Protein Expression Library

PEF uses a suite of expression host systems to enable the efficient production of recombinant proteins. Each system has benefits and drawbacks as summarised below.

E. coli is one of the most widely used expression hosts for the production of recombinant proteins.

The advantages of this system include:

Inexpensive setup and running costs
High recombinant protein production levels
Short timeframe for protein expression
Limited technical knowledge required for culturing
Scalability from small (1 mL) to very large culture (>10,000 L) volumes

One of the main drawbacks of this expression system however is its limited capability for post-translational modifications (PTMs).

How do I select an expression host?

A wide range of bacterial strains are available for recombinant protein production, each offering a novel advantage. Some common E. coli expression strains are listed below. PEF recommends screening multiple strains where possible to determine the optimal cell strain for producing your target protein. Strains and cells are available from the UQ resource centre.

Strain	Features
BL21 (DE3)	Most common host strain, enables high-level recombinant protein expression
BL21 (DE3) pLysS	Enables high-level expression and suppression of T7 RNA Polymerase basal level expression
BL21-CodonPlus	Improved expression of genes with codons rarely used in bacteria
Rosetta	Improved expression of genes with codons rarely used in bacteria
Arctic Express	Contains a plasmid encoding chaperonins to aid in folding and allow expression at low temperature (12°C)
Tuner	Enables uniform uptake of IPTG into each cell, finer control of expression can be achieved by varying IPTG concentration
Shuffle^® T7 Express	Expresses DsbC to enable cytoplasmic disulphide bond formation

How do I optimise my protein expression in bacteria?

Generating soluble, active protein is a major challenge for recombinant protein production in E. coli. Over-expression of eukaryotic proteins often leads to the formation of insoluble aggregates known as inclusion bodies which complicates the protein purification process. Several parameters can impact the success of protein production in E. coli, as described below.

Host strain selection

Selection of the right cell strain is critical for successful soluble protein production. Advances in host cell engineering have led to the development of several key capabilities that should be taken into consideration when choosing a strain for expression:

Rare codon usage: These strains carry an extra plasmid (e.g. pRARE) that encode a number of rare codon tRNAs. This allows more efficient expression of genes that contain these rare codons (generally those of eukaryotic origin)
Improved folding: These strains carry either a mutation in specific genes that inhibit the formation of disulphide bonds, or carry a chromosomal copy of disulphide bond isomerase. This enables cytoplasmic production of proteins requiring disulphide bonds
Induction control: These strains contain a mutation in the lac permease, allowing homogenous uptake of IPTG into all cells in the culture. This enables finer control over induction by varying the concentration of IPTG

Growth temperature

Although most bacterial strains are typically cultured at 37°C, lowering the temperature (e.g. to 30°C or 15°C) often improves protein folding and increases soluble protein production.

Co-expression

Co-expression of recombinant proteins with chaperonins and foldases can aid expression in multiple ways. Chaperonins such as cpn10 and cpn60 bind to and stabilise unfolded or partially folded proteins. Expression with foldases such as disulphide oxioreductase (DsbA) or disulphide isomerase (DsbC) can aid in producing soluble protein if formation of disulphide bonds is required.

Codon optimisation

Codon optimisation is the process of modifying codons in a gene sequence to match the codon usage bias of the host cell used for expression. This is frequently applied to heterologous proteins expressed in E. coli, as codon usage varies heavily between prokaryotes and eukaryotes. Optimising the codon usage may improve the protein expression.

Media

LB broth is the most commonly used media for molecular cloning and protein expression in E. coli. Although this media is simple and cheap to prepare, it does not support high-level biomass production. Other media formulations such as TB, 2x YT or chemically defined media enable higher density cultures in shake flasks and bioreactors, which may increase target protein yield.

Yeast are a single-celled eukaryotic organisms that combine high levels of recombinant protein production with eukaryotic post-translational modifications (PTMs).

Some advantages of using yeast over other host systems for protein expression include:

Relatively inexpensive setup and running costs
Very high levels of recombinant protein production
Able to perform many PTMs e.g. N-glycosylation
Simplified downstream processing for secreted proteins
Amenable to large-scale fermentation

Some drawbacks of yeast as a host cell expression system include:

PTMs are less complex when compared to higher eukaryotic systems (e.g. insect and mammalian cells)
Yeast’s tough cell wall is cumbersome to disrupt when processing intracellular proteins

Yeast expression hosts

There are two strains commonly used for protein production in yeast: Saccharomyces cerevisiae and Pichia pastoris. P. pastoris has a number of advantages over S. cerervisiae that make it the preferred host cell for expression: a strong, inducible promoter, high growth rates and PTM patterns more similar to higher eukaryotes.

