His-tagged GFP Research Articles

GFP fusion promotes the soluble and active expression of a pea actin isoform (PEAc1) in Escherichia coli

In the present study, we report that a GFP fusion tag facilitated the soluble expression of a pea actin isoform (PEAc1) in E. coli. To investigate the influence of a GFP fusion tag on PEAc1 structure and activity, PEAc1, His-tagged PEAc1 (His-PEAc1), His-tagged GFP (His-GFP), and His-tagged PEAc1 fusion with GFP (His-PEAc1-GFP) were expressed in E. coli. SDS-PAGE and western blot analyses reveal that the solubility of His-PEAc1-GFP was higher than that of PEAc1 and His-PEAc1. The His-PEAc1-GFP and His-GFP fusion proteins were purified from the supernatant of cell homogenate on a Ni-affinity column, and PEAc1 and His-PEAc1 were purified from inclusion bodies. CD spectrum analysis of the four purified proteins indicated that the proportion of α-helix and β-sheet in PEAc1 was closest to the predicted data in His-PEAc1-GFP (compared with His-PEAc1 or PEAc1). In addition, the actin-associated activities of His-PEAc1-GFP, including polymerization to microfilaments under specific ionic conditions and DNase I inhibition by monomers, were more similar to those of muscle actin (compared with PEAc1 and His-PEAc1). These improvements in PEAc1 solubility and activity are likely the result of correct PEAc1 folding mediated by GFP fusion.

Proteomics analysis of host cell proteins after immobilized metal affinity chromatography: Influence of ligand and metal ions

Different degrees of protein purity have been observed in immobilized metal affinity chromatography ranging from extremely high purity to moderate and low purity. It has been hypothesized that the host cell protein composition and the metal ligands are factors governing the purity of a protein obtained after immobilized metal affinity chromatography (IMAC). Ni nitrilotriacetic acid (NTA) has become the first choice for facile His-tagged protein purification, but alternative ligands such as iminodiacetic acid (IDA) with other immobilized metal ions such as Zn, Cu and Co are valuable options when the expected purity or binding capacity is not reached. Especially Cu and Zn are very attractive, due to their reduced environmental and safety concerns compared to Ni. Co and Zn are more selective than Ni and Cu. This increased selectivity comes at the cost of weaker binding. In this work, the influence of ligand choice on protein purity after IMAC was evaluated by several methods, including peptide mapping. His-tagged GFP was used as model protein. We found that host cell protein (HCP) content varies drastically between ligands, as IDA eluates generally showing higher HCP concentrations than NTA. The relative content of the key amino acids His, Cys and Trp in the sequence of the co-eluted protein does not suffice to explain co-eluting propensity. The co-elution of HCPs is mostly influenced by metal binding clusters on the protein surface and not by total content or surface concentration of metal interacting amino acids. Prediction of co-elution is not dependent on these clusters alone, due to protein-protein interactions, indicted by a relative low metal binding cluster score but high co-elution propensity and in a lot of cases these proteins are often part of complex such as ribosome and chaperones. The different co-eluting proteins were presented by a heatmap with a dendrogram. Ward's linkage method was used to calculate the distance between groups of co-eluting proteins. Clustering of co-eluting HCPs was observed according to ligand and by metal ions, with Zn and Co forming one cluster and Ni and Cu another. The co-elution of host cell proteins can be explained by clusters of metal interacting amino acids on the protein surface and by protein-protein interactions. While Ni NTA still appears to be highly advantageous, it might not be the cure-all for all applications.

Fe3O4@PAM@NTA-Ni2+ Magnetic Composite Nanoparticles for Highly Specific Separation of His-tagged Proteins

A facile approach has been developed to synthesize Fe3O4@PAM (polyacrylamide) nanoparticles (NPs) with carboxyl groups on the surfaces by copolymerization with acrylamide and acrylic acid in Fe3O4 NPs aqueous suspension. Nitrilotriacetic acid (NTA) was conjugated to the magnetic NPs via well-known carboniimide chemistry using EDC and NHS. The Ni2+ ions loaded on the surface of NPs provide abundant docking sites for immobilization of His-tagged green fluorescent proteins (His-tagged GFP). The high magnetic property of Fe3O4@PAM@NTA-Ni2+ allows an easy separation of the NPs from solution under an external magnetic field, with high His-tagged protein binding capacity (42 μg protein/mg of NPs). The NPs can be recycled for at least four times without significant loss of binding capacity to proteins. These materials show great potential to separate His-tagged protein with low-cost purification at industrial scale.

The development of a single molecule fluorescence standard and its application in estimating the stoichiometry of the nuclear pore complex

We report here an image-based method to quantify the stoichiometry of diffraction-limited sub-cellular protein complexes in vivo under spinning disk confocal microscopy. A GFP single molecule fluorescence standard was first established by immobilizing His-tagged GFP molecules onto the glass surface via nickel nitrilotriacetic acid functionalized polyethylene glycol. When endogenous nucleoporins were knocked down and replaced by the exogenously expressed and knockdown-resistant GFP-nucleoporins, the stoichiometry of the nucleoporin was estimated by the ratio of its fluorescence intensity to that of the GFP single molecules. Our measured stoichiometry of Nup35, Nup93, Nup133 and Nup88 is 23, 18, 14 and 9 and there are possibly16 copies of Nup107-160 complex per nuclear pore complex.

