engineering
- Transgene modification and codon optimization (see expanded points below)
- Expression vector selection and development (see expanded description below)
- Host cell selection, adaptation, reengineering, and development
- Optimization of plasmid backbone and expression cassette for gene expression
- Methods for DNA introduction into mammalian cells
- Lipid reagents for DNA transfer into mammalian cells
- Reporter genes for monitoring gene expression in mammalian cells
- Gene targeting techniques for efficient protein production
- Gene transfer and amplification in mammalian cells
- Viral-based vectors for gene expression in mammalian cells
- Adeno-associated virus
- Epstein-Barr virus
- Herpes simplex virus
- Semliki Forest virus
- Simian virus 40
- Sindbis virus
- Vaccinia virus
- Co-transfer of multiple plasmids/viruses to introduce several genes
- Matrix-attachment regions and protein production
- Chromatin insulators, position effects, and locus control regions
- Posttranslational processing, transport and secretion of proteins
- Pathways of mammalian protein glycosylation
- Metabolic engineering of mammalian cells for higher protein yield
- Translational regulation in mammalian cells
- Pathways of mammalian messenger RNA degradation
- Pathways of mammalian protein degradation
- Intracellular targeting of antibodies in mammalian cells
- Inducible gene expression in mammalian cells
- Inducible gene expression in mammalian cells
- Protein production in transgenic animals
- Protein purification in mammalian cell culture
Consideration of Transgene Sequence Modification and Codon Optimization
The following points can be considered for preparing transgene to conduct protein expression and functional studies:
- Codon optimization and G/C content adaptation
- Inhibition of internal splicing and premature polyadenylation
- Prevention of creation of stable RNA secondary
- Introduction/avoidance of immunomodulatory CpG motifs
- Avoidance of direct DNA repeats and thereby recombination events
- Domain shuffling, epitope scrambling and protein chimarization
- Obstruction of formation of stable dsRNAs with host transcriptom
- Avoidance of possible RNAi viable genes
- Optimization of tissue specific promoter/enhancer sequences
- Addition of RNA stabilizing and nuclear translocation supporting sequence elements
- Validation of various gene derivatives combining the optimized gene with
- Preselected P/E sequences
- RNA stabilization and export signal
- Development of the most efficient cell line/vector combination
- Quantification of gene expression by state-of-the-art analysis
Membrane Protein Expression
Integral membrane protein expression in mammalian cells is not difficult if one is trying to accomplish expression at low physiological levels. For example, integral membrane protein expression has been used for decades for drug and biological discovery. However, over-expression of integral membrane proteins presents a number of different challenges.
- Produce a large quantity of a membrane protein for purification and structural study
- Collect systematic and complete mammalian over-expression data with a variety of different integral membrane proteins using several different cell lines and different N/C termini tags.
- Perform oligonucleotide microarray analysis to determine gene expression profile of different mammalian cells overexpressing different integral membrane proteins.
- Automate the technologies to conduct mammalian expression.
- Using a directed evolution approach, mutate integral membrane proteins to increase expression and stability.
- Detergent extraction and purification of integral membrane protein targets derived from mammalian expression system.
- Biophysical characterization including functional assays for integral membrane proteins expressed in the mammalian expression system.
Production of Fusion Protein
This might at first seem a disadvantage because the natural product of the inserted gene is not made. However, the extra amino acids on the fusion protein can be a great help in purifying the protein product. Oligo-histidine is one of the commonly used fusion tags for protein expression (in both prokaryotic and eukaryotic system). Why would we want to attach six or ten histidines to our protein? Oligo-histidine regions like this have a high affinity for metals like nickel, so the expressed target proteins can be purifed using nickel affinity chromatography. The beauty of this method is its simplicity and speed. After the expression systems have made the fusion protein, it can be applied to a nickel affinity column, wash out all unbound proteins, and then release the fusion protein with histidine or a histidine analog called imidazole. This procedure allows harvesting essentially pure fusion in only one step. This is possible because very few if any natural proteins have oligo-histidine regions, so the fusion protein is essentially the only one that binds to the column. There are other tags have been used in protein expression in mammalian cells. There are mechanisms designed for removing tag from the fusion proteins. For example, enterokinase or Xa factor have been used for cleave tags from the fusion proteins that contain the cleave site for the enzymes.
GenWay specializes in mammalian expression system. To meet different needs for protein expression, mammalian cell protein expression can be carried out via the following different approaches:
- Transient burst expression
- Adenovirus-assisted expression.
- Stable mammalian cell line expression (CHO and HEK293)
Mammalian Expression System
A. Transient transfection. Proteins expressed were shown by SDS-PAGE and Western analyses; B. Stable transfection of green fluorescence protein (GFP); C. Adenoviral vector-mediated protein expression in mammalian cells. β-gal expression was shown by X-gal staining.
For other systems of protein expression, please visit the pages:
GenWay offers comprehensive custom services in recombinant protein expression.
For more detailed information, please see the specific description pages in protein expression