A Single-Component Multilayered Self-Assembling Protein Nanoparticle Vaccine Based on Extracellular Domains of Matrix Protein 2 against Both Influenza A and B.

The development of an effective and broadly protective influenza vaccine against circulating and emerging strains remains elusive. In this study, we evaluated a potentially universal influenza vaccine based on single-component self-assembling protein nanoparticles (1c-SApNPs) presenting the conserved matrix protein 2 ectodomain (M2e) from influenza A and B viruses (IAV and IBV, respectively). We previously designed a tandem antigen comprising three IAV M2e domains of human, avian/swine, and human/swine origins (termed M2ex3). The M2ex3-presenting 1c-SApNPs conferred complete protection in mice against sequential lethal challenges with H1N1 and H3N2. To broaden this protection to cover IBVs, we designed a series of antigens incorporating different arrangements of three IAV M2e domains and three copies of IBV M2e. Tandem repeats of IAV and IBV (termed influenza A-B) M2e arrayed on the I3-01v9a 60-mer 1c-SApNP, when formulated with an oil-in-water emulsion adjuvant, generated greater M2e-specific immunogenicity and protective efficacy than the soluble influenza A-B M2e trimer, indicated by higher survival rates and reduced weight loss post-challenge. Importantly, one of the influenza A-B M2e SApNP constructs elicited 100% protection against a lethal influenza A/Puerto Rico/8/1934 (H1N1) challenge in mice and 70% protection against a lethal influenza B/Florida/4/2006 (Yamagata lineage) challenge, the latter of which has not been reported in the literature to date. Our study thus provides a promising M2e-based single-component universal vaccine candidate against the two major types of influenza virus circulating in humans.

A tale of two fusion proteins: understanding the metastability of human respiratory syncytial virus and metapneumovirus and implications for rational design of uncleaved prefusion-closed trimers.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we designed uncleaved prefusion-closed (UFC) trimers for the fusion (F) proteins of both viruses by examining mutations critical to F metastability. For RSV, we assessed four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identified key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we developed a stable UFC trimer with a truncated F2-F1 linkage and an interprotomer disulfide bond. Tens of UFC constructs were characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F and one hMPV-F structures), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identified three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites Ø and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induced robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development.

Single-Component Multilayered Self-Assembling Protein Nanoparticles Displaying Extracellular Domains of Matrix Protein 2 as a Pan-influenza A Vaccine.

The development of a cross-protective pan-influenza A vaccine remains a significant challenge. In this study, we designed and evaluated single-component self-assembling protein nanoparticles (SApNPs) presenting the conserved extracellular domain of matrix protein 2 (M2e) as vaccine candidates against influenza A viruses. The SApNP-based vaccine strategy was first validated for human M2e (hM2e) and then applied to tandem repeats of M2e from human, avian, and swine hosts (M2ex3). Vaccination with M2ex3 displayed on SApNPs demonstrated higher survival rates and less weight loss compared to the soluble M2ex3 antigen against the lethal challenges of H1N1 and H3N2 in mice. M2ex3 I3-01v9a SApNPs formulated with a squalene-based adjuvant were retained in the lymph node follicles over 8 weeks and induced long-lived germinal center reactions. Notably, a single low dose of M2ex3 I3-01v9a SApNP formulated with a potent adjuvant, either a Toll-like receptor 9 (TLR9) agonist or a stimulator of interferon genes (STING) agonist, conferred 90% protection against a lethal H1N1 challenge in mice. With the ability to induce robust and durable M2e-specific functional antibody and T cell responses, the M2ex3-presenting I3-01v9a SApNP provides a promising pan-influenza A vaccine candidate.

Single-component multilayered self-assembling protein nanoparticles presenting glycan-trimmed uncleaved prefusion optimized envelope trimmers as HIV-1 vaccine candidates.

Uncleaved prefusion-optimized (UFO) design can stabilize diverse HIV-1 envelope glycoproteins (Envs). Single-component, self-assembling protein nanoparticles (1c-SApNP) can display 8 or 20 native-like Env trimers as vaccine candidates. We characterize the biophysical, structural, and antigenic properties of 1c-SApNPs that present the BG505 UFO trimer with wildtype and modified glycans. For 1c-SApNPs, glycan trimming improves recognition of the CD4 binding site without affecting broadly neutralizing antibodies (bNAbs) to major glycan epitopes. In mice, rabbits, and nonhuman primates, glycan trimming increases the frequency of vaccine responders (FVR) and steers antibody responses away from immunodominant glycan holes and glycan patches. The mechanism of vaccine-induced immunity is examined in mice. Compared with the UFO trimer, the multilayered E2p and I3-01v9 1c-SApNPs show 420 times longer retention in lymph node follicles, 20-32 times greater presentation on follicular dendritic cell dendrites, and up-to-4 times stronger germinal center reactions. These findings can inform future HIV-1 vaccine development.

Structural Properties of CXCL4L1 and Its Recognition of the CXCR3 N-Terminus.

Chemokine CXCL4L1, a homologue of CXCL4, is a more potent antiangiogenic ligand. Its structural property is correlated with the downstream receptor binding. The two chemokines execute their functions by binding the receptors of CXCR3A and CXCR3B. The receptors differ by an extra 51-residue extension in the CXCR3B N-terminus. To understand the binding specificity, a GB1 protein scaffold was used to carry different CXCR3 extracellular elements, and artificial CXCL4 and CXCL4L1 monomers were engineered for the binding assay. We first characterized the molten globule property of CXCL4L1. The structural property causes the CXCL4L1 tetramer to dissociate into monomers in low concentrations, but native CXCL4 adopts a stable tetramer structure in solution. In the titration experiments, the combination of the CXCR3A N-terminus and receptor extracellular loop 2 provided moderate and comparable binding affinities to CXCL4 and CXCL4L1, while sulfation on the CXCR3A N-terminal tyrosine residues provided binding specificity. However, the CXCR3B N-terminal extension did not show significant enhancement in the binding of CXCL4 or CXCL4L1. This result indicates that the tendency to form a chemokine monomer and the binding affinity together contribute the high antiangiogenic activity of CXCL4L1.

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli.

Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34-39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.

Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction.

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9-Asn28; α3, His34-Gly38; and α5, Glu62-Arg72), and a π-helix (π4, Cys43-Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7-Cys43, Cys23-Cys39, and Cys26-Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand-receptor binding specificity.

Structural Insight into the Contributions of the N-Terminus and Key Active-Site Residues to the Catalytic Efficiency of Glutamine Synthetase 2.

Glutamine synthetase (GS) catalyzes the condensation of ammonia and glutamate, along with ATP, to form glutamine. Despite extensive studies on GSs from eukaryotes and prokaryotes, the roles of the N-terminus and other structural features in catalysis remain unclear. Here we report the decameric structure of Drosophila melanogaster GS 2 (DmGS2). The N-terminal short helices, α1 and α2, constitute a meander region, and form hydrogen bonds with residues 3-5 in the N-terminal loop, which are not present in the GSs of other species. Deletion of α1 or α1-α2 inactivates DmGS2. Notably, the Arg4 in each monomer of one pentamer forms hydrogen bonds with Glu7, and Asp8 in the adjacent monomer of the other pentamer. Replacement of Arg4 with Asp (R4D) abolishes activity. Analytical ultracentrifugation revealed that Arg4 is crucial for oligomerization. Circular dichroism spectra revealed that R4D may alter the secondary structure. We mutated key residues to identify the substrate-binding site. As Glu140 binds glutamate and Glu311 binds ammonia, mutants E140A and E311A have little activity. Conversely, mutant P214A (P contributes to ATP binding) has higher activity than wild-type DmGS2. These findings expand the understanding of the structural and functional features of the N-terminal meander region of DmGS2 and the residues important for catalytic efficiency.

An essential role of acetyl coenzyme A in the catalytic cycle of insect arylalkylamine N-acetyltransferase.

Acetyl coenzyme A (Ac-CoA)-dependent N-acetylation is performed by arylalkylamine N-acetyltransferase (AANAT) and is important in many biofunctions. AANAT catalyzes N-acetylation through an ordered sequential mechanism in which cofactor (Ac-CoA) binds first, with substrate binding afterward. No ternary structure containing AANAT, cofactor, and substrate was determined, meaning the details of substrate binding and product release remain unclear. Here, two ternary complexes of dopamine N-acetyltransferase (Dat) before and after N-acetylation were solved at 1.28 Å and 1.36 Å resolution, respectively. Combined with the structures of Dat in apo form and Ac-CoA bound form, we addressed each stage in the catalytic cycle. Isothermal titration calorimetry (ITC), crystallography, and nuclear magnetic resonance spectroscopy (NMR) were utilized to analyze the product release. Our data revealed that Ac-CoA regulates the conformational properties of Dat to form the catalytic site and substrate binding pocket, while the release of products is facilitated by the binding of new Ac-CoA.

