Ni(II) functionalized polyhedral oligomeric silsesquioxane based capillary monolith for purification of histidine-tagged proteins by immobilized metal affinity micro-chromatography.

A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (µ-IMAC). For this purpose, mercaptosuccinic acid (MSA) linked-polyhedral oligomeric silsesquioxane [MSA@poly(POSS-MA)] monolith 300 µm in diameter was obtained by thiol-methacrylate polymerization using methacryl substituted-polyhedral oligomeric silsesquioxane (POSS-MA) and MSA as the thiol functionalized agent in a fused silica capillary tubing. Ni(II) cations were immobilized onto the porous monolith via metal-chelate complex formation with double carboxyl functionality of bound MSA segments. µ-IMAC separations aiming the purification of histidine tagged-green fluorescent protein (His-GFP) from Escherichia coli extract were carried out on Ni(II)@MSA functionalized-poly(POSS-MA) [Ni(II)@MSA@poly(POSS-MA)] capillary monolith. His-GFP was succesfully isolated by µ-IMAC on Ni(II)@MSA@poly(POSS-MA) capillary monolith with the isolation yield of 85 % and the purity of 92 % from E. coli extract. Higher His-GFP isolation yields were obtained with lower His-GFP feed concentrations and lower feed flow rates. The monolith was used for consecutive His-GFP purifications with a tolerable decrease in equilibrium His-GFP adsorption over five runs.

Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain.

Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins.

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH).

Three-Dimensional Interfacing of Cells with Hierarchical Silicon Nano/Microstructures for Midinfrared Interrogation of In Situ Captured Proteins.

Label-free optical detection of biomolecules is currently limited by a lack of specificity rather than sensitivity. To exploit the much more characteristic refractive index dispersion in the mid-infrared (IR) regime, we have engineered three-dimensional IR-resonant silicon micropillar arrays (Si-MPAs) for protein sensing. By exploiting the unique hierarchical nano- and microstructured design of these Si-MPAs attained by CMOS-compatible silicon-based microfabrication processes, we achieved an optimized interrogation of surface protein binding. Based on spatially resolved surface functionalization, we demonstrate controlled three-dimensional interfacing of mammalian cells with Si-MPAs. Spatially controlled surface functionalization for site-specific protein immobilization enabled efficient targeting of soluble and membrane proteins into sensing hotspots directly from cells cultured on Si-MPAs. Protein binding to Si-MPA hotspots at submonolayer level was unambiguously detected by conventional Fourier transform IR spectroscopy. The compatibility with cost-effective CMOS-based microfabrication techniques readily allows integration of this novel IR transducer into fully fledged bioanalytical microdevices for selective and sensitive protein sensing.

Novel luminescent affiprobes for molecular detection of Staphylococcus aureus using flow cytometry.

AIMS: Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus. METHODS AND RESULTS: To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA1 ) and fluorescent dimeric affibody (fA2 ) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA1 and fA2 had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that 'fA2 ' affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the 'fA1 ' affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results. CONCLUSIONS: The results show that 'fA1 ' affiprobe and 'fA2 ' affiprobe are two new efficient luminescent affiprobes for detecting S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits.

Direct recovery of enhanced green fluorescent protein from unclarified Escherichia coli homogenate using ion exchange chromatography in stirred fluidized bed.

A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10-3 mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed.

Expression and Purification of Membrane Proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is one of the most popular expression systems for eukaryotic membrane proteins. Here, we describe protocols for the expression and purification of mitochondrial membrane proteins developed in our laboratory during the last 15 years. To optimize their expression in a functional form, different promoter systems as well as codon-optimization and complementation strategies were established. Purification approaches were developed which remove the membrane protein from the affinity column by specific proteolytic cleavage rather than by elution. This strategy has several important advantages, most notably improving the purity of the sample, as contaminants stay bound to the column, thus eliminating the need for a secondary purification step, such as size exclusion chromatography. This strategy also avoids dilution of the sample, which would occur as a consequence of elution, precluding the need for concentration steps, and thus preventing detergent concentration.

