Expression of intein‐tagged fusion protein and its applications in downstream processing

The conventional methods of downstream purification of a recombinant protein are not only complicated and delicate but time consuming, and need to be improved. Since the intein, the protein splicing element, was discovered, this self‐cleaving element has been exploited and applied to the purification of recombinant proteins which could significantly simplify the purification procedure. Intein has the unique property that when it is combined with an affinity tag, it enables a target protein to be purified in a single chromatographic step. This review elucidates the properties of intein (the mechanism that unravels the intein‐based protein splicing), the advantages of an intein affinity expression system, the progress of intein‐based protein purification procedures, and recent advances in the applications of intein. Further development of the intein‐based purification system may lead to the applications of this system to industrial‐scale production of recombinant proteins. Copyright © 2009 Society of Chemical Industry     

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.     

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.     

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.     

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.     

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.     

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.     

Split Inteins: Nature’s Protein Ligases

Split inteins carry out a naturally occurring process known as protein trans-splicing, where two protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. In the past thirteen years since their discovery, chemists and biologists have utilized split inteins in exogenous contexts for a number of biotechnological applications centered around the formation of native peptide bonds. While many protein trans-splicing technologies have emerged and flourished in recent years, several factors still limit their wide-spread practical use. Here, we discuss the development, applications, and limitations of split intein-based technologies and propose that further advancement in this field will require a more fundamental understanding of split intein structure and function.     

Bacterial Cell-Surface Display of Semisynthetic Cyclic Peptides

Semisynthetic cyclic peptides containing both non-proteinogenic building blocks, as the synthetic part, and a genetically encoded sequence amenable to DNA-based randomization hold great potential to expand the chemical space in the quest for novel bioactive peptides. Key to an efficient selection of novel binders to biomacromolecules is a robust method to link their genotype and phenotype. A novel bacterial cell surface display technology has been developed to present cyclic peptides composed of synthetic and genetically encoded fragments in their backbones. The fragments were combined by protein trans-splicing and intramolecular oxime ligation. To this end, a split intein half and an unnatural amino acid were displayed with the genetically encoded part on the surface of Escherichia coli. Addition of the synthetic fragment equipped with the split intein partner and an aminooxy moiety, as well as the application of a pH-shift protocol, resulted in the onsurface formation of the semisynthetic cyclic peptide. This approach will serve for the generation of cyclic peptide libraries suitable for selection by fluorescence-activated cell sorting, and more generally enables chemical modification of proteins on the bacterial surface.     

Unambiguous Assignment of Short‐ and Long‐Range Structural Restraints by Solid‐State NMR Spectroscopy with Segmental Isotope Labeling

We present an efficient method for the reduction of spectral complexity in the solid‐state NMR spectra of insoluble protein assemblies, without loss of signal intensity. The approach is based on segmental isotope labeling by using the split intein DnaE from Nostoc punctiforme. We show that the segmentally ¹³C,¹⁵N‐labeled prion domain of HET‐s exhibits significantly reduced spectral overlap while retaining the wild‐type structure and spectral quality. A large number of unambiguous distance restraints were thus collected from a single two‐dimensional ¹³C,¹³C cross‐correlation spectrum. The observed resonances could be unambiguously identified as intramolecular without the need for preparing a dilute, less sensitive sample.     

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 “Quenchergenic” Chemoselective Protein Labeling Strategy

Dynamic changes in protein structure can be monitored by using a fluorescent probe and a dark quencher. This approach is contingent upon the ability to precisely introduce a fluorophore/quencher pair into two specific sites of a protein of interest. Despite recent advances, there is continued demand for new and convenient approaches to site-selectively label proteins with such optical probes. We have recently developed a chemoselectively rapid azo-coupling reaction (CRACR) for site-specific protein labeling; it relies on rapid coupling between a genetically encoded 5-hydroxytryptophan residue and various aromatic diazonium ions. Herein, it is reported that the product of this conjugation reaction, a highly chromophoric biarylazo group, is a potent fluorescence quencher. The absorption properties of this azo product can be tuned by systematically altering the structure of the aryldiazonium species. A particular “quenchergenic” aryldiazonium has been identified that, upon conjugation, efficiently quenches the fluorescence of green fluorescent protein, which is a widely used genetically encoded fluorescent probe that can be terminally attached to target proteins. This fluorophore/quencher pair was used to evaluate the protein-labeling kinetics of CRACR, as well as to monitor the proteolysis of a fusion protein.     

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.     

Protein‐Specific Imaging of O‐GlcNAcylation in Single Cells

Thousands of intracellular proteins are post‐translationally modified with O‐GlcNAc, and O‐GlcNAcylation impacts the function of modified proteins and mediates diverse biological processes. However, the ubiquity of this important glycosylation makes it highly challenging to probe the O‐GlcNAcylation state of a specific protein at the cellular level. Herein, we report the development of a FLIM–FRET‐based strategy, which exploits the spatial proximity of the O‐GlcNAc moiety and the attaching protein, for protein‐specific imaging of O‐GlcNAcylation in single cells. We demonstrated this strategy by imaging the O‐GlcNAcylation state of tau and β‐catenin inside the cells. Furthermore, the changes in tau O‐GlcNAcylation were monitored when the overall cellular O‐GlcNAc was pharmacologically altered by using the OGT and OGA inhibitors. We envision that the FLIM–FRET strategy will be broadly applicable to probe the O‐GlcNAcylation state of various proteins in the cells.     