N-terminal Backbone Pairing Shifts in CCL5-12AAA14 Dimer Interface: Structural Significance of the FAY Sequence.

CC-type chemokine ligand 5 (CCL5) has been known to regulate immune responses by mediating the chemotaxis of leukocytes. Depending on the environment, CCL5 forms different orders of oligomers to interact with targets and create functional diversity. A recent CCL5 trimer structure revealed that the N-terminal conversed F12-A13-Y14 (12FAY14) sequence is involved in CCL5 aggregation. The CCL5-12AAA14 mutant with two mutations had a deficiency in the formation of high-order oligomers. In the study, we clarify the respective roles of F12 and Y14 through NMR analysis and structural determination of the CCL5-12AAA14 mutant where F12 is involved in the dimer assembly and Y14 is involved in aggregation. The CCL5-12AAA14 structure contains a unique dimer packing. The backbone pairing shifts for one-residue in the N-terminal interface, when compared to the native CCL5 dimer. This difference creates a new structural orientation and leads to the conclusion that F12 confines the native CCL5 dimer configuration. Without F12 anchoring in the position, the interfacial backbone pairing is permitted to slide. Structural plasticity occurs in the N-terminal interaction. This is the first case to report this structural rearrangement through mutagenesis. The study provides a new idea for chemokine engineering and complements the understanding of CCL5 oligomerization and the role of the 12FAY14 sequence.

Integrative Model to Coordinate the Oligomerization and Aggregation Mechanisms of CCL5.

CC-type chemokine ligand 5 (CCL5) is involved in the pathogenesis of many inflammatory conditions. Under physiological conditions, CCL5 oligomerization and aggregation are considered to be responsible for its inflammatory properties. The structural basis of CCL5 oligomerization remains controversial because the current oligomer models contain no consensus interactions. In this study, NMR and biophysical analyses proposed evidence that the CC-type CCL5 dimer acts as the basic unit to constitute the oligomer and that CCL5 oligomerizes alternatively through E66-K25 and E66-R44/K45 interactions. In addition, a newly determined trimer structure, constituted by CCL5 and the E66S mutant, reported an interfacial interaction through the N-terminal 12FAY14 sequence. The interaction contributes to CCL5 aggregation and precipitation but not to oligomerization. In accordance with the observations, an integrative model explains the CCL5 oligomerization and aggregation mechanism in which CCL5 assembly consists of two types of dimer-dimer interactions and one aggregation mechanism. For full-length CCL5, the molecular accumulation triggers oligomerization through the E66-K25 and E66-R44/K45 interactions, and the 12FAY14 interaction acts as a secondary effect to derive aggregation and precipitation. In contrast, the E66-R44/K45 interaction might dominate in CCL5 N-terminal truncations, and the interaction would lead to the filament-like formation in solution.

Salt-sensitive intein for large-scale polypeptide production.

In this chapter, we propose to use salt-sensitive intein as a fusion protein to promote polypeptide expression; the removal of intein from the target sequence requires no enzyme, only a buffer change. The method will be particularly helpful for large-scale polypeptide preparations. Intein is an enzyme that can perform N- and C-terminal self-cleavage. Upon introduction of a mutation to eliminate the N-terminal cleavage activity, the C-terminal cleavage function can still be preserved. This feature was used to develop intein as a fusion protein through conjugation with a given sequence to promote protein expression in a biosynthesis system. Fused intein could later be separated from the target sequence through a C-terminal self-cleavage reaction. Here, a type of salt-sensitive intein is characterized in which ionic strength becomes an effector to control the self-cleavage activity. Low salt concentrations favor the cleavage reaction. Thus, using salt-sensitive intein as a fusion protein simply requires a buffer change to activate the self-cleavage mechanism, which makes it an enzyme-free process. This process has many advantages, including low cost, no extra residue remaining after cleavage, feasibility for preparing proteins starting from a non-Met codon and a special benefit for producing isotope-labeled peptides.

Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells.

