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Recombinant protein fraction in E. coli

Recombinant protein fraction in E. coli


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If a protein is heterologously expressed in E. coli under the T7 promoter, what fraction of the total protein concentration in the cell is the heterologously expressed protein? What could be its concentration at most? Does it strongly depend on the size of the protein or are there certain size thresholds for low/high expression?


Miroux and Walker (1996) Over-production of Proteins in Escherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels. J Mol Biol. 260: 289-298

The authors report on problems of over-expressing membrane proteins in E. coli using a T7 (pET) system. They isolate mutant strains which show improved levels of expression compared to the parent strain, BL21(DE3). In their Table 1 they document levels of expression of 17 proteins including, as a control, GFP. GFP was found as a combination of soluble and inclusion body protein at a level of 37 mg L-1 in BL21(DE3) and 140 mg L-1 in one of their mutant strains (C41(DE3)). The highest level of expression that they observed was 300 mg L-1 for bovine OSCP.

Unfortunately the authors document neither the total protein content, nor the cell density of the cultures that they analysed.

Let's assume that the cultures were 10e9 cells mL-1 = 10e12 cells L-1.

According to this source (citing Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. Vol 1. pp. 14, ASM Press 1996), the dry weight of an E. coli cell is 2.8e-13 g

The generally accepted figure for protein as a fraction of dry weight in E. coli = 0.55

I calculate from these numbers a value of 1540 mg protein L-1. For the three protein expression levels described above this means:

GFP in BL21(DE3) = 2.4%

GFP in C41(DE3) = 9.6%

bovine OSCP in C41(DE3) = 19.4%

I conclude (from these numbers, and from personal experience) that overexpression levels are highly variable, and are going to be strain and protein-dependent. Values in the range of 1-20% total cell protein may be anticipated.


From this study it is clear that hence for the high recovery of the active protein molecule, solubilization and refolding parts must be of high precision. Inclusion bodies consist of polypeptides of the recombinant protein. They are the inactive secondary &ndashlike structures. Thus, the isolation and purification of the protein are simple. The activity of the unfolded protein can be brought out by using mild solubilizing conditions. This will help in the high recovery of the bioactive protein than that compared to solubility using high chaotropic agent.

The following Downstream processing steps can be used for the production of the recombinant protein from the inclusion bodies The recombinant E.coli is grown in LB medium with antibiotics such as kanamycin or ampicillin or chloramphenicol based on the plasmid. The flasks are shaken at rpm around 150 -250 and the temperature is maintained at 37 degree Celsius. After the cells have reached the log phase, This study outlines that IPTG is added and further shaken for 4-6 hours. The cells are centrifuged and re-suspended in the 50 mM sodium phosphate buffer.

The cells are lysed using the homogenizer or sonicator. The cell suspension is centrifuged and filtered using 0.45 µm polyethersulfonate membrane. Many protein-specific methods are available for the increased solubility of the recombinant proteins in E.coli. To recover the soluble proteins, strong denaturants like urea, guanidinium hydrochloride are used. The solubilization is carried out under reducing conditions. These inclusion bodies must be washed well before solubilization. Solubilizing agents such as thioredoxin are known to improve the solubility of the proteins.

Gene fusion techniques are equally good for the separation of the proteins. Maltose binding protein and Glutathione-S- transferase are found to bind well with the protein and can be removed by using the affinity chromatography techniques. Refolding is performed using dilution or diafliltration in buffers of low.


Recombinant protein production in E. coli

Escherichia coli (E. coli) genetic has been studied during decades and this organism became an unavoidable key element in laboratories.
The development of genetic engineering and synthetic biology associated to a short term generation and a flexibility to exploit a given genetic information, define E. coli as a host of choice for recombinant protein production.
Based on our know-how in genetic engineering, in fermentation and in protein production, we developed technologies and services in recombinant protein production.

Our technology Staby®Express is an improvement of available technologies that we usually use. It allows the removal of antibiotic resistance gene in agreement with the regulatory agencies recommandations and a reduction of energy bioburden while ensuring a perfect plasmid stabilization.

Our recombinant protein production services are performed with or without our technology and include several adaptation/optimization for your production:

  • Sequence and codon optimization
  • Gene synthesis
  • Selection of a promoter
  • Addition of a Tag for protein purification or identification

After the genetic construction of your expression vector, we perform a small expression test in order to optimize the culture and expression condition. We evaluate then the presence of your protein in the soluble or insoluble fraction that will be determining for the purification process.

After the validation of the expression condition we will produce your protein in the scale of your choice and purify it according to a defined protocol.