Membrane Protein Expression in Insect Cells Using the Baculovirus Expression Vector System.

Integral membrane proteins have a critical role in fundamental biological processes; they are major drug targets and therefore of high research interest. Recombinant protein production is the first step in the protein tool generation for biochemical and biophysical studies. Here, we provide simplified protocols that facilitate the generation of high-quality virus and initial expression analysis for integral membrane protein targets utilizing the baculovirus-mediated expression system in insect cells. The protocol steps include generation of viruses, virus quality control, and initial expression trials utilizing standard commercial baculovirus vector systems and are exemplified for G protein-coupled receptor targets. The viral quality, quantity, and recombinant protein expression are evaluated by microscopy, flow cytometry, fluorimetry, and SDS-PAGE, using either covalently fused fluorescent proteins or co-expressed fluorescence markers. Moreover, integral membrane protein expression levels, approximate molecular mass, and stability can be evaluated from small-scale expression and purification trials.

Long-living and highly efficient bio-hybrid light-emitting diodes with zero-thermal-quenching biophosphors.

Bio-hybrid light-emitting diodes (Bio-HLEDs) based on color down-converting filters with fluorescent proteins (FPs) have achieved moderate efficiencies (50 lm/W) and stabilities (300 h) due to both thermal- and photo-degradation. Here, we present a significant enhancement in efficiency (~130 lm/W) and stability (>150 days) using a zero-thermal-quenching bio-phosphor design. This is achieved shielding the FP surface with a hydrophilic polymer allowing their homogenous integration into the network of a light-guiding and hydrophobic host polymer. We rationalize how the control of the mechanical and optical features of this bio-phosphor is paramount towards highly stable and efficient Bio-HLEDs, regardless of the operation conditions. This is validated by the relationships between the stiffness of the FP-polymer phosphor and the maximum temperature reached under device operation as well as the transmittance of the filters and device efficiency.

High-efficiency Ni2+-NTA/PAA magnetic beads with specific separation on His-tagged protein.

To effective capture and universal enrichment of His-tagged protein, polyacrylic acid (PAA) brushes were used to encapsulate Fe3O4 nanoparticles, connect NTA, and Ni2+ to prepare magnetic beads. These materials provide many advantages, such as excellent stability, tuneable particle size, and a surface for further functionalisation with biomolecules. His-tagged green fluorescence protein (GFP) was separated efficiently, and the binding capacity of Fe3O4/MPS@PAA/NTA-Ni2+ was 93.4 mg/g. Compared with High-Affinity Ni-NTA Resin and Ni-NTA Magnetic Agarose Beads, Fe3O4/MPS@PAA/NTA-Ni2+ nanocomposites exhibited higher separation efficiency and binding capacity towards His-tagged GFP. Moreover, the selectivity and recyclability of them for the target proteins were maintained well after six cycles. This study would widen the application of PAA in constructing multifunctional nanocomposites for biomedical fields.

Versatile modules enable automated multi-column purifications on the ÄKTA pure chromatography system.

Protein purification processes in basic research using ÄKTA™ liquid chromatography systems are often limited to single sample injections and simple one-column purifications. Because many target proteins in structural biology require complex purification protocols the work easily becomes laborious. To streamline and accelerate downstream protein production, an ALIAS™ autosampler and a modular sample in-line dilution process coupled to ion-exchange chromatography were incorporated into the workflow to automate two of the most commonly performed purification strategies - ion-exchange to size exclusion and nickel-ion metal affinity to size exclusion. The chromatographic setup enabled purification of a large array of cytosolic and membrane proteins from small-scale expression cultures produced in insect cells necessary to develop and optimize isotope-labeling strategies for nuclear magnetic resonance spectroscopy applications, resulting in a reduction in experiment time of about 20% per run for both cytosolic and membrane protein purification schemes. However, when queuing multiple samples the throughput increased by 66% and 75%, respectively. In addition, a novel system configuration is presented, where two column valves can be operated independently. This allows for the design of purification loops to increase purity of the target protein.