The delivery of active proteins into cells (protein transfection) for biological purposes offers considerable potential for clinical applications. Herein we demonstrate that, with a readily available, inexpensive organic agent, the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) method can be used for simple and efficient protein transfection. By mixing proteins with a pure HEPES solution before they are applied to live cells, proteins with various molecular weights (including antibodies, recombinant proteins, and peptides) were successfully delivered into the cytoplasm of different cell types. The protein transfection efficiency of the HEPES method was not inferior to that of commercially available systems that are both more expensive and time consuming. Studies using endocytotic inhibitors and endosomal markers have revealed that cells internalize HEPES-protein mixtures through endocytosis. Results that HEPES-protein mixtures exhibited a low diffusion coefficient suggest that HEPES might neutralize the charges of proteins and, thus, facilitate their cellular internalization. Upon internalization, the cytosolic antibodies caused the degradation of targeted proteins in TRIM21-expressing cells. In summary, the HEPES method is efficient for protein transfection and has potential for myriad clinical applications.

Understanding the Biomineralization Role of Magnetite-Interacting Components (MICs) From Magnetotactic Bacteria.

Biomineralization is a process that takes place in all domains of life and which usually helps organisms to harden soft tissues by creating inorganic structures that facilitate their biological functions. It was shown that biominerals are under tight biological control via proteins that are involved in nucleation initiation and/or which act as structural skeletons. Magnetotactic bacteria (MTB) use iron biomineralization to create nano-magnetic particles in a specialized organelle, the magnetosome, to align to the geomagnetic field. A specific set of magnetite-associated proteins (MAPs) is involved in regulating magnetite nucleation, size, and shape. These MAPs are all predicted to contain specific 17-22 residue-long sequences involved in magnetite formation. To understand the mechanism of magnetite formation, we focused on three different MAPs, MamC, Mms6 and Mms7, and studied the predicted iron-binding sequences. Using nuclear magnetic resonance (NMR), we differentiated the recognition mode of each MAP based on ion specificity, affinity, and binding residues. The significance of critical residues in each peptide was evaluated by mutation followed by an iron co-precipitation assay. Among the peptides, MamC showed weak ion binding but created the most significant effect in enhancing magnetite particle size, indicating the potency in controlling magnetite particle shape and size. Alternatively, Mms6 and Mms7 had strong binding affinities but less effect in modulating magnetite particle size, representing their major role potentially in initiating nucleation by increasing local metal concentration. Overall, our results explain how different MAPs affect magnetite synthesis, interact with Fe2+ ions and which residues are important for the MAPs functions.

The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors.

CXCR3 plays important roles in angiogenesis, inflammation, and cancer. However, the precise mechanism of regulation and activity in tumors is not well known. We focused on CXCR3-A conformation and on the mechanisms controlling its activity and trafficking and investigated the role of CXCR3/LRP1 cross talk in tumor cell invasion. Here we report that agonist stimulation induces an anisotropic response with conformational changes of CXCR3-A along its longitudinal axis. CXCR3-A is internalized via clathrin-coated vesicles and recycled by retrograde trafficking. We demonstrate that CXCR3-A interacts with LRP1. Silencing of LRP1 leads to an increase in the magnitude of ligand-induced conformational change with CXCR3-A focalized at the cell membrane, leading to a sustained receptor activity and an increase in tumor cell migration. This was validated in patient-derived glioma cells and patient samples. Our study defines LRP1 as a regulator of CXCR3, which may have important consequences for tumor biology.

Computational Prediction of New Intein Split Sites.

Split inteins have emerged as a powerful tool in protein engineering. We describe a reliable in silico method to predict viable split sites for the design of new split inteins. A computational circular permutation (CP) prediction method facilitates the search for internal permissive sites to create artificial circular permutants. In this procedure, the original amino- and carboxyl-termini are connected and new termini are created. The identified new terminal sites are promising candidates for the generation of new split sites with the backbone opening being tolerated by the structural scaffold. Here we show how to integrate the online usage of the CP predictor, CPred, in the search of new split intein sites.

NMR Study Reveals the Receiver Domain of Arabidopsis ETHYLENE RESPONSE1 Ethylene Receptor as an Atypical Type Response Regulator.