Proteins can be produced in shake flask or in fermenter (from 5 L to 50 L).
We provide purified protein from mg to several gramme scale.


Strategies where target modification is avoided

Some proteins directly influence the cellular metabolism of the host by their catalytic properties, but in general expression of recombinant proteins induces a "metabolic burden". The metabolic burden is defined as the amount of resources (raw material and energy), which are withdrawn from the host metabolism for maintenance and expression of the foreign DNA [6]. The formation of inclusion bodies occurs as a response to the accumulation of denatured protein. The metabolic burden and inclusion body formation are not directly linked but are both among the main factors to determine the ability of cells to produce soluble recombinant protein. Since the accumulation of denatured protein and the metabolic burden can be controlled by a number of environmental factors, we are partially able to control the formation of soluble protein in vivo.

Protein expression at reduced temperatures

A well known technique to limit the in vivo aggregation of recombinant proteins consists of cultivation at reduced temperatures [7]. This strategy has proven effective in improving the solubility of a number of difficult proteins including human interferon α-2, subtilisin E, ricin A chain, bacterial luciferase, Fab fragments, β-lactamase, rice lipoxygenase L-2, soybean lypoxygenase L-1, kanamycin nuclotidyltransferase and rabbit muscle glycogen phosphorylase (see [8] and references cited therein).

The aggregation reaction is in general favored at higher temperatures due to the strong temperature dependence of hydrophobic interactions that determine the aggregation reaction [9]. A direct consequence of temperature reduction is the partial elimination of heat shock proteases that are induced under overexpression conditions [10]. Furthermore, the activity and expression of a number of E. coli chaperones are increased at temperatures around 30°C [11, 12]. The increased stability and potential for correct folding at low temperatures are partially explained by these factors.

However, a sudden decrease in cultivation temperature inhibits replication, transcription and translation [13]. Traditional promoters used in vectors for recombinant protein expression are also strongly affected in terms of efficiency [14]. A similar transcriptional effect is achieved when a moderately strong or weak promoter is used or when a strong promoter is partially induced. Low induction levels have been found to result in higher amounts of soluble protein [15]. This is a result of the reduction in cellular protein concentration which favors folding. However, bacterial growth is decreased, thus resulting in a decreased amount of biomass.

Different strategies aimed at optimizing the expression of recombinant proteins at low temperature are as follows.

A system based on the cspA promoter was developed for the expression of proteins at low temperature [16]. The cspA promoter is highly induced at low temperature and is well repressed at and above 37°C. A sequence encoding the TolAI-β-lactamase fusion protein which is toxic to E. coli and rapidly degraded at 37°C was placed under the control of the cspA promoter. Temperature downshift to 15 or 23°C abolished degradation of the fusion protein and the toxic phenotype associated with expression at 37°C was suppressed. It was suggested that this system is a valuable tool for the production of proteins containing membrane-spanning domains or otherwise unstable gene products in E. coli.

A principle that allows for protein expression and folding at 4°C was presented recently [17]. This principle is based on co-expression of the target protein with chaperones from a psychrophilic bacterium. The two chaperones (Cpn60 and Cpn10 from Oleispira antarctica RB8 T ) allow E. coli to grow at high rates at 4°C [12]. An esterase from O. antarctica RB8 T was co-expressed with Cpn60 and Cpn10 in E. coli at 4°C. This procedure increased the specific activity of the purified esterase 180 fold as compared to enzyme prepared from cultivations at 37°C. It was concluded that the low temperature was beneficial to folding and the system was suggested as a tool for expression and correct folding of recombinant proteins in the cytoplasm of E. coli.

E. colistrains used to improve soluble expression

Numerous specialized host strains have been developed to overcome the metabolic burden related to high level protein expression.

Two E. coli mutant strains have contributed significantly to the soluble expression of difficult recombinant proteins. C41(DE3) and C43(DE3) are mutants that allow over-expression of some globular and membrane proteins unable to be expressed at high-levels in the parent strain BL21(DE3) [18]. Expression of the F1Fo ATP synthase subunit b membrane protein in these strains, in particular C43(DE3), is accompanied by the proliferation of intracellular membranes and inclusion bodies are absent [19]. These strains are now commercialized by Avidis http://www.avidis.fr and a high number of reports on their use in expression of difficult proteins have been published [20–23]. A recent work reports that the stability of plasmids encoding toxic proteins is increased in C41(DE3) and especially in C43(DE3) [24].

