Spatio-Temporal Photoactivation of Cytotoxic Proteins

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.     

Engineered pH-inducible intein Mtu ΔI-CM variants with markedly reduced premature cleavage activity

Inteins have been widely exploited for the purification of tagless proteins. Among them, pH-inducible C-terminal-cleavage inteins enable the preparation of proteins and peptides with an authentic N-terminus. However, a severe premature cleavage around neutral pH has limited the application of these inteins, especially when used in recombinant hosts such as Escherichia coli. By targeting the microenvironment of the two key histidine residues H429 and H439, we engineered Mtu ΔI-CM intein to markedly reduce its premature cleavage. Kinetic analyses suggested that although the variants retained the pH dependence, they indeed cleaved slower, especially at pH 7.6. These variants resulted in higher yields for two model polypeptides than the original Mtu ΔI-CM intein, when used in conjunction with a cleavable self-assembling tag. This work suggests that more controllable pH-inducible inteins can be obtained by manipulating the residues in the self-cleavage sites and provide better performance for tag-based protein preparation strategies.     

Efficient Generation of Hydrazides in Proteins by RadA Split Intein

Protein C-terminal hydrazides are useful for bioconjugation and construction of proteins from multiple fragments through native chemical ligation. To generate C-terminal hydrazides in proteins, an efficient intein-based preparation method has been developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method has been expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. It is expected that this versatile preparation method will expand the utilization of protein C-terminal hydrazides in protein preparation and modification.     

Cover Picture: Semisynthesis and Characterization of the First Analogues of Pro‐Neuropeptide Y (ChemBioChem 5/2003)

The cover picture shows how a combination of recombinant synthesis and chemical synthesis has been used to obtain chemically modified proteins. N‐terminal protein segments of pro‐neuropeptide Y (proNPY) were produced as intein‐fusion proteins in Escherischia coli in order to obtain thioesters. C‐terminal segments were synthesized by parallel automated peptide synthesis and derivatized to obtain carboxyfluorescein‐ (CF) and biotin‐labeled peptides. Native chemical ligation yielded chemically modified full‐length analogues of proNPY that can be used to monitor the biosynthesis of neuropeptide Y. Futher information can be found in the article by Beck‐Sickinger and co‐workers on p. 425 ff.     

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (K_a of 2.45×10⁵ M ^?1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.     

Affinity proteins and their generation

Engineered affinity proteins have, together with antibodies and antibody derivatives, become indispensable tools in many areas of life science and with an increasing number of applications. The need for high‐throughput methods for generation of these different affinity proteins is evident. Today, combinatorial protein engineering is the most successful strategy to generate novel affinity proteins of non‐immunoglobulin origin. In this approach, high‐complexity combinatorial libraries are constructed from which affinity proteins are isolated using appropriate selection methods, thus circumventing the need for detailed knowledge of the protein structure and the binding mechanism that is necessary in more rational approaches. Since the introduction of the phage display technology, several alternative selection systems have been developed for this purpose. This review presents briefly some of the more commonly used affinity proteins, and gives an overview of the different methods and challenges related to the generation of library diversity and the selection methods available for the isolation of affinity proteins with desired properties. Copyright © 2012 Society of Chemical Industry     

Starch-Coated Magnetic Iron Oxide Nanoparticles for Affinity Purification of Recombinant Proteins

Starch-coated magnetic iron oxide nanoparticles have been synthesized by a simple, fast, and cost-effective co-precipitation method with cornstarch as a stabilizing agent. The structural and magnetic characteristics of the synthesized material have been studied by transmission electron microscopy, Mössbauer spectroscopy, and vibrating sample magnetometry. The nature of bonds between ferrihydrite nanoparticles and a starch shell has been examined by Fourier transform infrared spectroscopy. The data on the magnetic response of the prepared composite particles have been obtained by magnetic measurements. The determined magnetic characteristics make the synthesized material a good candidate for use in magnetic separation. Starch-coated magnetic iron oxide nanoparticles have been tested as an affinity sorbent for one-step purification of several recombinant proteins (cardiac troponin I, survivin, and melanoma inhibitory activity protein) bearing the maltose-binding protein as an auxiliary fragment. It has been shown that, due to the highly specific binding of this fragment to the starch shell, the target fusion protein is selectively immobilized on magnetic nanoparticles and eluted with the maltose solution. The excellent efficiency of column-free purification, high binding capacity of the sorbent (100–500 µg of a recombinant protein per milligram of starch-coated magnetic iron oxide nanoparticles), and reusability of the obtained material have been demonstrated.     

Harnessing Split-Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells

Biochemical studies of integral membrane proteins are often hampered by low purification yields and technical limitations such as aggregation causing in vitro manipulations to be challenging. The ability of controlling proteins in live cells bypasses these limitations while broadening the scope of accessible questions owing to the proteins being in their native environment. Here we take advantage of the intein biorthogonality to mammalian systems, site specificity, fast kinetics, and auto-processing nature as an attractive option for modifying surface proteins. Using EGFR as a model, we demonstrate that the split-intein pair Ava^N/Npu^C can be used to efficiently and specifically modify target membrane proteins with a synthetic adduct for downstream live cell application.     

