An off-the-Shelf Approach for the Production of Fc Fusion Proteins by Protein Trans-Splicing towards Generating a Lectibody In Vitro.

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a "lectibody". Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine.

Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (-1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(-1) residue, including d-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N-S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (-1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(-1)Phe and Gly(-1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein-intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein-intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins.

FRET monitoring of a nonribosomal peptide synthetase.

Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine-thioester and phenylalanine-AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.

Mapping of the Communication-Mediating Interface in Nonribosomal Peptide Synthetases Using a Genetically Encoded Photocrosslinker Supports an Upside-Down Helix-Hand Motif.

Nonribosomal peptide synthetases (NRPSs) are large modular protein templates that assemble bioactive peptides, many of which possess therapeutic importance. Protein-protein interactions between subunits of bacterial NRPSs are essential for proper template formation. The structural basis of the typical subunit interface between epimerization (E) and condensation domains is only poorly understood. Conflicting helix-helix and helix-hand models were previously proposed. Here, the genetically encoded photocrosslinker p-benzoylphenylalanine (BpF) was incorporated into the C-terminal communication-mediating domain (COM) of GrsA. Using the partner elongation module TycB1 to form a dipeptide product, we could correlate the ability to form covalent crosslinks with the functional module interaction. Perturbation of the module interaction with the large side chain of BpF in a scan at 19 positions demonstrated the importance of three hydrophobic residues in an α-helical arrangement. Mapping of covalent crosslinks using tandem mass spectrometry revealed the residues from the interior of the condensation domain as part of the protein interface; a finding not predicted by the helix-helix model. The epimerization domain of GrsA was found to be important for the interaction. Together with multiple sequence analyses and structural modeling, our results suggest an upside-down helix-hand model in which the C-terminal COM-helix is embedded in a hand motif with a hydrophobic core in a reversed orientation compared to a previous proposal. Our results provide a more detailed and the first direct structural understanding of the COM domain interaction and will contribute to successful biocombinatorial engineering attempts in the design of artificial NRPS templates.

Ligation of synthetic peptides to proteins using semisynthetic protein trans-splicing.

Protein trans-splicing using split inteins is a powerful and convenient reaction to chemically modify recombinantly expressed proteins under mild conditions. In particular, semisynthetic protein trans-splicing with one intein fragment short enough to be accessible by solid-phase peptide synthesis can be used to transfer a short peptide segment with the desired synthetic moiety to the protein of interest. In this chapter, we provide detailed protocols for two such split intein systems. The M86 mutant of the Ssp DnaB intein and the MX1 mutant of the AceL-TerL intein are two highly engineered split inteins with very short N-terminal intein fragments of only 11 and 25 amino acids, respectively, and allow the efficient N-terminal labeling of proteins.

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.

Recent progress in intein research: from mechanism to directed evolution and applications.

Inteins catalyze a post-translational modification known as protein splicing, where the intein removes itself from a precursor protein and concomitantly ligates the flanking protein sequences with a peptide bond. Over the past two decades, inteins have risen from a peculiarity to a rich source of applications in biotechnology, biomedicine, and protein chemistry. In this review, we focus on developments of intein-related research spanning the last 5 years, including the three different splicing mechanisms and their molecular underpinnings, the directed evolution of inteins towards improved splicing in exogenous protein contexts, as well as novel applications of inteins for cell biology and protein engineering, which were made possible by a clearer understanding of the protein splicing mechanism.

Site-specific protein cleavage in vivo by an intein-derived protease.

Site-specific protein cleavage is a ubiquitous process in cellular protein metabolism, yet molecular tools to provide control of protein cleavage inside living cells remain scarce. Here, we show that the C-terminal intein fragment of the non-canonical Ssp (Synechocystis sp. PCC6803) DnaB S1 split-intein can be used as a site-specific protease for in vivo protein cleavage both in bacterial and eukaryotic cells. Mutagenesis data indicate a broad tolerance of the intein-derived protease (IP) toward the amino acid upstream of the cleavage site. Furthermore, deletion studies reveal that the recognition sequence for the IP can be as short as ten amino acids. The structural features underlying the cleavage reaction preclude unintended proteolysis of endogenous proteins, thus ensuring that negative effects on cell viability are minimal.

Intein lacking conserved C-terminal motif G retains controllable N-cleavage activity.

A split-intein consists of two complementary fragments (N-intein and C-intein) that can associate to carry out protein trans-splicing. The Ssp GyrB S11 split-intein is an engineered unconventional split-intein consisting of a 150-amino-acid N-intein and an extremely small six-amino-acid C-intein, which comprises the conserved intein motif G. Here, we show that fusion proteins containing the 150-amino-acid N-intein could be triggered to undergo controllable N-cleavage in vitro when the six-amino-acid C-intein or a derivative thereof was added as a synthetic peptide in trans. More importantly, we discovered, unexpectedly, that the 150-amino-acid N-intein could be induced by strong nucleophiles to undergo N-cleavage in vitro, and in Escherichia coli cells, in the absence of the motif G-containing six-amino-acid C-intein. This finding indicated that the first step of the protein splicing mechanism (acyl shift) could occur in the absence of the entire motif G. Extensive kinetic analyses revealed that both the motif G residues and the Ser+1 residue positively influenced N-cleavage rate constants and yields. The 150-amino-acid N-intein could also tolerate various unrelated sequences appended to its C-terminus without disruption of the N-cleavage function, suggesting that the catalytic pocket of the intein has considerable structural flexibility. Our findings reveal interesting insights into intein structure-function relationships, and demonstrate a new and potentially more useful method of controllable, intein-mediated N-cleavage for protein engineering applications.