Cysteines in the E. coli cytoplasm are actively kept reduced by pathways involving thioredoxin reductase and glutaredoxin. The disulfide bond dependent folding of heterologous proteins is improved in the Origami strains from Novagen. Disruption of the trxB and gor genes encoding the two reductases, allow the formation of disulfide bonds in the E. coli cytoplasm. The trxB (Novagen AD494) and trxB/gor (Novagen Origami) negative strains of E. coli have been selected in several expression situations [25–27]. Folding and disulfide bond formation in the target protein, is enhanced by fusion to thioredoxin in strains lacking thioredoxin reductase (trxB) [28]. Overexpression of the periplasmic foldase DsbC in the cytoplasm stimulates disulfide bond formation further [27].

Modification of cultivation strategies to obtain soluble protein

The simplest way to produce a recombinant protein is by batch cultivation. Here all nutrients required for growth are supplied from the beginning and there is a limited control of the growth during the process. This limitation often leads to changes in the growth medium such as changes in pH and concentration of dissolved oxygen as well as substrate depletion. Furthermore inhibitory products of various metabolic pathways accumulate. Cell densities and production levels are only moderate in batch cultivations.

In fed batch cultivations, the concentration of energy sources can be adjusted according to the rate of consumption. Several other factors can also be regulated in order to obtain the maximal production level in terms of target protein per biomass. The formation of inclusion bodies can be followed in fed batch cultivations by monitoring changes in intrinsic light scattering by flow cytometry [29]. This allows for real time optimization of growth conditions as soon as inclusion bodies are detected even at low levels and inclusion body formation can potentially be avoided [30].

Folding of some proteins require the existence of a specific cofactor. Addition of such cofactors or binding partners to the cultivation media may increase the yield of soluble protein dramatically. This was demonstrated for a recombinant mutant of hemoglobin for which the accumulation of soluble product was improved when heme was in excess [31]. Similarly, a 50% increase in solubility was observed for gloshedobin when E. coli recombinants were cultivated in the presence of 0.1 mM Mg 2+ [32]. An important factor in soluble expression of recombinant proteins is media composition and optimization. Although this is attained mostly by trial and error, it nevertheless may be beneficial.

Molecular chaperones drive folding of recombinant proteins

A possible strategy for the prevention of inclusion body formation is the co-overexpression of molecular chaperones. This strategy is attractive but there is no guarantee that chaperones improve recombinant protein solubility. E. coli encode chaperones, some of which drive folding attempts, whereas others prevent protein aggregation [4, 11, 33]. As soon as newly synthesized proteins leave the exit tunnel of the E. coli ribosome they associate with the trigger factor chaperone [34]. Exposed hydrophobic patches on newly synthesized proteins are protected by association with trigger factor from unintended inter- or intramolecular interactions thus preventing premature folding. Proteins can start or continue their folding into the native state after release from trigger factor. Proteins trapped in non-native and aggregation prone conformations, are substrates for DnaK and GroEL. DnaK (Hsp70 chaperone family) prevents the formation of inclusion bodies by reducing aggregation and promoting proteolysis of misfolded proteins [11]. A bi-chaperone system involving DnaK and ClpB (Hsp100 chaperone family) mediates the solubilization or disaggregation of proteins [35]. GroEL (Hsp60 chaperone family) operates the protein transit between soluble and insoluble protein fractions and participates positively in disaggregation and inclusion body formation. Small heat shock proteins lbpA and lbpB protect heat denatured proteins from irreversible aggregation and have been found associated with inclusion bodies [36, 37].

Simultaneous over-expression of chaperone encoding genes and recombinant target proteins proved effective in several instances. Co-overexpression of trigger factor in recombinants prevented the aggregation of mouse endostatin, human oxygen-regulated protein ORP150, human lysozyme and guinea pig liver transglutaminase [38, 39]. Soluble expression was further stimulated by the co-overexpression of the GroEL-GroES and DnaK-DnaJ-GrpE chaperone systems along with trigger factor [39]. The chaperone systems are cooperative and the most favorable strategies involve co-expression of combinations of chaperones belonging to the GroEL, DnaK, ClpB and ribosome associated trigger factor families of chaperones [40–42].

Interaction partners and protein folding

Protein insolubility in the E. coli cytoplasm is partially related to the distribution of hydrophobic residues on the surface of the protein. The soluble expression of subunits of hetero multimeric proteins therefore sometimes suffers from inclusion body formation in the absence of an appropriate binding partner.

Soluble expression in E. coli of the bacteriophage T4 gene 23 product (major capsid protein) required the co-expression of gene product 31 (phage co-chaperonin gp31) [43]. Expression of the correct interaction partner enabled gp23 to fold correctly and form long regular structures in the cytoplasm of E. coli.

