Semisynthesis and folding of the potassium channel KcsA

In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant peptide alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal peptide corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins.     

Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation

BACKGROUND: The site-specific chemical modification of proteins has proved to be extremely powerful for generating tools for the investigation of biological processes. Although a few elegant methods exist for engineering a recombinant protein at a unique position, these techniques cannot be easily extended to allow several different chemical probes to be specifically introduced into a target sequence. As such multiply labeled proteins could be used to study many biological processes, and in particular biomolecular interactions, we decided to investigate whether such protein reagents could be generated using an extension of the semisynthesis technique known as expressed protein ligation. RESULTS: A solid-phase expressed protein ligation (SPPL) technology is described that enables large semisynthetic proteins to be assembled on a solid support by the controlled sequential ligation of a series of recombinant and synthetic polypeptide building blocks. This modular approach allows multiple, different chemical modifications to be introduced site-specifically into a target protein. This process, which is analogous to solid-phase peptide synthesis, was used to dual-label the amino and carboxyl termini of the Crk-II adapter protein with the fluorescence resonance energy transfer pair tetramethylrhodamine and fluorescein, respectively. The resulting construct reports (through a fluorescence change) the phosphorylation of Crk-II by the nonreceptor protein tyrosine kinase, c-Abl, and was used to probe the protein-protein interactions that regulate this important post-translational process. CONCLUSIONS: SPPL provides a powerful method for specifically modifying proteins at multiple sites, as was demonstrated by generating a protein-based biosensor for Crk-II phosphorylation. Such protein derivatives are extremely useful for investigating protein function in vitro and potentially in vivo. This modular approach should be applicable to many different protein systems.     

Generation of a Genetically Encoded,Photoactivatable Intein for the Controlled Production of Cyclic Peptides

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split‐intein‐mediated SICLOPPS technology provides a generic access to fully genetically encoded head‐to‐tail cyclized peptides and large libraries thereof (SICLOPPS=split‐intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light‐dependent intein using the photocaged tyrosine derivative ortho‐nitrobenzyltyrosine incorporated at an internal, non‐catalytic position is now reported. Stable intein precursors were purified from the E. coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.     

Generation of a Genetically Encoded,Photoactivatable Intein for the Controlled Production of Cyclic Peptides

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split‐intein‐mediated SICLOPPS technology provides a generic access to fully genetically encoded head‐to‐tail cyclized peptides and large libraries thereof (SICLOPPS=split‐intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light‐dependent intein using the photocaged tyrosine derivative ortho‐nitrobenzyltyrosine incorporated at an internal, non‐catalytic position is now reported. Stable intein precursors were purified from the E. coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.     

Design of an intein that can be inhibited with a small molecule ligand

Protein splicing is a process in which an intervening sequence, the intein, catalyzes its own excision out of a larger polypeptide precursor by joining the flanking sequences, the exteins, with a native peptide bond. Inteins are almost completely promiscuous toward the nature of their extein sequences and can be inserted into virtually any host protein. The intein-mediated formation of a peptide bond between two polypeptides offers great potential to modulate protein structure and, hence, protein function on the post-translational level. In this work, we report the design of an intein that can be inhibited by the addition of a specific small molecule ligand. Our design strategy involved the generation of a trans-splicing intein, in which the intein domain is split into two-halves that are located on two separate polypeptides, each joined with the respective N- or C-terminal extein. To turn these fragments into an active intein with an incorporated "off" switch, each was fused at its newly created terminus with the F36M mutant of FKBP12, referred to as the FM domain. The F36M substitution was reported to effect a homodimerization of the usually monomeric FKBP12 protein; however, addition of the small molecule ligand, rapamycin, or synthetic derivatives thereof leads to a dissociation of the dimer. This phenomenon was exploited by first reconstituting the active intein on the basis of FM domain dimerization. Second, addition of the small molecule ligand prevented formation of the active intein complex and inhibited protein trans-splicing. This intein exhibited unexpected kinetic properties and provides a new and potentially very general means to control protein function on the post-translational level.     

Synthesis of photoactive analogues of a cystine knot trypsin inhibitor protein

We describe the preparation of a recently described photoactive amino acid analogue (photoMethionine) by two novel synthetic routes, one of which is flexible and enantiospecific, and the site-specific chemical incorporation of photoMethionine into a defined and functionally active protein using a combination of solid-phase peptide synthesis and modern chemical ligation methodology. Site-specific labeling of proteins with this amino acid analogue through chemical synthesis provides valuable probes for photoaffinity cross-linking studies.     