Protein trans-splicing as a protein ligation tool to study protein structure and function.

Protein trans-splicing (PTS) exerted by split inteins is a protein ligation reaction which enables overcoming the barriers of conventional heterologous protein production. We provide an overview of the current state-of-the-art in split intein engineering, as well as the achievements of PTS technology in the realm of protein structure-function analyses, including incorporation of natural and artificial protein modifications, controllable protein reconstitution, segmental isotope labeling and protein cyclization. We further discuss factors crucial for the successful implementation of PTS in these protein engineering approaches, and speculate on necessary future endeavours to make PTS a universally applicable protein ligation tool.

PRP8 intein in Ajellomycetaceae family pathogens: sequence analysis, splicing evaluation and homing endonuclease activity.

Inteins are intervening sequences that are transcribed and translated with flanking host protein sequences and then self-excised by protein splicing. Bi-functional inteins also contain a homing endonuclease responsible for their genetic mobility. The PRP8 intein, the most widespread among fungi, occurs in important pathogens such as Histoplasma capsulatum and Paracoccidioides brasiliensis, from the Ajellomycetaceae family. Herein, we describe the bi-functional PRP8 intein in two other Ajellomycetacean pathogens, Blastomyces dermatitidis and Emmonsia parva. Sequence analysis and experimental evidence suggest that the homing endonuclease from PbrPRP8 is inactive. The splicing activity of the PRP8 intein from the B. dermatitidis, E. parva and P. brasiliensis species complex was demonstrated in a non-native protein context in Escherichia coli. Since the PRP8 intein is located in a functionally essential nuclear protein, it can be considered a promising therapeutic target for anti-fungal drugs, because inhibition of intein splicing should inhibit proliferation of intein-containing pathogens.

Protein trans-splicing and its use in structural biology: opportunities and limitations.

Obtaining insights into the molecular structure and dynamics of a protein by NMR spectroscopy and other in-solution biophysical methods relies heavily on the incorporation of isotopic labels or other chemical modifications such as fluorescent groups into the protein of interest. These types of modifications can be elegantly achieved with the use of split inteins in a site- and/or region-specific manner. Split inteins are split derivatives of the protein splicing element intein, and catalyze the formation of a peptide bond between two proteins. Recent progress in split intein engineering provided the opportunity to also perform peptide bond formation between a protein and a chemically synthesized peptide. We review the current state-of-the-art in preparing segmental isotope-labeled proteins for NMR spectroscopy, and highlight the importance of split intein orthogonality for the ligation of a protein from multiple fragments. Furthermore, we use split intein-mediated site-specific fluorescent labeling as a framework to illustrate the general usefulness of split inteins for custom protein modifications in the realm of structural biology. We also address some limitations of split intein technology, and offer constructive advice to overcome these shortcomings.

Intein-mediated cyclization of bacterial acyl carrier protein stabilizes its folded conformation but does not abolish function.

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.

Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein.

BACKGROUND: Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. METHODOLOGY: A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. CONCLUSIONS: We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.

Controllable protein cleavages through intein fragment complementation.

Intein-based protein cleavages, if carried out in a controllable way, can be useful tools of recombinant protein purification, ligation, and cyclization. However, existing methods using contiguous inteins were often complicated by spontaneous cleavages, which could severely reduce the yield of the desired protein product. Here we demonstrate a new method of controllable cleavages without any spontaneous cleavage, using an artificial S1 split-intein consisting of an 11-aa N-intein (I(N)) and a 144-aa C-intein (I(C)). In a C-cleavage design, the I(C) sequence was embedded in a recombinant precursor protein, and the small I(N) was used as a synthetic peptide to trigger a cleavage at the C-terminus of I(C). In an N-cleavage design, the short I(N) sequence was embedded in a recombinant precursor protein, and the separately produced I(C) protein was used to catalyze a cleavage at the N-terminus of I(N). These N- and C-cleavages showed >95% efficiency, and both successfully avoided any spontaneous cleavage during expression and purification of the precursor proteins. The N-cleavage design also revealed an unexpected and interesting structural flexibility of the I(C) protein. These findings significantly expand the effectiveness of intein-based protein cleavages, and they also reveal important insights of intein structural flexibility and fragment complementation.

Novel split intein for trans-splicing synthetic peptide onto C terminus of protein.

Conventional split inteins have been useful for trans-splicing between recombinant proteins, and an artificial S1 split intein is useful for adding synthetic peptide onto the N terminus of recombinant proteins. Here we have engineered a novel S11 split intein for trans-splicing synthetic peptide onto the C terminus of recombinant proteins. The C-intein of the S11 split intein is extremely small (6 amino acids (aa)); thus it can easily be produced together with a synthetic C-extein to be added to the C terminus of target proteins. The S11 intein was derived from the Ssp GyrB intein after deleting the homing endonuclease domain and splitting the remaining intein sequence near the C terminus, producing a 150-aa N-intein (IN) and a 6-aa C-intein (IC). Its trans-splicing activity was demonstrated first in Escherichia coli cells and then in vitro for trans-splicing between a synthetic peptide and a recombinant protein. The in vitro trans-splicing reaction exhibited a typical rate constant of (6.9+/-2.2)x10(-5) s(-1) and reached a high efficiency of approximately 80%. This S11 split intein can be useful for adding any desirable chemical groups to the C terminus of a protein of interest, which may include modified and unnatural amino acids, biotin and fluorescent labels, and even drug molecules.