Another study reports the purification of a heterodimeric complex by expression of each subunit (pheromaxein A and C) as a fusion to thioredoxin [44]. Each subunit remained soluble in solution, when thioredoxin was proteolytically removed, only in the presence of the other.

Conclusively, interaction partners potentially favour in vivo solubility of target proteins. New systems for co-expression of multiple proteins involved in complex structures enable such strategies [1].


MATERIALS AND METHODS

Plasmid construction, cloning, and E. coli transformation

After codon usage analysis, a 320 bp-long construct containing codon-optimized RMCP1 gene sequence (NM_001082294.1) flanked by two restriction sites, NcoI and XhoI in pUC57 (RMCP1/pUC57), was generated by GeneCust (Dudelange, Luxembourg) (Fig. 1). RMCP1/pUC57 was then used to transform E. coli TOP10F' strain (TOP10) cells to amplify the plasmid DNA. The cells were grown in Luria-Bertani (LB) broth (Sigma-Aldrich, St. Louis, MO) containing tetracycline (10 μg/mL) at 37°C and 200 rpm. The plasmid was then extracted using the SolGent Plasmid Mini Prep kit, according to the manufacturer's instructions (Seoul, Korea), and digested with XhoI and NcoI. The digestion products were separated on agarose gel and the RMCP1-containing fragment was purified by gel extraction using a GeNet Bio Kit (Daegeon, Korea). The purified fragment was ligated with an XhoI- and NcoI-digested pET28a vector (Pasteur Institute, Tehran, Iran), and used to transform TOP10 cells. All the TOP10 transformations were done using the calcium chloride and heat shock protocol. A sequence encoding a His6-tag is present immediately after the XhoI restriction site on the pET28a plasmid, and the generated recombinant protein was therefore produced with a fused C-terminal His6-tag.

(a) Schematic diagram of the construct containing rabbit MCP1 coding sequence. (b) Nucleotide (lower) and translated amino acid (upper) sequences of the recombinant rabbit MCP1. Position 1 in the amino acid sequence corresponds to the first amino acid (methionine) of the mature rabbit MCP1 protein.

(a) Schematic diagram of the construct containing rabbit MCP1 coding sequence. (b) Nucleotide (lower) and translated amino acid (upper) sequences of the recombinant rabbit MCP1. Position 1 in the amino acid sequence corresponds to the first amino acid (methionine) of the mature rabbit MCP1 protein.

The resulted positive clones were evaluated by colony PCR and digestion. The PCR reaction mixtures contained Taq DNA polymerase (Thermo Scientific, Waltham, MA) (0.3 μL, 1.25 U), 10X buffer (2.5 μL Thermo Scientific), 10 mM dNTPs (1 μL), 1.25 mM MgCl2 (0.5 μL Thermo Scientific), double distilled water (17.75 μL), and 1 μL of 10 mM T7 forward (F, 5΄-TAATACGACTCACTATAGGG-3΄) and reverse (R, 5΄-GCTAGTTATTGCTCAGCGG-3΄) primers. The PCR program was started with a 4-min incubation at 96°C followed by 30 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 50 s and ending with a step at 72°C for 10 min. Bio-Rad thermal cycler (Hercules, CA) was used for PCR. PCR product sizes were verified by agarose gel (1%) electrophoresis and comparison with a DNA Mix ladder (Fermentase).

Recombinant protein expression

The constructed plasmid (RMCP1/pET28a) was extracted from positive clones and used to transform E. coli BL21 (DE3) (BL21) (Pasteur Institute). Single transformants were selected on LB agar (Sigma-Aldrich) containing 25 μg/mL of kanamycin, and verified by colony PCR and XhoI/NcoI restriction enzyme digestion. The exact sequence of the inserted fragment in the resultant clones (two clones) was confirmed by DNA sequencing, which was performed by Bioneer Company (Korea).

The two positive clones (clones 2 and 3) were selected for rRMCP1 protein production. To induce protein expression, the clones were cultured in LB broth supplemented with 25 μg/mL of kanamycin, with isopropyl-β- d -1-thiogalactopyranoside (IPTG) induction. To determine the optimal conditions allowing an intermediate level of protein expression, the following induction parameters were investigated: OD600 of bacterial culture at induction (0.4–0.5, 1.0–1.2 and 2.0–2.5), temperature (26°C, 30°C and 37°C), induction duration (5–24 h), shaking rate (180–250 rpm) and final concentration of IPTG (0.5, 1 and 2 mM). The effect of media replacement every 4–6 h during induction was also investigated namely, every 4–6 h, the bacteria were collected by centrifugation and fresh culture medium with the same antibiotic and IPTG concentrations was added.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with protein visualization by Coomassie Brilliant Blue R-250 staining and western blotting with conjugated horseradish peroxidase/anti-His antibody (Thermo Scientific) was performed to verify the expression of rRMCP1.