Reactivity of intein thioesters: appending a functional group to a protein

The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein-mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small-molecule thioester revealed that the alpha-hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an alpha-hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3-dipolar azide-alkyne cycloaddition. Thus, the mechanism of intein-mediated protein splicing provides the means to install a useful functional group at a specific site-the C terminus-of virtually any protein.     

Site-specific chemical modification of proteins with a prelabelled cysteine tag using the artificially split Mxe GyrA intein

The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine-containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl-reactive probe. In a second step, protein trans-splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein-labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site-specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine-reactive probes with a straightforward route for their site-specific incorporation even into complex and cysteine-rich proteins.     

Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.     

Preparation of a modified crosslinked chitosan/polyvinyl alcohol blended affinity membrane for purification of His-tagged protein

Based on a crosslinked chitosan (CS)/polyvinyl alcohol (PVA) matrix membrane, an immobilized metal ion affinity membrane (IMAM) using Cu²⁺ and Ni²⁺ ions as affinity ligands was prepared for purification of the His-tagged recombinant protein. The affinity membrane possessed a favorable membrane structure including 1.39 μm average pore size and 0.33 mL·cm⁻²·s⁻¹ water flux under 0.08 MPa pressure at 25 °C. The Cu²⁺ and Ni²⁺ ions capacities immobilized on the IMAM were 155.6 and 137.3 μmol·disk⁻¹, respectively. The IMAM had an excellent specific affinity to His-tagged protein. About 10-fold purification factor for the model protein was obtained in a batch adsorption, and serine hydroxymethyl transferase could be purified to a single band in sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis from its crude extract solution with an affinity membrane cartridge by a dynamic purification process. This work provides a promising IMAM for the purification of His-tagged recombinant proteins. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47347.     

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.     

Recent Progress in Strategies for the Creation of Protein‐Based Fluorescent Biosensors

The creation of novel bioanalytical tools for the detection and monitoring of a range of important target substances and biological events in vivo and in vitro is a great challenge in chemical biology and biotechnology. Protein‐based fluorescent biosensors—integrated devices that convert a molecular‐recognition event to a fluorescent signal—have recently emerged as a powerful tool. As the recognition units various proteins that can specifically recognize and bind a variety of molecules of biological significance with high affinity are employed. For the transducer, fluorescent proteins, such as green fluorescent protein (GFP) or synthetic fluorophores, are mostly adopted. Recent progress in protein engineering and organic synthesis allows us to manipulate proteins genetically and/or chemically, and a library of such protein scaffolds has been significantly expanded by genome projects. In this review, we briefly describe the recent progress of protein‐based fluorescent biosensors on the basis of their platform and construction strategy, which are primarily divided into the genetically encoded fluorescent biosensors and chemically constructed biosensors.     

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of non-redundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.     

A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants

A novel gene fusion system to express and purify small recombinantproteins in Escherichia coli has been constructed. The conceptallows for affinity purification of soluble gene products bysequential albumin- and Zn²+-affinity chromatography. The dual-affinitysystem is well suited for expression of unstable proteins asonly full-length protein is obtained after purification andproteins gain proteolytic stability in the fusion protein. Herewe show that the dual-affinity approach can be used for theexpression of various unstable derivatives of a single IgG-bindingdomain based on staphylococcal protein A. Analysis of the proteolyticstabilities and the IgG-binding properties of the differentmutant proteins suggest that the model for the structure ofan IgG-binding domain must be re-evaluated.     

A Split‐Intein‐Based Method for the Efficient Production of Circularized Nanodiscs for Structural Studies of Membrane Proteins

Phospholipid nanodiscs are a native‐like membrane mimetic that is suitable for structural studies of membrane proteins. Although nanodiscs of different sizes exist for various structural applications, their thermal and long‐term stability can vary considerably. Covalently circularized nanodiscs are a perfect tool to overcome these limitations. Existing methods for the production of circularized nanodiscs can be time‐consuming and technically demanding. Therefore, an easy in vivo approach, in which circularized membrane scaffold proteins (MSPs) can be directly obtained from Escherichia coli culture, is reported herein. Nostoc punctiforme DnaE split‐intein fusions with MSPs of various lengths are used and consistently provide circularized nanodiscs in high yields. With this approach, a large variety of circularized nanodiscs, ranging from 7 to 26 nm in diameter, that are suitable for NMR spectroscopy and electron microscopy (EM) applications can be prepared. These nanodiscs are superior to those of the corresponding linear versions in terms of stability and size homogeneity, which affects the quality of NMR spectroscopy data and EM experiments. Due to their long‐term stability and homogeneity, the presented small circular nanodiscs are suited for high‐resolution NMR spectroscopy studies, as demonstrated with two membrane proteins of 17 or 32 kDa in size. The presented method will provide easy access to circularized nanodiscs for structural studies of membrane proteins and for applications in which a defined and stable nanodisc size is required.     

Chemical Protein Modification through Cysteine

The modification of proteins with non‐protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill‐defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site‐selective modification, including a unique nucleophilicity, and a low natural abundance—both allowing chemo‐ and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.     

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples.