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1. Introduction
Contents:
  1. Introduction to protein expression
  2. Overview of Protein Expression
  3. Subscribe to the PEACE 12222 newsletter
  4. Current Uses of Synthetic Biology – BIO

To enhance recombinant protein expression, the IE-1 promoter can be combined with the homologous region 5 hr5 enhancer [ 48 ]. The late protein p6. The expression of enhanced GFP under the control of the p6. The very-late p10 and polh genes produce the very-late 10 kDa protein and polyhedrin, respectively.

These are highly active promoters and are used in many BEVS kits, but they depend on proteins translated in earlier phases of the infection for their activity. The activity of the polh promoter can be increased even further if p10 expression is abolished [ 50 ]. Protein tags can be used to simplify the detection and purification of recombinant proteins produced using BEVS. Common tags include His 6 and GST. The His 6 tag comprises six histidine residues and can be fused to either the N-terminus or C-terminus of proteins.

This tag facilitates protein purification using nickel or cobalt ions or anti-histidine antibodies immobilized on a chromatography resin [ 51 ]. GST is a 26 kDa protein which is highly soluble and folds rapidly after translation. The tag is often used to increase protein solubility in prokaryotes. The affinity between this enzyme and its substrate means that immobilized glutathione can also be used for protein purification by affinity chromatography.

The purification tag is often removed from the recombinant protein after purification because its size and unique chemical properties can interfere with protein functions. For this purpose, a TEV protease cleavage site is often placed between the tag and the mature recombinant protein, allowing the tag to be released in vitro [ 52 , 53 ]. Alternatively, transfer plasmids with thrombin cleavage sites are marketed by BD Biosciences and Oxford Expression Technologies. To improve the secretion of recombinant proteins produced in insect cells, secretion signals consisting of 15—30 amino acids can be fused to the N-terminus.

Common secretion signals include those native to honeybee melittin HBM or the baculovirus envelope surface glycoprotein 67 gp Transfer plasmids containing these secretion signals are available from BD Biosciences and LifeSensors. Further, signal peptides that enhance protein secretion have also been described [ 54 ]. The effect of low and high MOI on protein expression has been addressed in multiple experiments [ 55 — 59 ] and simulations [ 60 — 63 ].

The infection of a cell by a virus particle can be modelled using a Poisson distribution [ 61 , 64 ]. The probability that a cell will absorb an infectious particle is therefore given in Equation 1 , with n representing the number of absorbed baculovirus particles:.

To guarantee the simultaneous infection of all cells, the MOI should be 5 As described in Section 2. A low MOI is easier to achieve and requires smaller volumes of virus stock, which is advantageous in large-scale cell cultures [ 56 , 57 ]. Fewer virus amplification steps are required thus limiting the negative effects of passaging, such as the increasing proportion of defective viruses after each round of amplification [ 58 ].

Infection with a low MOI also results in the proliferation of cells not infected during primary infection, increasing the number of cells available for secondary infection and thus the number of cells producing the recombinant protein [ 58 , 65 ]. On the other hand, the need for secondary infection also prolongs the process, but this drawback is outweighed by the advantages listed above. The final protein yield is not necessarily lower when starting with a low MOI compared to a high MOI [ 41 , 62 , 63 , 66 ]. The early or mid-exponential phase is the optimal TOI when using a low MOI [ 56 , 60 , 61 ] because infection during the late exponential phase can lead to substrate limitation [ 67 ].

For laboratory-scale processes, centrifugation as a clarification step can produce virus stocks of sufficient purity and quantity. However, a virus concentration step is necessary for larger-scale processes, or for processes featuring a high MOI or high cell density. Very pure virus stocks are required for pharmaceutical applications such as the manufacture of vaccines [ 68 ] or the use of baculovirus vectors for in vivo gene therapy [ 69 , 70 ], and it is particularly important to reduce host cell proteins and DNA to acceptable levels [ 71 ].

