Periplasmic Extraction Research Articles

Triton™ X-100 Behaves Similarly to Tyrosine-Containing and Tryptophan-Free Proteins in UV-Vis Spectroscopy

As a known non-ionic, -denaturing detergent and emulsifier, Triton™ X-100 is often used in various biochemical studies including in the isolation of membrane-protein complexes for solubilizing membrane proteins, in the process of periplasmic protein extraction as a component of the lysis buffer, in both indirect immunofluorescence staining and flow cytometry as a permeabilization reagent, etc. It has been shown that the diluted solution of Triton™ X-100 with the optimal pH range of 6.0-8.0 has a significant absorption of UV light. In the present project, we show that the absorption spectrum of Triton™ X-100, when dissolved in 1X phosphate-buffered saline, is similar to that of α-synuclein, as a representative of those proteins lack tryptophan but contain tyrosine as their main UV absorber. These results show that whenever the use of Triton™ X-100 for extracting membrane and periplasmic proteins is inevitable, scavenging it before the characterization of the proteins by UV-Vis spectroscopy, especially the determination of their concentration using Beer-Lambert Law, would be necessary.

Improving the yield of recalcitrant Nanobodies® by simple modifications to the standard protocol

Nanobodies are single-domain antibody constructs derived from the variable regions of heavy chain only (VHH) camelid IgGs. Their small size and single gene format make them amenable to various molecular biology applications that require a protein affinity-based approach. These features, in addition to their high solubility, allows their periplasmic expression, extraction and purification in E. coli systems with relative ease, using standardized protocols. However, some Nanobodies are recalcitrant to periplasmic expression, extraction and purification within E. coli systems. To improve their expression would require either a change in the expression host, vector or an increased scale of expression, all of which entail an increase in the complexity of their expression, and production cost. However, as shown here, specific changes in the existing standard E. coli culture protocol, aimed at reducing breakdown of selective antibiotic pressure, increasing the initial culture inoculum and improving transport to the periplasmic space, rescued the expression of several such refractory Nanobodies. The periplasmic extraction protocol was also changed to ensure efficient osmolysis, prevent both protein degradation and prevent downstream chelation of Ni2+ ions during IMAC purification. Adoption of this protocol will lead to an improvement of the expression of Nanobodies in general, and specifically, those that are recalcitrant.

Periplasmic expression of EGFRvIII extracellular domain as a fusion protein in Escherichia coli NiCo21(DE3)

Epidermal growth factor receptor variant III is a mutant variant of EGFR that has a deletion on its DNA encoding extracellular ligan-binding domain. EGFRvIII is only found in cancer cells and not in normal cells, make it an ideal target as a biomarker for antibody-based cancer therapy. This study performed the expression and characterization of EGFRvIII extracellular domain conjugated with a blue fluorescent protein (BFP) as a fusion protein in Escherichia coli periplasmic space. Endoxylanase signal peptide was employed to guide the recombinant protein through the membrane. IPTG as inducer was added into expression medium with various concentrations of 0; 0.1; 0.25; 0.5; 1 mM, followed by periplasmic extraction using the hypertonic solution. Total proteins and periplasmic proteins were characterized using SDS-PAGE and slot blot analyses. Extracellular domain of EGFRvIII-BFP fusion protein was detected using confocal fluorescence microscopy. This study showed that the ∼43 kDa target protein was successfully expressed on E. coli NiCo21(DE3) periplasmic space with optimum IPTG concentration of 0.1 mM and and showed a blue fluorescence color under the microscope..

Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris–HCl concentration, buffer’s pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli. The results indicated that the Tris–HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris–HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min.