Some common P. pastoris expression strains are listed below.

Strain	Features
PichiaPink Strain 1	Adenine auxotroph
PichiaPink Strain 2	Adenine auxotroph, Protease A knockout
PichiaPink Strain 3	Adenine auxotroph, Protease B knockout
PichiaPink Strain 4	Adenine auxotroph, Protease A and Protease B knockout
X-33
GS115r	Histidine auxotroph
KM71H	Strain uses weaker AOX2 promoter

How do I optimise my protein expression in yeast?

Several parameters can impact the success of protein production in the yeast expression system, as described below.

Promoter selection

Selection of the right promoter is critical for successful secreted protein production. Two common promoters used for recombinant protein production in yeast is the highly inducible P_AOX1 and constitutively expressive P_GAP promoters.

The alcohol oxidase 1 promoter (P_AOX1) supports a two-step process for gene regulation, repression and derepression. Commonly, glycerol is used to grow Pichia cells to maximise cell biomass until it is depleted. Methanol, the inducer, is then added into the culture medium for derepression of the promoter which allows for transcription of the gene of interest downstream of AOX1. Although very popular, the use of P_AOX1may not be optimal for commercial-scale fermentation productions due to risks associated with the storage of large quantities the toxic and highly flammable methanol.
The glyceraldehyde-3-phosphate dehydrogenase promoter (P_GAP) takes advantage of selecting a specific carbon source (e.g. glucose, glycerol, maltose methanol, oleic acid, sorbitol, or sucrose) for increasing cell biomass and recombinant target protein expression at the same time.

Clonal variability

Electroporation is the most common method to transform your cloned plasmid DNA into your yeast cell strain of choice. Homologous recombination between your linearised plasmid DNA and the Pichia genome occurs, denoted as a crossover “insertion” event. As this is a chromosomally-integrated system, clonal variability can occur i.e. some clones may have one single crossover event and other clones may have multiple crossovers. It is a general misconception that having more ‘insertions’ may lead to increased target protein expression however due to gene-dosage this varies from protein to protein. Identifying the ‘best” or “optimal” clone is recommended prior to any scale-up productions.

Codon optimisation

Codon optimisation is the process of modifying codons in a gene sequence to match the codon usage bias of the host cell used for expression. This is frequently applied to heterologous proteins expressed in P. pastoris, as codon usage can vary heavily between different organisms. Optimising the codon usage may improve the protein expression.

The insect cell/baculovirus expression vector system (BEVS) is becoming increasingly popular for the production of recombinant proteins.

The system offers a number of advantages, including:

Ability to perform complex post-translational modifications (PTMs)
High success rate of soluble protein recovery
Suitable for the production of large protein complexes, e.g. virus like particles (VLPs)
High protein expression levels compared to other higher eukaryotes

One of the main drawbacks of the system is longer timeframes for protein expression in comparison to other host systems.

How do I optimise my protein expression in the baculovirus/insect cell system?

Insect cell/BEVS expression technologies

There are a number of different technologies that can be used to generate a recombinant baculovirus containing the gene of interest. Some of the most commonly used technologies are listed below. Screening protein expression using multiple baculovirus technologies can determine the most suitable for producing your target protein.

System	Transfer Vectors	Features
Bac-to-Bac^TM	pFastbac-1, pFastBac Dual	Requires integration with baculovirus shuttle vector (Bacmid) in bacterial host cell before transfection
flashBAC^TM	pBac-1	Transfection with linear DNA, recombination occurs within host insect cells. Deletion of chiA enables better secretion of recombinant protein
BaculoGOLD^TM	pVL1392, 1393	Transfection with linear DNA, recombination occurs within host insect cells
BaculoDirect^TM	pENTR	Transfer vector recombines with linear DNA before transfection into host cell. Requires Ganciclovir as selection agent

Cell lines

The most commonly used cell lines are Sf9 and High Five^TM (see table below). Both can be grown as adherent or suspension cultures and scaled up from microplates to shake flasks and rocking motion bioreactors. They are also able to be grown in serum-free media (SFM) and do not require the addition of CO₂ gas. Some commonly used insect cell lines are listed in the table below.

Selection of the right cell line is critical for successful protein production and PEF recommends screening at least two insect cell lines. Plasmids, strains and cells are available from the UQ resource centre.

Cell Line	Origin	Features
Sf9	Spodoptera frugiperda	Adapted to SFM, commonly used to express all types of recombinant proteins
Mimic Sf9	Spodoptera frugiperda	Adapted to SFM, expresses mammalian glycosyltransferases to generate proteins with more complex N-glycan structures
High Five^TM	Trichoplusia ni	Adapted to SFM, commonly used for secretion of recombinant proteins

Growth temperature

Although insect cells are typically cultured at 27°C, lowering the temperature (e.g. to 21˚C) can improve protein folding and target protein production.