If the objective is to purify active virus particles with minimal loss, then the purification method must consider the stability of the virus.

Bioprocessing Part 3: Purification

The virus is sensitive to high temperatures, i. The virus is insensitive to shear forces in a peristaltic pump and is stable over the pH range 6—8. The stability of the virus against shear forces allows it to be concentrated by tangential filtration. Polyethersulfone membranes with cut-offs in the range , kDa can be used to achieve a fold concentration of virus particles at an average of 0.

For medical applications, virus concentration alone does not meet the requirement to reduce the levels of host cell protein and DNA. These contaminants can be removed using ion exchange membranes, e. Figure 6 shows the virus and protein concentrations at each step of the purification process: adsorption of the viral particle to the column, washing and elution [ 73 ]. Steps in the purification of AcMNPV using membrane chromatography, showing the virus titer and host cell protein concentration [73].

The most suitable method is therefore a matter of the required purity, process duration and scale. Although the BEVS platform is highly versatile and probably the most popular insect-based expression system, it is not the best choice for all products. Factors such as protein complexity, post-translational modifications and process mode must be considered during process development [ 70 ].

Recent studies show that recombinant D. This system is based on an embryonic D. Stable transformation of S2 cells with plasmid vectors facilitates the production of heterologous proteins. The general procedure used to generate stable rS2 cell lines is the same for all packages and is summarized in Figure 7. A plasmid carrying the GOI is used to transfect S2 cells, and stable transformants that have integrated the expression construct are propagated under selection to yield a stable cell line.

This line can be used for the isolation of a highly productive clone. Depending on the amount of protein required and the time available, several starting points can be used for protein production. For high-throughput screening or when small amounts of protein are sufficient, transient expression may produce enough and the selection of cell lines is unnecessary [ 79 ]. If higher protein yields are required, stable cell lines can be established [ 80 ] and even single cell cloning may be necessary [ 81 ]. Although protein production in stable cell lines usually takes longer than the BEVS platform, the rS2 system retains some flexibility.

Overview of the general procedure to produce stably transformed D. Protein expression can be initiated at different points, starting with transient expression immediately after transfection followed by stable expression in a polyclonal cell line and finally the selection of a highly productive monoclonal cell line. Stable rS2 cell lines are recommended for protein expression when the following aspects are important for the production process [ 76 , 82 ]:.

Introduction to protein expression

Both, stable rS2 cells and BEVS share the advantage of minimal risk of contamination with human viruses because most human viruses cannot replicate in insect cells. This is particularly important for the production of pharmaceutical proteins intended for administration to humans. Stable rS2 cells are ideal when bacterial expression systems yield an inactive protein [ 83 , 84 ] and where even BEVS is not efficient [ 84 — 86 ]. Because of the non-lytic and stable nature of protein production, different bioprocessing modes such as batch or fed-batch cultures [ 87 ], chemostat cultures [ 88 ] and perfusion cultures [ 81 ] can be used.

Perfusion mode in particularly achieves high protein yields [ 81 ] [1] -. No cell lysis occurs, so less host cell protein is released and the recombinant target protein is protected from proteolytic degradation. Therefore process-integrated product recovery is also conceivable. Stable rS2 cells also achieve high batch-to batch reproducibility between manufacturing runs, and generate a homogeneous glycan profile. Several rS2-derived products have already entered clinical development, confirming that rS2 cells are not only used routinely in research but also for the commercial production of high value pharmaceutical proteins Table 3.

Stable rS2 cell lines are produced by transformation with suitable plasmid vectors carrying the GOI in an expression cassette and a selectable marker. Five general strategies have been developed, as summarized in Figure 8. Classically, separate expression and selection cassettes with their own promoters can be combined in a single plasmid [ 97 , 98 ] Figure 8b. Alternatively, both features can be placed in one expression cassette, separated by an internal ribosome entry site IRES or a 2A-like sequence T2A , resulting in bicistronic vectors with heterologous protein production and antibiotic resistance under the control of the same constitutive promoter [ 99 , ] Figure 8c.