A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus

BackgroundPorcine epidemic diarrhea virus (PEDV), which is characterized by severe watery diarrhea, vomiting, dehydration and a high mortality rate in piglets, leads to enormous economic losses to the pork industry and remains a large challenge worldwide. Thus, a rapid and reliable method is required for epidemiological investigations and to evaluate the effect of immunization. However, the current diagnostic methods for PEDV are time-consuming and very expensive and rarely meet the requirements for clinical application. Nanobodies have been used in the clinic to overcome these problems because of the advantages of their easy expression and high level of stability. In the present work, a novel biotinylated nanobody-based blocking ELISA (bELISA) was developed to detect anti-PEDV antibodies in clinical pig serum.ResultsUsing phage display technology and periplasmic extraction ELISA (PE-ELISA), anti-PEDV N protein nanobodies from three strains of PEDV were successfully isolated after three consecutive rounds of bio-panning from a high quality phage display VHH library. Then, purified Nb2-Avi-tag fusion protein was biotinylated in vitro. A novel bELISA was subsequently developed for the first time with biotinylated Nb2. The cutoff value for bELISA was 29.27%. One hundred and fifty clinical serum samples were tested by both newly developed bELISA and commercial kits. The sensitivity and specificity of bELISA were 100% and 93.18%, respectively, and the coincidence rate between the two methods was 94%.ConclusionsIn brief, bELISA is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PEDV vaccines efficacy and indirect diagnosis of PEDV infection.

Pharmacological studies of pegylated anti-TNF-α antibodies

Objective Rheumatoid arthritis (RA) is a systemic disease with chronic inflammation of the joints, and is caused by increased inflammatory cytokines such as TNF-α. The inhibition of TNF-α was found to effectively control inflammation, and block the progress of the disease. This study designed a new and efficient strategy to develop TNF-α antibodies. Methods Prokaryotic engineered cells were growing in a small scale bioreactor, and Fab' products (TMP5) were purified by affinity chromatography. Then TMP5 was modified by 20 kDa site-specific pegylation, and purified by SP sepharose column. Activities and affinities of TMP5 and PEG-TMP5 in vitro were studied. Results TMP5 was effectively produced after fermentation, periplasmic extraction and purification. The purity of TMP5 was more than 96%. After pegylation by 20 kDa PEG, the affinity and activity of PEG-TMP5 were (6.48±0.86)×10-9 M and (7.58±0.61)×10-11 M respectively, and were in the same range as positive controls. Conclusion PEG-TMP5 is a promising drug candidate for treating rheumatoid arthritis. Key words: Antibodies; Tumor necrosis factor α; Pegylation; pharmacology in vitro

Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing's sarcoma.

Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60% soluble fraction, we obtained a yield of 25mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75% soluble fraction 35mg C7 dAbd per L of cell culture grown in a synthetic media containing 3g/L of vegetable peptone and 1g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells.

The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli.

Background:Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process.Objectives:In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b).Materials and Methods:The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd.Results:Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band.Conclusions:The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step.

Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library

The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, are time-consuming, and require appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage display vector pMECS with diversity of 2.8 × 109. Based on phage display technology and periplasmic extraction ELISA, H7N2-specific VHH antibodies were successfully isolated. According to a pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo, and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin–streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/ml. Given the above, the newly developed VHH antibody-based double sandwich ELISA (DAS–ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.

Cloning and expression of Pteropine Orthoreovirus sigma C protein in bacterial cells