Recombinant protein purification

BL21 cells producing the rRMCP1 protein were lysed in 4 mL of lysis buffer [100 mM NaH2PO4, 10 mM Tris-HCl (pH 8.0) and 8 M urea] per 1 g of bacterial pellet, and this process was completed by sonication (five pulses of 50 s), according to QIAGEN (Hilden, Germany) protein purification protocol. The lysates were centrifuged at 8500 × g at 4°C for 3 min, and the supernatant was then incubated with Nickel-nitrilotriacetic acid (Ni-NTA) resin (QIAGEN) at room temperature (22°C–25°C) for 45 min. The protein/resin complex was applied to a miniature QIAGEN column, washed, and eluted using buffers B to E and the fractions were separately collected. Following SDS-PAGE analysis of the collected fractions, the purified protein fractions were pooled and concentrated using Amicon Ultra-4 centrifugal filters (Merck Millipore, Billerica, MA). Protein concentration was determined using the Bradford assay (Bradford 1976), with bovine serum albumin (Sigma-Aldrich) as a standard.

SDS-PAGE and western blotting

SDS-PAGE

BL21, BL21/pET28a and BL21/pET28a/RMCP1 cells were cultured in LB broth medium. At OD600 = 0.4–0.5, BL21/pET28a/RMCP1 culture was split into two tubes, and one half was induced by IPTG for 24 h. Samples were withdrawn 5, 16 and 24 h after induction and the amount of bacteria was normalized between the three conditions by measuring their OD600s and equaling them by dilution, and prepared for electrophoresis. The samples and a pre-stained protein marker (10–270 kDa, Thermo Scientific) were loaded onto a 15% SDS-PAGE gel, then stained in a Coomassie R-250 solution, for 3 h, and destained.

Western blotting

Following SDS-PAGE, the proteins were transferred to a polyvinylidene difluoride membrane using transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, pH 8.3), at 90 V for 90 min. (Feizollahzadeh et al. 2016). The rRMCP1 protein was detected using mouse monoclonal anti-poly-histidine peroxidase conjugated antibody (working dilution of 1:2000 Sigma-Aldrich).

Biological activity of rRMCP1

Monocyte culture

Nearly 3 mL blood from ears of two male New Zealand white rabbits (2.3 ± 0.2 kg) (purchased from Pasteur Institute) was taken. The experiment was approved by the animal ethics committee of Isfahan University of Medical Sciences, Isfahan, Iran.

The monocytes were separated by Ficoll-Paque (Sigma-Aldrich) according to the manufacturer's recommendations. The separated monocytes were maintained in Roswell Park Memorial Institute medium (RPMI) (Biosera, Nuaille, France) supplemented with 10% of fetal bovine serum (Sigma-Aldrich), 100 U/mL of penicillin and 100 μg/mL of streptomycin (Invitrogen, Carlsbad, CA), in a humid incubator at an atmosphere of 5% CO2 at 37°C.

RRMCP1-mediated monocyte migration assay

Two six-well cells with a 0.4-μm pore polycarbonate (PCTE) membrane (Sterlitech Corporation, Kent, OH) were used. Different concentrations of purified rRMCP1 (5, 10, 15 or 20 μg per 2 mL of RPMI medium supplemented with 0.5% of fetal bovine serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin) were prepared, and placed in the bottom chamber of each well, in duplicate. Two wells were the controls, with no protein added. Monocytes (2.5 × 10 6 cells) were suspended in 10 mL of RPMI, and 1 mL of the suspension was placed in the top chamber. The plates were then incubated at 37°C in a humid incubator, 5% CO2, overnight. The media in the top chambers were transferred to separate tubes. The media from the bottom chambers were transferred to separate tubes, centrifuged at 1500 × g for 5 min at room temperature, the pellets suspended in 1 mL of RPMI and the cells counted after trypan blue staining. The migration of monocytes was evaluated based on the number of cells that migrated from the top chamber, through the membrane, into the bottom chamber in each well which contained 0.18, 0.36 or 0.72 ng/mL, compared with control. The number of monocytes that migrated through the filter was proportional to chemotaxis.

Statistical analysis

The data of monocyte migration assay were analyzed in IBM SPSS software (version 20.0) using the independent t test and one-way ANOVA. P-values lower than 0.05 were considered significant.