However, the most common approach is the use of two separate plasmids, the first containing the expression cassette and the second containing the selectable marker Figure 8a. This exploits the ability of dipteran cell lines to recombine different plasmids in long tandem arrays [ 82 , ]. The transfection or co-transfection of S2 cells with such plasmids followed by selection generally leads to the integration of multiple plasmid copies into the genome [ , 82 ].

The co-transfection of two plasmids using the calcium phosphate precipitation method Section 2. The ratio of integrated plasmid sequences is similar to their proportions in the transfection mixture. When using two separate plasmids, it is therefore advisable to increase the initial ratio in favor of the expression plasmid. The random integration of multiple plasmids generates heterogeneous cell populations and it is possible that rearrangements occur within the integrated array [ ].

In contrast to the random transgene arrays described above, transposable elements can be used to insert a single copy of the GOI and selectable marker into the S2 cell genome Figure 8d [ , ]. For this purpose, the expression construct and selectable marker are flanked by transposase recognition sites, i. Minos inverted repeats [ ] or P element terminal repeats [ , ]. Co transfection together with a helper plasmid encoding the corresponding transposase causes the GOI and marker to be inserted at a more or less random site.

Transposition-mediated insertion events can occur more than once in the same genome, but the mutagenic nature of each insertion generally limits the number of integration events to between one and 10 copies [ 82 ]. This method is cumbersome because it is necessary to map the transposon insertion site and identify clones with single-copy insertions, and the low copy number limits the yield of recombinant protein. This method is more suitable when the goal is functional analysis rather than protein production, and can be very useful when combined with the technique of recombinase mediated cassette exchange RMCE as shown in Figure 8e.

In this system, a single docking cassette flanked by the first recognition site att P is stably integrated into the genome by transposition. A second helper plasmid is then used to transiently express the integrase. The subsequent introduction of a plasmid with an expression cassette flanked by the corresponding recognition site att B promotes cassette exchange. Based on one parental cell line containing the docking cassette, different comparable clones can easily be generated, which is particular helpful in comparative studies e. Although the classification of different plasmid types can simplify the principles underlying the generation of rS2 cells, it is not a fixed dogma.

For example, the bicistronic system can be used to express two different proteins of interest, while co-transfection with a second plasmid provides the selection cassette [ ]. Furthermore, co-transfection is not restricted to two plasmids. Indeed, up to four different proteins have been expressed simultaneously by co transfecting S2 cells with multiple vectors [ ].

Although baculoviruses cannot replicate in S2 cells, infection achieves successful protein production from the non-replicating vector [ — ]. Accordingly, rS2 cells appear to be a powerful and versatile tool for protein expression. Detailed protocols and more background information on the different techniques have been published [ — , 82 ].

As described above, the expression plasmid contains various features required for protein production and the best combinations must be assessed for each process. Several constitutive and inducible promoters have been used for the production of recombinant proteins in rS2 cells Table 4. Strong constitutive promoters are generally favored for transient expression, because the protein must be expressed immediately after transfection.

Inducible promoters can be more suitable in stable rS2 cell lines, particularly if the overexpressed protein is toxic to the host cell. Inducible promoters allow the decoupling of cell proliferation and protein expression, which can be appropriate for advanced process designs i.

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The most widely used promoters in rS2 cells are the constitutive actin 5C Ac5 promoter [ ] and the copper-inducible metallothionein Mt promoter [ ]. Other constitutive promoters, such as the copia long terminal repeat LTR promoter, can be used for the GOI but are usually paired with the resistance marker gene. The inducible hsp70 promoter can also be used, but unlike the preferred Mt promoter it has a relatively high basal activity, and the heat shock required for induction can also induce endogenous heat-shock genes causing changes in gene expression and cell behavior that inhibit protein production [ ].

To rationalize all the different factors that affect protein expression inducer concentration, time of induction, culture medium , a structured approach such as statistically designed experiments DoE may yield valuable information. As a future prospect, the doxycycline-inducible TRE promoter may also be useful because it can achieve good yields in D.

The Kozak consensus sequence is required for the efficient initiation of translation. It is important to note that D. Signal peptides are used to mediate protein trafficking, initiate proper folding or to ensure protein secretion to the supernatant. The signal peptides are cleaved off during or after translation. The most common signal peptide used in S2 cells is D.

Signal peptides from human tissue plasminogen activator tPa [ ] and from Galleria mellonella gloverin GmGlv also work in rS2 cells [ ]. The proper folding of a dopamine receptor and its insertion into the cell membrane has been achieved using an influenza virus hemagglutinin signal sequence [ ].

As discussed above, fusion tags for protein detection and purification can be attached to either the C-terminus or N-terminus of a protein, with or without an additional protease cleavage site e. The His 6 tag can be detected with an antibody, and purification can be achieved with the same antibody or by immobilized metal ion affinity chromatography IMAC [ , ].

As usual for eukaryotic organisms, mRNA must be polyadenylated in rS2 cells to maintain stability and support efficient protein synthesis [ , ]. The late SV40 polyA signal from simian virus 40 achieved the best performance, indicating that the polyadenylation mechanism is conserved between mammalian and insect cells. Several selection systems have been tested in rS2 cells as summarized in Table 5. Each system comprises a cytotoxic agent and a corresponding marker that confers resistance. The marker may encode an enzyme that catalyses the transformation of a selective agent into a harmless product, e.

Alternatively, the marker may encode a mutated enzyme that replaces an endogenous enzyme inhibited by the cytotoxic agent, e. Only cells that have integrated the selection cassette can survive and proliferate in the presence of the selective agent. The most appropriate selection system depends on time, cost and risk. Blasticidin and puromycin are more expensive than hygromycin but also work faster. A high rate of spontaneous resistance has been reported for G Commonly used selective agents are indicated with an asterisk. Lower case letters indicate delivery methods:.

Several strategies for the transfection of insect cells were described in Section 2. All of these methods are appropriate for the production of stable rS2 cell lines, but it is important to consider that transfection causes stress to the cells and a recovery period may be necessary before selection commences. As described above for Sf cells, calcium phosphate-DNA co-precipitation was also one of the first methods used to generate rS2 cells [ ] and was used extensively in the past [ 83 ].

Calcium phosphate-DNA co-precipitation has therefore been largely replaced by more flexible techniques that achieve greater reproducibility [ ]. Electroporation is a convenient method for the transfection of S2 cells [ 38 ] that can achieve efficiencies comparable to calcium phosphate—DNA co-precipitation [ ]. This method also allows the uptake of DNA over a very large concentration range, making it useful for the transposon-mediated generation of cells with single-copy inserts [ ].

To illustrate the generation of a stable rS2 cell line, this section describes the expression of the reporter protein GFP and its time resolved detection during cell line establishment. Figure 9 shows the GFP expression profile monitored by flow cytometry. Within the first 10 days, most of the non-transfected cells died and only low levels of GFP were detected.

After 20 days under selection, the cell population became increasingly GFP-positive, and a population producing high levels of GFP was established within 30 days. Further sub-culturing revealed the existence of different sub-populations with varying GFP expression profiles and different growth properties. By day 77, a population characterized by moderate levels of GFP became prevalent, suggesting that polyclonality is detrimental for long-term protein production.

This can be overcome by single cell cloning, as discussed in the next section. Time course showing the establishment of a stable rS2 cell line expressing GFP under the control of the constitutive Ac5 promoter. GFP activity was recorded by flow cytometry. Once a stable cell line is established, it can be used as a straightforward basis for subsequent up-scaling and protein expression even at the bioreactor scale. However, the expression profile within the polyclonal cell population is heterogeneous.