Expression duration

The timeframe of protein expression can also play a role in successful target protein production. For example, protein harvested 48 hours post infection (hpi) may have a different expression profile to protein harvested at 72 hpi. Screening protein expression at multiple time points may determine the optimal condition for your target protein.

The dominant host system for producing therapeutic recombinant proteins over the last few decades has been the mammalian expression system.

The benefit of this system includes:

Highest capacity for generating complex post-translational modifications (PTMs) compared to other expression systems
High success rate of soluble protein recovery
Suitable for antibody production

One of the main drawbacks of the system is the increased expense when compared to other systems such as bacteria.

Transient vs stable cell culture

There are two main methods for recombinant protein production in mammalian cells i.e. stably transfected and transiently transfected cell lines.

Stably transfected cell lines enable continuous expression of the recombinant protein at high yields. The creation of a stable cell line can take up to several months to complete.

A transient transfection can be used for small- to large-scale expression cultures. Recent improvements to the method have been able to increase yields from milligram to gram quantities. The advantages of a transient transfection method lie in the shorter timeframe from DNA delivery to protein harvest.

How do I optimise my protein expression in the mammalian system?

Several parameters can impact the success of protein production in the mammalian system, as described below:

Cell lines

Commonly used mammalian cell lines for protein production, in particular biotherapuetics, are the Chinese hamster ovary (CHO), Human embryonic kidney (HEK-293) and Mouse myeloma (NS0) cell lines. PEF recommends screening multiple cell lines when determining the optimal condition for producing your target protein. Cell lines are available from the UQ resource centre.

Growth temperature

Although mammalian cells are typically cultured at 37°C, using a temperature shift during protein expression (e.g. decreasing the culture temperature from 37˚C to 32˚C following transfection) can improve target protein production.

Expression duration

The timeframe of protein expression can also play a role in target protein production. For example, protein harvested 4 days post transfection may have a different expression profile to protein harvested at 5 days post transfection. Screening protein expression at multiple time points may determine the optimal condition for your target protein.

Listed below are a number of articles that provide a broad overview about using different expression systems for the production of recombinant proteins.

Bacterial expression system

Papaneophytou, C.P. et al. Statistical approaches to maximize recombinant protein expression in Escherichia coli: A general review. Protein Expression and Purification 94, 22–32 (2014)

Kudla, G. Coding-Sequence Determinants of Gene Expression in Escherichia coli. Science 324, 255-258 (2014)

Muntari, B., et al. Recombinant bromelain production in Escherichia coli: process optimization in shake flask culture by response surface methodology. AMB Express 2:12 (2012)

Rouet, R., et al. Expression of high-affinity human antibody fragments in bacteria. Nature Protocols 364VOL.7 NO.2 (2012)

Vincentelli, R. et al. High-throughput protein expression screening and purification in Escherichia coli, Methods 55, 65-72 (2011)

Unger, T., et al. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression. Journal of Structural Biology 172, 34-44 (2010).

Schein, C.H. Soluble Protein Expression in Bacteria. Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology 1-20 (2010).

Francis, D.M. et al. Strategies to Optimize Protein Expression in E. coli. Current protocols in protein science Chapter 5, Unit 5 24 (2010).

Brondyk, W.H. Selecting an Appropriate Method for Expressing a Recombinant Protein. Guide to Protein Purification, Second Edition 463, 131-147 (2009).

Rosano G.L. et al. Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain. Microbial Cell Factories 8:41 (2009)

Selleck, W. et al. Recombinant protein complex expression in E. coli. Current protocols in protein science Chapter 5, Unit 5 21 (2008).

Komar A.A A pause for thought along the co-translational folding pathway. Trends in Biochemical Sciences Vol.34 No.1 (2008)

Berrow, N.S. et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Research 35, e45 (2007).

Cabrita, L.D., et al. A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production. BMC Biotechnology 6, 12 (2006).

Duetz, W.A., et al. Methods for Intense Aeration, Growth, Storage, and Replication of Bacterial Strains in Microtiter Plates. Applied and Environmental Microbiology, Vol 66, No. 6, 2641–2646 (2006)

Waugh, D. Making the most of affinity tags. TRENDS in Biotechnology Vol.23 No.6 (2005)

Goulding C.W. et al. Protein production in Escherichia coli for structural studies by X-ray crystallography. Journal of Structural Biology 142 133–143 (2003

Yeast expression system

Spadiut, O. et al. Microbials for the production of monoclonal antibodies and antibody fragments. Trends in Biotechnology 1:54-60 (2014)

Vogl, T. et al. New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris. Current Opinion in Biotechnology 6:1094-101 (2013)

Jiang Y. et al. Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris. Protein Expression and Purification 76 7–14 (2011)

Abad, S. et al. Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst. Microbial Cell Factories, 9:24 (2010)

Bollok, M., Resina, D., Valero, F. & Ferrer, P. Recent patents on the Pichia pastoris expression system: expanding the toolbox for recombinant protein production. Recent Patents on Biotechnology 3, 192-201 (2009).