In Cellulo Protein Semi-Synthesis from Endogenous and Exogenous Fragments Using the Ultra-Fast Split Gp41-1 Intein

Protein semi-synthesis inside live cells from exogenous and endogenous parts offers unique possibilities for studying proteins in their native context. Split-intein-mediated protein trans-splicing is predestined for such endeavors and has seen some successes, but a much larger variety of established split inteins and associated protocols is urgently needed. We characterized the association and splicing parameters of the Gp41-1 split intein, which favorably revealed a nanomolar affinity between the intein fragments combined with the exceptionally fast splicing rate. Following bead-loading of a chemically modified intein fragment precursor into live mammalian cells, we fluorescently labeled target proteins on their N- and C-termini with short peptide tags, thus ensuring minimal perturbation of their structure and function. In combination with a nuclear-entrapment strategy to minimize cytosolic fluorescence background, we applied our technique for super-resolution imaging and single-particle tracking of the outer mitochondrial protein Tom20 in HeLa cells.     

Intracellular protein labeling with prodrug-like probes using a mutant β-lactamase tag

Intracellular protein labeling with small molecular probes that do not require a washing step for the removal of excess probe is greatly desired for real-time investigation of protein dynamics in living cells. Successful labeling of proteins on the cell membrane has been performed using mutant β-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on β-lactam prodrugs is described. The prodrug-based probes quickly permeated the plasma membranes of living mammalian cells, and efficiently labeled intracellular proteins at low probe concentrations. Because these cell-permeable probes were activated only inside cells, simultaneous discriminative labeling of intracellular and cell surface BL-tag fusion proteins was attained by using cell-permeable and impermeable probes. Thus, this technology enables adequate discrimination of the location of proteins labeled with the same protein tag, in conjunction with different color probes, by dual-color fluorescence. Moreover, the combination of BL-tag technology and the prodrug-based probes enabled the labeling of target proteins without requiring a washing step, owing to the efficient entry of probes into cells and the fast covalent labeling achieved with BL-tag technology after bioactivation. This prodrug-based probe design strategy for BL-tags provides a simple experimental procedure with application to cellular studies with the additional advantage of reduced stress to living cells.     

Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins

In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.     

蛋白质的化学全合成

含有非天然氨基酸的蛋白质（如翻译后修饰蛋白质、修饰有探针分子的蛋白质等）是化学生物学中重要的生理活性分子。这些分子难以通过生物表达来获取，而必须使用化学方法来合成。半胱氨酸肽片段连接方法是目前应用于蛋白质化学全合成中的一种重要方法，该方法能够在温和的水溶液中高效地实现肽片段的连接，从而生成天然或者非天然的蛋白质。本文系统地综述了半胱氨酸肽片段连接方法的基本原理，详细讨论了近年来人们对该方法的一些重要改进。最后又介绍了该方法在几类重要的蛋白质分子合成中的代表性应用。     

Selenolysine: A New Tool for Traceless Isopeptide Bond Formation

Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion by using conventional expression systems. Chemical protein synthesis or semisynthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of any cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and chemoselective deselenization in the presence of unprotected cysteine residues. Selenolysine's straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target.     

Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system

Inteins are self-cleavable proteins that under reducing conditions can be cleaved from a recombinant target protein. Industrially, an intein-based system could potentially reduce production costs of recombinant proteins by facilitating a highly selective affinity purification using an inexpensive substrate such as chitin. In this study, SuperPro Designer was used to simulate the large-scale recovery of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification process based on the commercially available IMPACT system. The intein process was also compared with a conventional process simulated by SuperPro. The intein purification process initially simulated was significantly more expensive than the conventional process, primarily owing to the properties of the chitin resin and high reducing-agent (dithiothreitol [DTT]) raw material cost. The intein process was sensitive to the chitin resin binding capacity, cleavage efficiency of the intein fusion protein, the size of the target protein relative to the intein tag, and DTT costs. An optimized intein purification process considerably reduced costs by simulating an improved chitin resin and alternative reducing agents. Thus, to realize the full potential of intein purification processes, research is needed to improve the properties of chitin resin and to find alternative, inexpensive raw materials.     

A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles

A novel protein purification method was developed using microbial polyhydroxyalkanoates (PHA) granule-associated protein phasin, a pH-inducible self-cleaving intein and PHA nanoparticles. Genes for the target proteins to be produced and purified were fused to genes of intein and phasin, the genes were jointly over-expressed in vivo, such as in E. coli cells in this study. The fused proteins containing target protein, intein and phasin produced by the recombinant E. coli were released together with all other E. coli proteins via a bacterial lysis process. They were then adsorbed in vitro to the surfaces of the hydrophobic polymer nanoparticles incubated with the cell lysates. The nanoparticles attached with the fused proteins were concentrated via centrifugation. Then, the reasonably purified target protein was released by self-cleavage of intein and separated with nanoparticles by a simple centrifugation process. Using this system, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and beta-galactosidase were successfully purified in their active forms with reasonable yields, respectively, demonstrating the effectiveness and reliability of this purification system. This method allows the production and purification of high value added proteins in a continuous way with low cost.     

Self-cleavable stimulus responsive tags for protein purification without chromatography

A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle.     

多肽片段连接方法及肽硫酯和肽醛的合成*

本文综述了多肽和蛋白质合成中的片段连接方法,这是近年来多肽和蛋白质合成领域中方法学上的重要进展.该方法使用非保护的多肽片段,无需酶或化学活化试剂,在缓冲溶液中能够高产率地获得多肽和蛋白质.还介绍了与多肽片段连接有关的肽硫酯和肽醛的合成方法.     

Modification of transmembrane and GPI-anchored proteins on living cells by efficient protein trans-splicing using the Npu DnaE intein

The naturally split Npu DnaE intein can be used for ligation of an exogenous polypeptide to membrane proteins on living cells. No reducing agents or other factors are required. The approach is rapid and virtually traceless, because the intein removes itself during the reaction.     

An Atypical Naturally Split Intein Engineered for Highly Efficient Protein Labeling

Protein trans‐splicing catalyzed by split inteins is a powerful technique for assembling a polypeptide backbone from two separate parts. However, split inteins with robust efficiencies and short fragments suitable for peptide synthesis are rare and have mostly been artificially created. The novel split intein AceL‐TerL was identified from metagenomic data and characterized. It represents the first naturally occurring, atypically split intein. The N‐terminal fragment of only 25 amino acids is the shortest natural intein fragment to date and was easily amenable to chemical synthesis with a fluorescent label. Optimal protein trans‐splicing activity was observed at low temperatures. Further improved mutants were selected by directed protein evolution. The engineered intein variants with up to 50‐fold increased rates showed unprecedented efficiency in chemically labeling of a diverse set of proteins. These inteins should prove valuable tools for protein semi‐synthesis and other intein‐related biotechnological applications.     

Oligo-Asp tag/Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins

To accomplish the selective labeling of a specific protein in complicated biological systems, a peptide tag incorporated into the protein and a complementary small molecular probe are required. Although a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n = 1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4 tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction.     

Chemical Protein Synthesis by Native Chemical Ligation and Variations Thereof

For a long time, the total synthesis of proteins was considered as a “mission impossible” because of the tedious and complex synthetic steps and demanding purification processes. However, with the development of modern synthetic methodologies, many protein syntheses have now been reported. More importantly, through chemical synthesis, desired modifications can be installed to target proteins precisely, which is a major advantage over traditional bio‐synthesis approaches. This review summarizes the techniques developed for protein assembly, including native chemical ligation, Se‐mediated ligation, and a range of other ligation methods. A few synthetic examples, whereby synthetic proteins with desired modifications have been utilized for related biological research, are also included. We believe that chemical synthesis can provide alternative pathways to solve problems that have hitherto proved insurmountable by traditional biological approaches.     

Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro

Production of recombinant proteins is an important prerequisite for biotechnology and life sciences in general. However, there is a paucity of methods for production of posttranslationally modified recombinant proteins or proteins with non-native functional groups, such as fluorophores, spin labels, and so forth. In this work we have used a combination of organic synthesis and in vitro protein ligation to construct monoprenylated Rab7 GTPase. The protein was prepared from a recombinant N-terminal portion and a peptide mimicking the C terminus of Rab7. For construction of a synthetic six-amino-acid-long fluorescent monoprenylated peptide, we used a block condensation strategy. Ligation was achieved with a yield of >70%. The resulting protein was purified from the unligated peptide by a combination of organic extraction and phase partitioning and refolding. The refolded monoprenylated semisynthetic Rab7 protein (Rab7GG) formed a stable complex with its natural chaperone REP-1 (Rab escort protein 1) and could serve as an acceptor of the second prenyl group in the enzymatic prenylation reaction. Using fluorescence spectroscopy, we characterized the interaction of the Rab7GG:REP-1 complex with Rab geranylgeranyl transferase and came to the conclusion that it functioned as a genuine intermediate of the prenylation reaction. Thus, we present the first example of the in vitro generation of a semisynthetic lipidated protein using the native chemical ligation method.