Kinetic Master Equation Modeling of an Artificial Protein Pump

Biological ion channels and pumps have inspired many efforts to generate artificial counterparts due to their fundamental importance and practical applications. Recent work by Hinds et al. (Adv. Funct. Mater. 2014, 24, 4317–4323) demonstrated a selective protein pump based on an artificial nanopore in which the pump cycles are based on selective binding and release of His-tagged GFP protein ions to a NTA–Ni2+ transition metal complex. In this work, a kinetic master equation method is presented to model this pump and to establish the origin of the variation of selectivity with concentration of imidazole that is involved in the release mechanism. A two-site model with multiple species of proteins is used to mimic the pump mechanism under nonequilibrium conditions. Numerical simulation qualitatively reproduces the experimental selectivity, and uncovers essential ingredients of the pump mechanism. In addition, the binding mechanism between His-tags and NTA–Ni2+ is investigated using molecular dynamics simulat...

Facial synthesis of nickel(II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins.

Immobilized metal affinity chromatography (IMAC) technique is frequently used in the purification of histidine-tagged (His-tagged) recombinant proteins. In this study, nickel(II)-immobilized carboxyl cotton chelator (CCC-Ni2+) fibers was synthesized by a simple method based on the coordination effect between Ni2+ and carboxyl group. The nickel content of the CCC-Ni2+ fibers was determined to be 5 times larger than that of Ni2+-immobilized sulfhydryl cotton fiber (SCF-Ni2+) fibers developed in our previous work. The prepared CCC-Ni2+ fibers were then applied for the selective and rapid separation of His-tagged protein from escherichia coli (E. coli) cell lysates on the basis of the high affinity of Ni2+ to 6×His with a lab-in-syringe format. Benefiting from the good biological compatibility and high nickel content, the results showed that CCC-Ni2+ fibers were able to selectively capture His-tagged proteins from complex E. coli cell lysates and exhibited a relatively large adsorption capacity toward His-tagged protein. The recoveries of His-tagged GFP in E. coli cell lysates were in the range of 89.8%-106.7% with the relative standard deviations (RSDs) less than 9.4% (intra-day) and 10.3% (inter-day). Taken together, this efficient approach for the purification of recombinant proteins extends the application of CCC-based fibrous materials in biological analysis.

The 5 kDa protein NdhP is essential for stable NDH-1L assembly in Thermosynechococcus elongatus.

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from Thermosynechococcus elongatus two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ∼450 kDa NDH-1L complex, that is involved in respiration and CET at high CO2 concentration, and not detectable in the NDH-1MS and NDH-1MS' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a ΔndhP::Km mutant.

Multivalent (Nitrilotriacetic Acid)-End-Functionalized Polystyrenes by ATRP and Their Self-Assembly

The synthesis of tri-(nitrilotriacetic acid) (NTA)-end-functionalized polystyrenes using an initiator containing tert-butyl protected NTA moieties, by atom transfer radical polymerization (ATRP) of styrene is described. First, a suitable ATRP initiator is prepared and subsequently characterized by 1H and 13C NMR spectroscopy, gel-permeation chromatography (GPC), and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectro­scopy. The structures of the tri-NTA-end-functionalized polystyrenes (tri-NTA-PS) are confirmed by 1H and 13C NMR spectroscopy. Tri-NTA-PS itself produces self-assembled spherical aggregates with ≈40–60 nm diameters in water/THF, whereas nickel-complexed tri-NTA-PS produces spherical core–shell hybrid aggregates with ≈90–115 nm diameters with His-tagged GFP in water/DMF (DMF 4 vol%), as confirmed by transmission electron microscopy and dynamic light scattering measurements.

In situ formation of polymer–protein hybrid spherical aggregates from (nitrilotriacetic acid)-end-functionalized polystyrenes and His-tagged proteins

Polymer–protein hybrid nanostructures are important for their potential applications in pharmaceuticals and biotechnology, and new methods to generate hybrid nanostructures would be advantageous in the above fields. In this study, we present a simple method for the construction and control of polymer–protein hybrid spherical aggregates in situ from nickel complexed (nitrilotriacetic acid)-end-functionalized polystyrene (Ni-NTA-PS) and histidine-tagged green fluorescent protein (His-tagged GFP) through NTA-Ni–His interaction in water–DMF (DMF 4 vol.%) at physiological pH. The generality of the approach was demonstrated using His-tagged GFP and His-tagged lipases. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements revealed that hybrid aggregates of His-tagged GFP were stable up to 15 days. The size of hybrid aggregates depended on the molecular weight of polymers, concentration of polymers, concentration of protein, and also the rate of addition of polymer to the solvent containing protein. After formation of hybrid aggregates, removal of DMF by dialysis from the system resulted in the aggregation and phase separation of aggregates over time. The size of polymer–protein hybrid aggregates decreased with excess of imidazole addition. A possible mechanism for the formation of such polymer–protein hybrid aggregates is also described.

Development of a Novel Membrane Aerated Hollow‐Fiber Microbioreactor

A new challenge in biotechnological processes is the development of flexible bioprocessing platforms, allowing strain selection, facilitating scale-up and integrating separation steps. Miniaturization of such a cultivation system allows parallel use and the saving of resources but makes the supply of oxygen to the cells difficult. In this work we present a membrane aerated hollow-fiber microbioreactor (HFMBR) which consists of an acrylic glass module equipped with two different types of membrane fibers. Fibers of polyethersulfone and polyvinyldifluoride were used for substrate and oxygen supply, respectively. Cultivation of E. coli as model organism and production of His-tagged GFP were carried out in the extracapillary space of the membrane aerated HFMBR and compared with cultivations in shaking flask which are commonly used for screening experiments. The measurement of the oxygen transfer capacity and the online monitoring of the dissolved oxygen during the cultivation were performed using a fiber optic oxygen sensor. Online measurement of the optical density was also integrated to the bioreactor. Due to efficient oxygen transfer, a better cell growth than in the shaking flask experiments was achieved, while no negative influence on the GFP productivity was observed in the membrane aerated bioreactor. Thus the feasibility of a future integrated downstreaming could also be demonstrated.