Cloning, High Expression and Purification of Recombinant Human Intereferon-β-1b in Escherichia coli

Sequential evaluation and process control strategy were employed for impurity profile and high recovery with quality of rhIFN-β-1b expressed in Escherichia coli. The high-level expression was achieved by using codon substitution (AT content of 52.6% at N-terminal region) and optimization of culture conditions. The addition of rifampicin at a concentration of 200 μg/ml has increased the specific product yield of 66 mg optical density −1 l −1 (43.5% of total cellular protein). Eighty-three percent of lipopolysaccharides, 32% of host deoxyribonucleic acid (DNA), and 78% of host cell proteins were removed by 0.75% Triton X-100 and 2 M urea wash. Eleven percent of lipopolysaccharides, 39% of host DNA, and 12% of host cell proteins were removed at the solubilization step. Ninety-two percent of protein refolding was achieved by high-pressure diafiltration method. Refolding by high-pressure diafiltration, bed height, and height equivalent to the theoretical plate value in chromatography column were identified as key parameters for high recovery with purity. Finally, the established process yielded 34% of purified protein with greater than 99% purity and is acceptable for preclinical toxicological studies. The purified rhIFN-β-1b obtained in this study is the highest that has been reported so far.

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Protein Expression in E. Coli

Protein expression in the bacterium E. coli is the most popular means of producing recombinant protein. E. coli is a well-established host that offers short culturing time, easy genetic manipulation and low cost media. Additionally, E. coli has a long history of being capable of producing a wide variety of different types of proteins. Even proteins that contain disulfide bonds can be expressed within the cytoplasm of NEB SHuffle ® strains.

Within the realm of E. coli expression, the T7 system is the most popular approach for producing proteins. In this system, an expression vector containing a gene of interest cloned downstream of the T7 promoter is introduced into a T7 expression host. T7 expression hosts such as DE3 strains or T7 Express strains carry a chromosomal copy of the phage T7 RNA polymerase gene. When inducer is added, T7 RNA polymerase is expressed and becomes dedicated to transcription of the gene of interest.

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This article provides an overview of the advances in protein expression and purification methodology over the past 40 years.

Disulfide bond formation in the cytoplasm of wild type E. coli is not favorable, while SHuffle is capable of correctly folding proteins with multiple disulfide bonds in the cytoplasm.

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Lysis and Purification of Recombinant Protein

Traditionally, protein purification from E. coli includes four phases: harvest, bacterial cell lysis, lysate clarification, and protein purification. Bacterial lysis typically requires several time-consuming steps, including freeze/thaw cycles and sonication, which may negatively impact protein quality and contribute to sample variability. To maintain protein activity and integrity, detergent-based lysis buffers are used to avoid mechanical protein extraction methods. Centrifugation is traditionally required to pellet unwanted cell debris and permit clarified lysate recovery.

Purification is frequently performed using affinity media specific for expressed epitope tags. Agarose-based media are typically used, either as a slurry in microcentrifuge tubes or packed into columns. While easier to manipulate, columns are affected by lysate consistency and carryover of cell debris, which can cause clogging.

This article will demonstrate a new protocol for streamlining the traditional recombinant protein purification workflow by combining the enzymatic lysis and purification steps. This approach results in significantly less hands-on time and greater than two-hour time savings over the traditional workflow (Figure 1).

Incorporation of magnetic beads into the protocol eliminates the need to clarify lysates by centrifugation. Magnetic beads have been adopted where agarose beads have been used, reducing processing time and increasing sample throughput. Magnetic beads are generally used in batch mode and isolated on a magnet to allow for buffer exchange.

Magnetic beads facilitate automation of the protocol using a particle processor, resulting in increased sample throughput while reducing hands-on processing time to less than 10 minutes. To further improve the workflow, we used BugBuster® Master Mix reagent (EMD Millipore), which allows for nonmechanical extraction of soluble protein from bacterial cells. This extraction reagent combines detergent-based lysis with the enzymatic agent Benzonase® nuclease and the enzyme rLysozyme™ in a ready-to-use formulation.


Figure 1. One-step lysis combined with purification (right) saves considerable time compared to traditional recombinant protein purification, which requires separate lysis, lysate clarification, and purification steps (left).

Materials and Methods

Histidine-tagged recombinant glyceraldehyde phosphate dehydrogenase (GAPDH) was purified with PureProteome™ Nickel Magnetic Beads (EMD Millipore), using a traditional purification workflow (mechanical lysis) and the new condensed protocol (combined detergent-based lysis and purification). These magnetic beads capture histidine-tagged proteins they isolate recombinant proteins at high purity and can be used manually or on automated systems. We compared the manual processing results with those obtained using the KingFisher® automated particle processor (Thermo Scientific).