Affinity Based Nano-Magnetic Particles for Purification of Recombinant Proteins in Form of Inclusion Body

Background: Protein purification is the most complicated issue in the downstream processes of recombinant protein production; therefore, improved selective purification methods are important. Affinity-based protein purification method using polyhistidine-tag (His-tag) and nickel-nitrilotriacetic acid (Ni-NTA) resins is one of the most common strategies. Magnetic nanoparticles (MNPs) can be used as a beneficial alternative for Ni-NTA resins. However, there is no data on the capability of MNPs for protein purification from inclusion bodies; this issue is studied here. Methods: Recombinant His-tagged proteins of enhanced green fluorescent protein (EGFP)-His and streptokinase (SK)-His were expressed in E. coli BL-21 (DE3) in soluble and inclusion body forms, respectively. MNPs including Fe3O4 magnetic core, SiO2 shell, and Ni2+ on the surface were synthesized by sol-gel and hydrothermal reactions and then characterized by X-ray powder diffraction, vibrating sample magnetometer, and scanning electron microscopy imaging. Both synthesized Fe3O4@NiSiO3 and Fe3O4@NixSiOy MNPs were employed to purify EGEP-His and SK-His under native and denaturing conditions, respectively. The quantity and purity of purified proteins were analyzed by micro-Bradford assay and SDS-PAGE, respectively. Results: Both synthesized MNPs were spherical and well-dispersed with the size ranging from 290 to 415 nm. Synthesized MNPs contained Fe3O4, SiO2 shell, and Ni2+ on their structures with suitable magnetization properties. Using Fe3O4@NiSiO3 and Fe3O4@NixSiOy yielded 192 and 188 µg/mg of SK-His, as compared to 207 and 195 µg/mg of EGFP-His, respectively. Conclusion: MNPs containing magnetic Fe3O4 core, SiO2 shell, and Ni2+on their surface are versatile alternatives for Ni-NTA resins in protein purification for proteins expressed in both soluble and inclusion body forms.

Purification of the recombinant green fluorescent protein from tobacco plants using alcohol/salt aqueous two-phase system and hydrophobic interaction chromatography.

BACKGROUND: The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. RESULTS: The recombinant GFP was transiently expressed in an active form in agoinoculated Nicotiana benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~ 60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~ 4 h and can recover 34.1% of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics. CONCLUSIONS: The strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP.

Genetically Encoded Fluorescence/Bioluminescence Bimodal Indicators for Ca2+ Imaging.

Fluorescent and bioluminescent genetically encoded Ca2+ indicators (GECIs) are an indispensable tool for monitoring Ca2+ dynamics in numerous cellular events. Although fluorescent GECIs have a high spatiotemporal resolution, their application is often confined to short-term imaging due to the external illumination that causes phototoxicity and autofluorescence from specimens. Bioluminescent GECIs overcome these pitfalls with enhanced compatibility to optogenetic manipulation and photophysiological processes; however, they are compromised for spatiotemporal resolution. Therefore, there has been a push toward the use of Ca2+ indicators that possess the advantages of both fluorescent and bioluminescent GECI for a wide range of applications. To address this, we developed a high-affinity bimodal GECI, GLICO, using a single fluorescent protein-based GECI combined with a split luciferase. Through this novel design, the fusion protein becomes bimodal and possesses Ca2+ sensing properties similar to those of its fluorescent ancestor and confers bioluminescence-based Ca2+ imaging. GLICO in bioluminescence mode has the highest dynamic range (2200%) of all bioluminescent GECIs. We demonstrated the performance of GLICO in studying cytosolic Ca2+ dynamics in different cultured cells in each mode. With the purpose of Ca2+ imaging in high Ca2+ content organelle, we also created a low-affinity variant, ReBLICO and performed Ca2+ imaging of the ER in both fluorescence and bioluminescence modes. The ability to switch between fluorescence and bioluminescence modes with a single indicator would benefit transgenic applications by presenting an opportunity for a wide range of live Ca2+ imaging in physiological and pathophysiological conditions.

Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli.

Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the ε-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfish Aequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems.

Designing an ELP-intein system: toward a more realistic outlook.