Cregg, J.M. et al. Expression in the yeast Pichia pastoris. Methods in Enzymology 463, 169-189 (2009).

Gong B. et al. Characterization of N-Linked Glycosylation on Recombinant Glycoproteins Produced in Pichia pastoris Using ESI-MS and MALDI-TOF. Methods in Molecular Biology, Glycomics: Methods and Protocols, vol. 534 Chapter 16 (2009)

Ghosalkar A. et al. Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production. Bioresource Technology 99 7906–7910 (2008)

Hamilton, S.R. & Gerngross, T.U. Glycosylation engineering in yeast: the advent of fully humanized yeast. Current Opinion in Biotechnology 18, 387-392 (2007).

Lin-Cereghino, J., et al., Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques 38(1): 44–48. (2005)

Suga M. et al. Cryopreservation of competent intact yeast cells for efficient electroporation. Yeast 16: 889-896. (2000)

Insect cell / BEVS expression system

Fernandes, F. et al. Insect cells as a production platform of complex virus-like particles Expert Rev. Vaccines12(2), 225–236 (2013)

Kost, T. Baculovirus Gene Delivery: A Flexible Assay Development Tool. Current Gene Therapy 10 168-173 (2010)

Airenne, K.J., et. al. In Vivo Application and Tracking of Baculovirus. Current Gene Therapy, 2010

Madhan, S. et al. Baculovirus as Vaccine Vectors. Current Gene Therapy 10 201-213 (2010)

Trowitzsch, S., et al. New baculovirus expression tools for recombinant protein complex production. Journal of Structural Biology 172, 45-54 (2010).

Aucoin, M.G., et al. Bioprocessing of baculovirus vectors: a review. Current Gene Therapy 10, 174-186 (2010).

Jarvis, D.L. Baculovirus-insect cell expression systems. Methods in Enzymology 463, 191-222 (2009).

Chung Y.C., et al., Expression, purification and characterization of enterovirus-71 virus-like particles. World J Gastroenterol; 12(6): 921-927 (2006)

Kost, T.A., et al. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology 23, 567-575 (2005).

Weber, W. Optimisation of protein expression and establishment of the Wave Bioreactor for Baculovirus/insect cell culture. Cytotechnology 38: 77–85, (2002)

Mammalian cell expression system

Almo S.C., et al. Better and faster: improvements and optimization for mammalian recombinant protein production. Current Opinion in Structural Biology. 26:39-43 (2014)

Bandaranayake A.D. et al. Recent advances in mammalian protein production. FEBS Letters. 588 2 253-60 (2014)

Zhang, J. Mammalian Cell Culture for Biopharmaceutical Production. Fermentation and Cell Culture Chapter 12 157-178 (2012)

Baldi. L., et al. Large-scale transfection of mammalian cells. Methods in Molecular Biology Volume 801 13-26 (2012)

Rodrigues, M.E. Technological Progresses in Monoclonal Antibody Production Systems. Biotechnol. Prog. Vol. 26, No. 2 (2010)

Hacker D.L. 25 years of recombinant proteins from reactor-grown cells—Where do we go from here? Biotechnology Advances 27 1023–1027 (2009)

Nettleship, J. et al., The Production of Glycoproteins by Transient Expression in Mammalian Cells. Methods in Molecular Biology: High Throughput Protein Expression and Purification, vol. 498 Chapter 16 (2009)

Lackner, A. A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells. Analytical Biochemistry 380 146–148 (2008)

Matasci, M. et al. Recombinant therapeutic protein production in cultivated mammalian cells:current status and future prospects. Drug Discovery Today: Technologies 5, e37-e42 (2008).

Baldi, L., et al. Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnology Letters 29, 677-684 (2007).

Barnes, L.M., et al. Mammalian cell factories for efficient and stable protein expression. Current Opinion in Biotechnology 17 381–386 (2006)

Birch, J.R. et al. Antibody production. Advanced Drug Delivery Reviews 58 671 – 685 (2006)

Kunaparaju, R., et al., Epi-CHO, an Episomal Expression System for Recombinant Protein Production in CHO Cells. Wiley Periodicals, (2005)

Wurm, F.M. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology 22 No. 11 (2004)