The gaseous plant hormone ethylene, recognized by plant ethylene receptors, plays a pivotal role in various aspects of plant growth and development. ETHYLENE RESPONSE1 (ETR1) is an ethylene receptor isolated from Arabidopsis and has a structure characteristic of prokaryotic two-component histidine kinase (HK) and receiver domain (RD), where the RD structurally resembles bacteria response regulators (RRs). The ETR1 HK domain has autophosphorylation activity, and little is known if the HK can transfer the phosphoryl group to the RD for receptor signaling. Unveiling the correlation of the receptor structure and phosphorylation status would advance the studies towards the underlying mechanisms of ETR1 receptor signaling. In this study, using the nuclear magnetic resonance technique, our data suggested that the ETR1-RD is monomeric in solution and the rigid structure of the RD prevents the conserved aspartate residue phosphorylation. Comparing the backbone dynamics with other RRs, we propose that backbone flexibility is critical to the RR phosphorylation. Besides the limited flexibility, ETR1-RD has a unique γ loop conformation of opposite orientation, which makes ETR1-RD unfavorable for phosphorylation. These two features explain why ETR1-RD cannot be phosphorylated and is classified as an atypical type RR. As a control, phosphorylation of the ETR1-RD was also impaired when the sequence was swapped to the fragment of the bacterial typical type RR, CheY. Here, we suggest a molecule insight that the ETR1-RD already exists as an active formation and executes its function through binding with the downstream factors without phosphorylation.

Structure and function of chicken interleukin-1 beta mutants: uncoupling of receptor binding and in vivo biological activity.

Receptor-binding and subsequent signal-activation of interleukin-1 beta (IL-1ß) are essential to immune and proinflammatory responses. We mutated 12 residues to identify sites important for biological activity and/or receptor binding. Four of these mutants with mutations in loop 9 (T117A, E118K, E118A, E118R) displayed significantly reduced biological activity. Neither T117A nor E118K mutants substantially affected receptor binding, whereas both mutants lack the IL-1ß signaling in vitro but can antagonize wild-type (WT) IL-1ß. Crystal structures of T117A, E118A, and E118K revealed that the secondary structure or surface charge of loop 9 is dramatically altered compared with that of wild-type chicken IL-1ß. Molecular dynamics simulations of IL-1ß bound to its receptor (IL-1RI) and receptor accessory protein (IL-1RAcP) revealed that loop 9 lies in a pocket that is formed at the IL-1RI/IL-1RAcP interface. This pocket is also observed in the human ternary structure. The conformations of above mutants in loop 9 may disrupt structural packing and therefore the stability in a chicken IL-1ß/IL-1RI/IL-1RAcP signaling complex. We identify the hot spots in IL-1ß that are essential to immune responses and elucidate a mechanism by which IL-1ß activity can be inhibited. These findings should aid in the development of new therapeutics that neutralize IL-1 activity.

1H, 15N and 13C resonance assignments of light organ-associated fatty acid-binding protein of Taiwanese fireflies.

Fatty acid-binding proteins (FABPs) are a family of proteins that modulate the transfer of various fatty acids in the cytosol and constitute a significant portion in many energy-consuming cells. The ligand binding properties and specific functions of a particular type of FABP seem to be diverse and depend on the respective binding cavity as well as the cell type from which this protein is derived. Previously, a novel FABP (lcFABP; lc: Luciola cerata) was identified in the light organ of Taiwanese fireflies. The lcFABP was proved to possess fatty acids binding capabilities, especially for fatty acids of length C14-C18. However, the structural details are unknown, and the structure-function relationship has remained to be further investigated. In this study, we finished the (1)H, (15)N and (13)C chemical shift assignments of (15)N/(13)C-enriched lcFABP by solution NMR spectroscopy. In addition, the secondary structure distribution was revealed based on the backbone N, H, Cα, Hα, C and side chain Cß assignments. These results can provide the basis for further structural exploration of lcFABP.

A streamlined method for preparing split intein for NMR study.

A protein ligase, intein, mediates a protein-splicing reaction. It can be split into two complementary fragments and reconstituted as a whole intein scaffold to perform protein trans-splicing. To understand the association of intein fragments and the splicing mechanism, it is necessary to produce a large quantity of split intein for structural study. Conventionally, two fragments are prepared separately and assembled in solution, but severe aggregation of intein fragments occurs, and precise control of the relative concentration of each fragment is difficult. Here, we present a streamlined method to incorporate a circular permutation concept into the production of split intein. By circular permutation of the native split Nostoc punctiforme DnaE intein (NpuInt), a new backbone opening is relocated to the native split site at residue 102. As the protein splicing activity is preserved, the expressed NpuInt can immediately self-cleave into a two-piece split NpuInt. Because of a tight association between the two complementary fragments, split NpuInt can be purified in one step. The idea is simple and applicable to other split inteins. Employing the new preparation, we use NMR spectra to assign the backbone and side chain resonances for the native split NpuInt.