Introduction: Pteropine Orthoreovirus (PRV) was first cloned in 2011 to represent a collective of orthoreoviruses isolated from bat species of the genus Pteropus . Humans, especially infants or children, infected with Mammalian Orthoreovirus (MRV), a genetically closely related ‘ relative’ to PRV, commonly present with mild upper respiratory tract illness or enteritis. However, this does not hold true for PRV, as acute respiratory syndrome is commonly seen in patients infected with PRV. Objectives: The focus of this study was to clone the sigma C gene in two vectors (pMal-C5X and pMal-P5X) and express it in different bacterial cells (ER2523 and BL21). However, the targeted receptor will be identified in a future study. Materials and methods: The sigma C gene of PRV1K (Kampar virus), PRV2P (Pulau virus) and PRV3M (Melaka virus) were amplified and cloned into (pMal-C5X and pMal-P5X) vectors. Colony polymerase chain reaction (PCR) was preformed to screen for positive clones prior to being expressed in BL21 and ER2523 bacteria. Periplasmic and total lysis extraction was performed on both bacterial cells prior to separation by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and visualization with Coomassie Blue stain. Results: The PCR of sigma C gene for three different viruses, namely Pulau, Melaka and Kampar, were performed. PCR of Pulau and Kampar viruses were successful but Melaka virus was unsuccessful because it did not reach the desired band and was excluded. The protein receptors were confirmed by a restriction enzyme reaction. Conclusions: Sigma C of PRV2P was expressed in bacterial cells without any lethal effect on the bacteria. The protein will be purified and targeted receptor identified in a future study. Acknowledgements: We would like to acknowledge our University and our Dean, Dr Khalid Al Qumaizy, for giving us this great opportunity, and we acknowledge Dr Keeny Voon from International Medical University of Malaysia for helping us conduct this research.

Integration of host strain bioengineering and bioprocess development using ultra-scale down studies to select the optimum combination: an antibody fragment primary recovery case study.

An ultra scale-down primary recovery sequence was established for a platform E. coli Fab production process. It was used to evaluate the process robustness of various bioengineered strains. Centrifugal discharge in the initial dewatering stage was determined to be the major cause of cell breakage. The ability of cells to resist breakage was dependant on a combination of factors including host strain, vector, and fermentation strategy. Periplasmic extraction studies were conducted in shake flasks and it was demonstrated that key performance parameters such as Fab titre and nucleic acid concentrations were mimicked. The shake flask system also captured particle aggregation effects seen in a large scale stirred vessel, reproducing the fine particle size distribution that impacts the final centrifugal clarification stage. The use of scale-down primary recovery process sequences can be used to screen a larger number of engineered strains. This can lead to closer integration with and better feedback between strain development, fermentation development, and primary recovery studies. Biotechnol. Bioeng. 2014;111: 1971–1981. © 2014 Wiley Periodicals, Inc.

COMPARISON OF THE CYTOPLASMIC AND PERIPLASMIC PRODUCTION OF RETEPLASE IN Escherichia coli

Reteplase is the recombinant type of tissue plasminogen activator variant. In this study, preplasmic and cytoplasmic (as inclusion body: IBs) production and activity of recombinant reteplase in E. coli were investigated and compared using a pET system (pET22b and pET15b). The cDNA of reteplase was cloned by polymerase chain reaction (PCR) amplification, sequenced, inserted into the vector pET 22b and pET15b, and expressed using isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant plasmid was expressed in the form of inclusion body in pET 15b and in periplasmic space in pET22b. The obtained results of inclusion body extraction from recombinant pET22b (rpET22b) and recombinant pET15b (rpET15b) plasmids using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a band of ∼39 kD. However, the obtained results of periplasmic space extraction from rpET22b plasmid showed a very weak band, while cytoplasmic expression of reteplase (pET15b) produced a strong protein band confirmed with Western blotting. Consequently, our results demonstrated that the cytoplasmic expression system is efficient for the production of reteplase protein in prokaryote systems and a high amount of reteplase was obtained from the expressed proteins in the form of IBs. The obtained activity of rpET15b plasmid showed a higher enzyme absorbance in comparison to rpET22b plasmid. This suggests rpET15b as an appropriate candidate for reteplase production.

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfide-bond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white Lysozyme (HEWL) antibody fragment was carried out at 37oC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/L after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain the majority of the target protein in their periplasmic space ( i.e. 4 hrs post induction).