Site-specific protein modification on living cells catalyzed by Sortase

The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incubation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides a powerful tool for cell biology and cell surface engineering.     

Considerations on Probe Design for Affinity-Guided Protein Conjugation

The plethora of methods developed for the creation of protein conjugates often differs significantly with regard to the heterogeneity of the resulting products, in the degree of genetic manipulation of the protein required, and in the technical skills required to perform the conjugation procedure. Affinity-guided protein conjugation is a protein labeling methodology based on noncovalent binding interactions between a labeling probe and the protein of interest. These interactions increase the local concentration of a reactive group in the probe on the protein surface thus facilitating the conjugation in proximity of the complexation site. The ability to produce high-quality conjugates from nongenetically modified proteins both in vitro, but also in cells, demonstrates the power of affinity-guided protein conjugation. Here, we present the progress of affinity-guided protein conjugation in relation to selective protein labeling in living systems and the formation of high-quality protein conjugates. Furthermore, the probe design will be discussed in relation to the utility of the probe for labeling in vitro or in living systems.     

Cyclic Peptides Made by Linking Synthetic and Genetically Encoded Fragments

Cyclic peptides can be highly valuable as bioactive molecules, both for biomedical applications and in basic research. We introduce a new fragment‐based approach to access cyclic peptide structures in which one fragment is of synthetic origin and the other is genetically encoded. The synthetic peptide, which can contain one or more non‐proteinogenic building blocks, is coupled to the recombinantly expressed peptide through two bonds, one formed by protein trans‐splicing with a split intein and the other by oxime ligation. Semisynthetic macrocycles were obtained with high efficiency for various sequences and ring sizes; they can be prepared in quantities sufficient for initial bioactivity tests. We also prepared lipidated and d ‐amino‐acid‐containing peptides that were inspired by the peptide antibiotic daptomycin. Such structures are not accessible by other methods that harness the power of simple genetic diversification in the DNA‐encoded part of the peptide.     

Heterodimer Formation of the Homodimeric ABC Transporter OpuA

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.     

Protein Ligation-Assisted Reconstitution of Split HRP Fragments for Facile Production of HRP Fusion Proteins in E. coli

Horseradish peroxidase (HRP) is a pivotal biocatalyst for biosensor development and fine chemical synthesis. HRP proteins are mostly extracted and purified from the roots of horseradish because the solubility and productivity of recombinant HRP in bacteria are significantly low. In this study, we investigate the reconstitution system of split HRP fragments to improve its soluble expression levels in E. coli allowing the cost-effective production of bioactive HRPs. To promote the effective association between two HRP fragments (HRPn and HRPc), we exploit SpyTag-SpyCatcher chemistry, a versatile protein coupling method with high affinity and selectivity. Each HRP fragment was genetically fused with SpyTag and SpyCatcher, respectively, exhibiting soluble expression in the E. coli cytoplasm. The engineered split HRPs were effectively and irreversibly reconstituted into a biologically active and stable assembly that can catalyze intrinsic enzymatic reactions. Compared to the chaperone co-expression system, our approach shows that the production yield of soluble HRP is comparable, but the purity of the final product is relatively high. Therefore, our results can be applied to the high-yield production of recombinant HRP variants and other difficult-to-express proteins in bacteria without complex downstream processes.     

Chimeras of the homing endonuclease PI-SceI and the homologous Candida tropicalis intein: a study to explore the possibility of exchanging DNA-binding modules to obtain highly specific endonucleases with altered specificity

Homing endonucleases are extremely specific endodeoxyribonucleases. In vivo, these enzymes confer mobility on their genes by inducing a very specific double-strand cut in cognate alleles that lack the cooling sequence for the homing endonuclease; the cellular repair of the double-strand break with the endonuclease-containing allele as a template leads to integration of the endonuclease gene, completing the homing process. As a result of their extreme sequence specificity, homing endonucleases are promising tools for genome engineering. For this purpose, it is desirable to design enzymes with defined new specificities. To analyse which DNA-binding elements are potential candidates for use in the design of enzymes with modified or even new specificity, we produced several chimeric proteins derived from the Saccharomyces cerevisiae VMA1 intein (PI-SceI) and the related Candida tropicalis VMA1 intein. Although the mature Candida intein is devoid of endonucleolytic activity, the exchange of two DNA-binding modules of PI-SceI with the homologous elements from the Candida intein results in an active endonuclease. The low sequence homology in these modules indicates that different protein-DNA contacts are responsible for the recognition of related DNA sequences. This flexibility in DNA recognition should, in principle, allow endonucleases to be produced with new specificities useful for genome engineering.