Traditional Protein Purification
E. coli culture was pelleted into microcentrifuge tubes and the supernatant was discarded. Lysis/wash buffer containing lysozyme was added to each pellet. The pellet was resuspended and incubated with end-over-end mixing, followed by sonication using a microtip. The lysate was frozen, followed by quickly thawing the sonication/freeze-thaw cycle was repeated once more. To reduce viscosity, Benzonase endonuclease was added to the lysate and clarified by centrifugation.

The clarified lysate was added to PureProteome Nickel Magnetic Beads. The beads were incubated with E. coli lysate with end-over-end mixing. After removal of the lysate, the beads were washed with lysis/wash buffer by vortexing, capturing the beads on the magnet and removing the buffer by pipette. The wash step was repeated two more times prior to eluting the captured histidine-tagged GAPDH. The beads were mixed, and an additional elution was performed to achieve maximum yield. Both elution fractions were combined into one microcentrifuge tube for future analysis.

One-Step Protein Purification
Manual Processing
E. coli culture was pelleted into microcentrifuge tubes and the supernatant discarded. Suspended PureProteome Nickel Magnetic Bead slurry was added. Using the PureProteome Magnetic Stand, the preservative was removed, and the beads were washed with lysis/wash buffer. The beads were collected on the magnet, and the buffer was removed by pipette. The beads were resuspended in BugBuster Master Mix and the suspension was added to each E. coli pellet. Each tube was briefly vortexed, then incubated with end-over-end mixing. The unbound fractions were discarded and beads were washed using the PureProteome Magnetic Stand to capture beads. Elution was performed as previously described.

Automated Processing
E. coli culture was pelleted into a KingFisher Microtiter Deepwell 96 plate and the supernatant discarded. Reagents and samples were pipetted into the KingFisher Duo plates and the suspended PureProteome Nickel Magnetic Bead slurry was brought up to sufficient volume with wash buffer. Wash buffer was used for both equilibration and wash steps, and the elution buffer was pipetted into the elution strips. The protocol was executed and the plates were loaded into the KingFisher Duo System.

The PureProteome Nickel beads were equilibrated in wash buffer to remove preservatives. The beads were collected, and cell lysis and His-tagged protein capture was performed. The beads were then washed and recombinant protein was eluted. After the run ended, the eluted sample fractions were collected for further analysis.

Protein Analysis
Additional lysates were prepared using the traditional mechanical lysis approach as well as using the BugBuster Master Mix protocol no magnetic beads were added during the lysis step. The lysate was clarified by centrifugation, and total protein concentration was determined with the Direct Detect®
IR spectrometer (EMD Millipore).

A quantitative Bradford assay was also performed to assess the efficiency of lysis and protein purification.

Finally, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Purified samples were reduced, denatured and loaded onto NuPAGE® Bis-Tris gels (Life Technologies). Following electrophoresis, the gels were stained with Coomassie® blue to visualize protein bands.

The results demonstrated that the new protocol delivered more total protein in less time and with greater consistency than the traditional method. Traditional mechanical lysis of six samples took about two hours the average total protein collected was 3.54 mg/mL and the CV% was 15.55. In contrast, enzymatic lysis using the new protocol took about 30 minutes the average total protein collected was 4.09 mg/mL and the CV% was 2.05.

The eluted fractions were then tested for protein yield using a Bradford assay. All workflows demonstrated roughly equivalent yields of purified protein. However, the traditional method exhibited greater inter-sample variability than did either condensed protocol. Workflow automation offered the highest degree of reproducibility (Table).

Finally, the SDS-PAGE gel showed all workflows provided similar sample purity (Figure 2). As previously noted, greater sample-to-sample variability was observed for the traditional workflow.


Yield of His-tagged GAPDH using the traditional and condensed E. coli lysis and purification protocol as determined by Bradford assay.

For extraction of recombinant protein from E. coli, traditional mechanical lysis is a time-consuming manual process. Mechanical lysis and manual processing can also result in variable yield due to sample-to-sample variations in the sonication step. By combining gentle nonmechanical lysis with magnetic affinity capture beads for His-tagged protein purification, the traditional recombinant protein purification workflow has been condensed.

Even when samples were manually processed, a one-step lysis and purification protocol reduced processing time by 75% without sacrificing yield or purity. Due to reduced sample manipulation, the simplified protocol also provides greater sample-to-sample consistency. Moreover, by eliminating centrifugation requirements, the condensed workflow can be automated using particle processors to further reduce sample variability and increase throughput while reducing hands-on time to less than 10 minutes.