Furthermore, high-copy-number transgenic loci confer an additional metabolic burden that may inhibit cell growth. Long-term subculturing therefore enriches subpopulations that have lost copies of the transgene, and protein expression declines [ ] as illustrated with eGFP in Figure 9. As well as maintaining the cells under selection pressure, single cell cloning is necessary to minimize these effects and should commence before the highly-productive cells become overpopulated by their less-productive peers [ , ].

However, the productive cells should only be chosen once they have recovered from the stress of transfection because the success of a single cell cloning is highly dependent on clones that proliferate well. This reflects the demand for autocrine growth factors, which accumulate to sufficient levels only at high cell densities [ 77 ].

For example, the adenosine deaminase-related growth factor ADGF family is known to promote the growth of S2 cells [ , ] but no studies have yet shown that ADGF alone can stimulate growth at very low cell densities. Augmenting the culture with conditioned medium or heat inactivated FBS can improve proliferation, but is also not sufficient to stimulate propagation of single cells. Feeder cells are therefore required to facilitate the proliferation of a single rS2 cell.

There are two traditional cloning methods: cloning in soft agar and cloning by dilution. T-flask before co-culture [ 82 ]. Irradiated feeder cells do not divide, but remain able to secrete growth-promoting substances and provide an additional source of nutrients when they die [ ]. The cells grow in this semi-solid support and form colonies in the agar within 2 weeks. Cloning by dilution requires the mixing of feeder cells and transformants in such a way that approximately one clone per well can be seeded in a microtiter plate.

The corresponding protocols have been described in detail [ 82 , 38 , , ]. It is also necessary to ensure that the feeder cells are permanently unable to divide yet still survive at least 1 week post-irradiation to condition the medium during the early growth of the clones [ ].

Robustness against ionizing radiation is cell line-dependent, so parameters such as radiation dose and distance from source must be empirically standardized to achieve the requirements described above. As an alternative to irradiation, feeder cells can be treated with mitomycin C to chemically block mitosis, but this method must fulfil the same requirements and empirical testing is still necessary [ ].

A modified version of the limiting dilution protocol was recently reported that does not use radiation and therefore simplifies the cloning workflow [ 81 , 95 ]. The method is based on the co-cultivation of single transformants with living, non-transfected feeder cells followed by antibiotic selection of the clones. Because the procedure is not yet well established, the steps in the protocol are summarized below:. Co-cultivation for 1—3 days allows all cells to proliferate and ensures proper conditioning of the medium.

Adding the antibiotic e. Feeder cells will slowly decay, while colonies from antibiotic resistant clones will expand during the next 2 weeks. If necessary, medium can be added to renew the antibiotic and avoid desiccation. Checking colony growth using a microscope: wells with multiple colonies should be discarded because they are not monoclonal Figure Baculovirus-insect cell expression systems. Chung Y.

Overview of Protein Expression

Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology 23, Weber, W. Almo S. Better and faster: improvements and optimization for mammalian recombinant protein production. Bandaranayake A. Recent advances in mammalian protein production. FEBS Letters. Zhang, J. Mammalian Cell Culture for Biopharmaceutical Production. Large-scale transfection of mammalian cells.

Rodrigues, M. Hacker D. Nettleship, J. Lackner, A. A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells. Matasci, M. Recombinant therapeutic protein production in cultivated mammalian cells:current status and future prospects. Drug Discovery Today: Technologies 5, ee42 Baldi, L. Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnology Letters 29, Barnes, L.

Mammalian cell factories for efficient and stable protein expression. Birch, J. Antibody production. Kunaparaju, R. Wurm, F. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology 22 No. Kovtun O , et al. Skip to menu Skip to content Skip to footer.

Site search Search. Site search Search Menu. Protein Expression. Home Facilities. The figure below illustrates some of the key advantages and disadvantages of the four major systems used in PEF: Small-scale Expression Screen PEF offers a small-scale expression screen in each system that is designed to test different expression parameters to find an optimal condition. Large-scale Expression PEF offers large-scale expression in bacteria, yeast, insect and mammalian cell systems. Training PEF offers training for staff and students in all aspects of recombinant protein production. Bacteria Services E.

General Information - Bacteria Expression E. Bacterial Expression Hosts A wide range of bacterial strains are available for recombinant protein production, each offering a novel advantage. Host strain selection Selection of the right cell strain is critical for successful soluble protein production. Co-expression Co-expression of recombinant proteins with chaperonins and foldases can aid expression in multiple ways.

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. Media LB broth is the most commonly used media for cloning and bacterial expression.

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Antibiotics Antibiotics enable selection of recombinant clones as well as preventing contamination during cloning and expression. Yeast Services Yeast is a single-celled eukaryotic organism capable of producing very large quantities of recombinant protein. General Information - Yeast Expression 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 systems for protein expression include: Relatively inexpensive setup and running costs Very high levels of recombinant protein production Able to perform many PTMs e. Some commonly used insect cell lines are listed below. Mammalian Services Mammalian cell-based expression is the dominant system for the production of therapeutic recombinant proteins.

Services available from PEF include: Developing an effective strategy for molecular cloning and mammalian expression Custom or high throughput cloning into a range of mammalian expression vectors, including: pcDNA3. General Information - Mammalian Cell Expression The dominant system to produce therapeutic recombinant proteins over the last few decades has been mammalian cell-based expression systems.

Expression Vectors Mammalian expression vectors have been engineered to contain elements that improve expression and are generally more complex than vectors from other systems. Why Leishmania? How it works: The system is remarkably simple and allows single step production of recombinant protein from either plasmid or PCR-generated DNA templates. Related Articles on Protein Expression Listed below are a number of articles that provide a broad overview about using different expression systems for the production of recombinant proteins.

Link Schein, C. Link Brondyk, W. Link Rosano G. Link Komar A. Link Cabrita, L. Link Duetz, W. Link Cregg, J. Link Gong B. Link Lin-Cereghino, J. Link Aucoin, M. Link Jarvis, D.


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Link Chung Y. Link Weber, W. To date, one-point addition of inducer is the most prevailing process technological method to achieve expression tuning. However, due to inherent problems such as degradation of inducer and a lack of process technological control over the tuning process, we anticipate that other process technological methods such as the use of metabolizable inducers, inducer titration, and mixed-feed strategies will gain importance in the future.

We anticipate that expression tuning will unfold its full benefits only in combination with adequate control strategies. Hence, these methods will be essential for the industrial exploitation of tunable expression systems. Considering the broad spectrum of mature methods and technologies as well as the broad scientific knowledge available, we anticipate that expression tuning will soon be adapted by industry as generically applicable tool to enable and optimize the production of a broad spectrum of products in E. By enabling online controllability of protein expression, we believe that expression tuning is able to tackle the issues of constant product quality and culture long-term stability and therefore will pave the way for continuous production of biopharmaceuticals.

This in turn will further progress E. This article does not contain any studies with human participants or animals performed by any of the authors. Skip to main content Skip to sections. Advertisement Hide. Download PDF. Tunable recombinant protein expression in E. Open Access.

Current Uses of Synthetic Biology – BIO

First Online: 12 May Introduction The relevance of the gram-negative bacterium Escherichia coli for the basic biotechnological research as well as for industrial exploitation is outstanding. The main challenges of recombinant protein production in E. The mentioned challenges are either fully or to a great extent caused by recombinant protein expression.

The level of recombinant protein expression is affected by the strength of the expression system which involves the strength of the promoter used and the plasmid copy number Keasling as well as the process technological parameters such as temperature and the specific growth rate Hellmuth et al. It is frequently observed that a reduction of the protein expression level leads to increased end product titers, since the cells can be maintained in a productive state for a longer time Sagmeister et al.

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source When exploiting tunable systems for industrial bioprocesses with extended production phase, a continuous inducer supply is necessary to ensure a constant inducer concentration within the cell Fig. As this criterion is hard to meet, a possible simplification is to neglect the inducer consumption and transporting rates in and out of the cell and simply adjust the inducer amount to the biomass concentration.