Despite the ever-growing demand for proteins in pharmaceutical applications, downstream processing imposes many technical and economic limitations to recombinant technology. Elastin-like polypeptides tend to aggregate reversibly at a specific temperature. These biopolymers have been joined with self-cleaving inteins to develop a non-chromatographic platform for protein purification without the need for expensive enzymatic tag removal. Following the design and expression of an ELP-intein-tagged GFP, herein, we report certain complications and setbacks associated with this protein purification system, overlooked in previous studies. Based on our results, a recovery rate of 68% was achieved using inverse transition cycling. Fluorescence intensity analysis indicated a production yield of 11 mg GFP fusion protein per liter of bacterial culture. The low expression level is attributable to several factors, such as irreversible aggregation, slipped-strand mispairing or insufficiency of aminoacyl tRNAs during protein translation of the highly repetitive ELP tag. While the goals we set out to achieve were not entirely met, a number of useful tips could be gathered as a generic means for implementing ELP-intein protein purification. Overall, we believe that such reports help clarify the exact capacity of emerging techniques and build a fairly realistic prospect toward their application.

CRISPR/Cas9-mediated generic protein tagging in mammalian cells.

Systematic protein localization and protein-protein interaction studies to characterize specific protein functions are most effectively performed using tag-based assays. Ideally, protein tags are introduced into a gene of interest by homologous recombination to ensure expression from endogenous control elements. However, inefficient homologous recombination makes this approach difficult in mammalian cells. Although gene targeting efficiency by homologous recombination increased dramatically with the development of designer endonuclease systems such as CRISPR/Cas9 capable of inducing DNA double-strand breaks with unprecedented accuracy, the strategies still require synthesis or cloning of homology templates for every single gene. Recent developments have shown that endogenous protein tagging can be achieved efficiently in a homology independent manner. Hence, combinations between CRISPR/Cas9 and generic tag-donor plasmids have been used successfully for targeted gene modifications in mammalian cells. Here, we developed a tool kit comprising a CRISPR/Cas9 expression vector with several EGFP encoding plasmids that should enable tagging of almost every protein expressed in mammalian cells. By performing protein-protein interaction and subcellular localization studies of mTORC1 signal transduction pathway-related proteins expressed in HEK293T cells, we show that tagged proteins faithfully reflect the behavior of their native counterparts under physiological conditions.

zGrad is a nanobody-based degron system that inactivates proteins in zebrafish.

The analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane and cytosolic proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity, while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.

Protein fishing from single live cells.

Intracellular protein and proteomic studies using mass spectrometry, imaging microscopy, flow cytometry, or western blotting techniques require genetic manipulation, cell permeabilization, and/or cell lysis. We present a biophysical method that employs a nanoaspirator to 'fish' native cytoplasmic or nuclear proteins from single mammalian cells, without compromising cell viability, followed by ex cellulo quantitative detection. Our work paves the way for spatiotemporally-controlled, quantitative, live, single-cell proteomics.

Selective release of overexpressed recombinant proteins from E. coli cells facilitates one-step chromatographic purification of peptide-tagged green fluorescent protein variants.

The need for fast and convenient protein purification is enormous, not only on the industrial scale for the production of therapeutics but also in life science research. Most protein purification strategies require multiple steps, which are time consuming, cost and resource intensive. Selective release of the intracellular target product can considerably facilitate downstream processing by minimizing the HCP concentration before subsequent purification processes. Here, we present a simple method to selectively release soluble overexpressed proteins from E. coli with minimal laboratory equipment requirements. This method is based on the creation of pores in the cell envelope by a single freeze-thaw cycle. Proteins of various sizes were released by several 50 mM Tris pH 9.5 buffer washes with high yields. Consequently, the need for detergents and other additives were circumvented. Green fluorescent protein (GFP), was tagged with peptides of various charges and polarity. Releasing these GFP variants with our method enabled the successful one-step chromatographic purification on a quaternary amine resin despite the different isoelectric points of the target proteins. Up to 90% of the target protein elution peak during anion exchange chromatography was of >95% purity. This optimized process is more productive compared to multi-step purification procedures currently available in literature.