Figure 2. SDS-PAGE analysis of His-tagged GAPDH purified using the traditional and condensed recombinant protein purification workflows. The condensed protocol was performed manually as well as on the KingFisher Duo System. (Molecular weight standards are included in the rightmost lane.)


E. coli

NEB offers two protein expression systems in E.coli. The pMAL&trade Protein Fusion & Purification System (NEB #E8200) is used to express an MBP-fusion protein which is then purified by affinity purification. This system enhances solubility and results in reliable E.coli expression in either the cytoplasm or periplasm.

The IMPACT&trade Kit (NEB #E6901) allows fusion of a tag consisting of the intein and the chitin binding domain (CBD), to either the C-terminus (pTXB1) or the N-terminus (pTYB21) of the target protein. In the presence of thiols, such as DTT, the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag resulting in purification in a single chromatographic step with no need for proteases.

pMAL&trade and IMPACT&trade are trademarks of New England Biolabs, Inc.

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Read how to avoid common obstacles in protein expression that prevent interactions with cellular machinery.

Cell-free protein synthesis has the potential to become one of the most important high throughput technologies for functional genomics and proteomics.

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Lane 1: uninduced cell extract.
Lane 2: induced cell extract showing expressed fusion protein.
Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C.
Lane 4: MBP ligated to a peptide containing an N-terminal cysteine. Marker M is the Protein Ladder (NEB #P7703).

Protein expression with Lemo21(DE3) is very similar to BL21(DE3), with only a few minor changes.

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.


Example 4

Improvement in the Expression Level and Cellular In Vivo Folding of the Recombinant HSA with the Assistance of Molecular Chaperone System

Recombinant E. coli origami DE3 cells co-transformed with both the plasmids (pETHSA and pTF16) were inoculated in the primary culture of 5 mL LB medium containing both the antibiotics-ampicillin (100-300 μg/mL) and chloramphenicol (20-40 μg/mL). They were then inoculated from primary culture into a secondary culture in ZY autoinduction growth medium containing both ampicillin (100-300 μg/mL) and chloramphenicol (20-40 μg/mL) antibiotics. The secondary culture was grown at a temperature between 30-40° C. and the samples were collected and checked for optical density at regular time intervals. The growth of the cells was monitored and the cells were induced with L-Arabinose (Final conc. 0.3-0.6 mg/mL) when the OD600 reached 0.2 to 0.5 (which took between 6-12 hours) and left to grow again until the OD600 reached 0.6-1.0.

Δt this point, the incubation temperature of the recombinant host cell culture was lowered to a temperature in the range of 10-20° C. and cells were harvested after 8-12 hours of induction.

In order to obtain the expressed protein, the cells were resuspended in the lysis buffer containing the osmolyte trehalose in the concentration range of 0.5-1.0M. Cell lysate was obtained after the lysis step using conventional means. To summarize the steps above briefly, induced E. coli Origami2 (DE3) cells expressing both the Trigger factor (chaperone) and rHSA were harvested and pelleted down by centrifugation. The cell pellet obtained was resuspended in cell lysis buffer and incubated for 15-30 minutes. The resuspended cells in lysis buffer were then exposed to an ultrasonic cell disruptor to release the intracellular components in the lysis buffer. The sonicated cell lysate was fractionated by high speed centrifugation. The supernatant containing soluble fraction of rHSA was carefully aspirated without disturbing the pellet which contained the insoluble aggregated fraction of rHSA. Cells were then fractionated and analyzed for solubility and activity assay and was compared with the rHSA production in E. coli without Trigger factor expression (chaperone assistance).

Overall, the present disclosure provides a process for obtaining soluble and functional rHSA protein, in the presence of the molecular chaperone Trigger Factor. The process results in 1.5-2.0 fold increase in expression level of rHSA protein as compared to protein levels obtained without the presence of the molecular chaperone. Further, it is observed that though the soluble fraction of the rHSA expressed in the E. coli remains same in both the conditions (with or without chaperone assistance) i.e. 60%, there exists marked difference in the activity of the soluble fraction's functional content (FIG. 5). As has been demonstrated, the present process leads to 20-30% increase in functionally active soluble rHSA protein in the soluble fraction of rHSA when co-expressed along with the molecular chaperones as compared to when expressed alone in the E. coli host system (Recently filed patent application, IP no. 201611027096).

The enhancement in the activity is credited to the employment of trigger factor system for rHSA production as Trigger factor (molecular chaperone) acts to prevent misfolding and aggregation reaction by transiently shielding the hydrophobic regions exposed in non-native polypeptides during and after translation. (Agashe et al., Cell (2009) 117:199-209).

Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.


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