A method that compensates for these effects, is the continuous feeding of inducer in order to achieve a constant inducer-to-biomass ratio Striedner et al. A possible way to achieve that is to use an exponential feeding profile and adjust the inducer concentration to the calculated value according to the feeding profile. Therefore, a more accurate method is to estimate the biomass based on online accessible data with soft sensors Luttmann et al.

The type of inducer metabolizable or non-metabolizable results in several consequences with respect to the controllability of the system. When using a non-metabolizable inducer, the induction rate can be independently controlled from the substrate uptake rate or respectively the specific growth rate. When using metabolizable inducers as sole carbon source, the induction rate is tightly coupled to the sugar uptake rate and therefore cannot be controlled independently.

As described, expression tuning has been technologically implemented in various ways, which significantly differ in the degree of freedoms they leave with respect to process control. Two methods have to be highlighted for providing the highest degree of freedom while containing full controllability of the process: inducer titration with gratuitous inducer and mixed-feeding strategy with a metabolizable inducer as second carbon source. Both methods enable the independent control of specific growth rate and induction rate Fig.

A short outline of currently applied induction methods is given in Table 1. Table 1 Process technological methods for expression tuning. Verification and evaluation of expression tuning With respect to the definition as the adjustment of the recombinant gene transcription rate on cellular level, several things have to be considered in order to demonstrate expression tuning. In order to define comparable criteria when working with tunable systems we recommend the following strategy: 1.


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Specific cellular productivity as target variable. In the field of enzyme control analysis, it is necessary to use promoters of different strengths in order to investigate different molecular fluxes within the cell Jensen et al. This approach involves the construction of different constructs for different concentrations of observed enzymes. We anticipate that the construction of only one tunable construct to cover all cases would be a great benefit for the investigation and optimization of metabolic pathways.

Possible fields of applications for expression tuning are summarized in Table 2. Table 2 Use cases for expression tuning. Use case Mode of action Reference Increase soluble protein titer Prevent unwanted inclusion body formation through downregulation of expression Baig et al. Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare that they have no conflict of interest.

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Biotechnol J 6 6 — Springer Netherlands, Dordrecht, pp. Gene 1—2 — PLoS One 9 2 :e Biotechniques 40 3 — Glick BR Metabolic load and heterologous gene expression. Biotechnol Adv 13 2 — Curr Opin Biotechnol — J Biotechnol 32 3 — Bioprocess technology 12 16 — PubMed Google Scholar. Keasling JD Gene-expression tools for the metabolic engineering of bacteria. Trends Biotechnol 17 11 — Kelley B Industrialization of mAb production technology: the bioprocessing industry at a crossroads.

J Bacteriol 24 — J Ind Microbiol Biotechnol 29 1 — Appl Microbiol Biotechnol 75 2 — Microbiology 8 — Appl Environ Microbiol 71 11 — J Pharm Innov 10 3 — Biotechnol J 7 8 — Mairhofer J, Scharl T, Marisch K, Cserjan-Puschmann M, Striedner G Comparative transcription profiling and in-depth characterization of plasmid-based and plasmid-free Escherichia coli expression systems under production conditions. Appl Environ Microbiol 79 12 — J Mol Biol 73 1 — Biotechnol Prog 24 6 — J Biotechnol 1 — Biophys J 95 4 — BMC Biotechnology 7 1 doi May 20—21, Continuous Manufacturing Symposium. J Pharm Sci 3 — Nancib N, Boudrant J Effect of growth rate on stability and gene expression of a recombinant plasmid during continuous culture of Escherichia coli in a non-selective medium.

Biotechnol Lett 14 8 — Novick A, Weiner M Enzyme induction as all-or-none phenomenon. Nature — Paulsson D, Gustavsson R, Mandenius CF A soft sensor for bioprocess control based on sequential filtering of metabolic heat signals.