Biotin Carboxyl Carrier Protein Co-Purifies as a Contaminant in Core-Streptavidin Preparations

We have successfully cloned and expressed core-streptavidin in Escherichia coli. Core-streptavidin was expressed in shaker flask culture as a soluble protein, isolated by periplasmic extraction, purified by immobilized metal affinity chromatography column, and analyzed for its size, thermal stability, and biotin-binding activity. In Western blots using streptavidin-horseradish peroxidase (HRP) as a probe, we identified a contaminant that co-purified with core-streptavidin, identified as biotin carboxyl carrier protein (BCCP). Although BCCP cannot be detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it appears as a prominent band in Western blot when probed with streptavidin peroxidase conjugate. Based on the results from in vitro gel digestion, mass spectrometry and Mascot database search results, we confirmed the presence of BCCP. It was found that BCCP can complex with core-streptavidin and can dissociate when heated above 80 degrees C. BCCP could be successfully removed and recovered by using core-streptavidin immobilized magnetic beads under mild conditions. In addition, the enriched fractions of core-streptavidin oligotetramers were separated, which may be the by-products of BCCP binding to core-streptavidin in various ratios. Finally, enzyme linked immunosorbent assay results have shown that the amount of biotin-HRP binding to core-streptavidin was higher compared to commercially available streptavidin.

Immobilized metal-ion affinity chromatography of recombinant Fab protein OPG C11 in the presence of EDTA-Mg(II).

Undesired adsorption of host cell proteins poses a big challenge for immobilized metal-ion affinity chromatography (IMAC) purification. In this study, by using His6-tagged protein Fab OPG C11 from Escherichia coli fermentation as a model, we found that the presence of low concentrations of EDTA-Mg2+ in feed streams weakens the adsorption but makes it more specific towards polyhistidine tag. By combining EDTA-Mg2+ treatment and periplasmic extraction, we developed a one-step purification procedure for His6-tagged recombinant Fab OPG C11 using Ni-IDA (iminodiacetic acid) chromatography. This procedure eliminated the buffer exchange step after periplasmic extraction, which is usually required before IMAC in order to remove EDTA. In addition to savings on time and cost, this procedure eliminates undesired adsorption of most host cell proteins thus significantly improves the purity of polyhistidine-tagged recombinant proteins. The strategy of EDTA-Mg2+ treatment may have general application potentials.

TraC of IncN plasmid pKM101 associates with membranes and extracellular high-molecular-weight structures in Escherichia coli.

Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402-410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coli allowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery, traM, traB, traD, and traE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essential tra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.

Biochemical and EPR characterization of a high potential iron-sulfur protein inThiobacillus ferrooxidans

A soluble acid-stable high potential iron-sulfur protein (HiPIP) was purified from Thiobacillus ferrooxidans using the periplasmic extraction method. It was isolated in the form of a tetramer consisting of four subunits with a molecular mass of 5582 Da, and its biochemical and biophysical properties were characterized. The N-terminal amino acid sequence (15 residues) was compared with the nucleotide sequence of the iro gene isolated from another strain and the two sequences were found to be identical. The iron content measurement together with optical and EPR spectroscopic studies of the purified protein were consistent with the presence of one [4Fe-4S] cluster per subunit. The EPR spectrum recorded in the oxidized state was attributed to a [4Fe-4S]3+ cluster and the redox potential has been determined to be +380 mV.

Biochemical and EPR characterization of a high potential iron-sulfur protein in Thiobacillus ferrooxidans

A soluble acid-stable high potential iron-sulfur protein (HiPIP) was purified from Thiobacillus ferrooxidans using the periplasmic extraction method. It was isolated in the form of a tetramer consisting of four subunits with a molecular mass of 5582 Da, and its biochemical and biophysical properties were characterized. The N-terminal amino acid sequence (15 residues) was compared with the nucleotide sequence of the iro gene isolated from another strain and the two sequences were found to be identical. The iron content measurement together with optical and EPR spectroscopic studies of the purified protein were consistent with the presence of one [4Fe-4S] cluster per subunit. The EPR spectrum recorded in the oxidized state was attributed to a [4Fe-4S] 3+ cluster and the redox potential has been determined to be +380 mV.