Phase separation drives the folding of recombinant collagen.

Recombinantly produced collagens present a sustainable, ethical, and safe substitute for collagens derived from natural sources. However, controlling the folding of the recombinant collagens, crucial for replicating the mechanical properties of natural materials, remains a formidable task. Collagen-like proteins from willow sawfly are relatively small and contain no hydroxyprolines, presenting an attractive alternative to the large and post-translationally modified mammalian collagens.

Triblock Proteins with Weakly Dimerizing Terminal Blocks and an Intrinsically Disordered Region for Rational Design of Condensate Properties.

Condensates are molecular assemblies that are formed through liquid-liquid phase separation and play important roles in many biological processes. The rational design of condensate formation and their properties is central to applications, such as biosynthetic materials, synthetic biology, and for understanding cell biology. Protein engineering is used to make a triblock structure with varying terminal blocks of folded proteins on both sides of an intrinsically disordered mid-region.

Catcher/Tag Toolbox: Biomolecular Click-Reactions For Protein Engineering Beyond Genetics.

Manipulating protein architectures beyond genetic control has attracted widespread attention. Catcher/Tag systems enable highly specific conjugation of proteins in vivo and in vitro via an isopeptide-bond. They provide efficient, robust, and irreversible strategies for protein conjugation and are simple yet powerful tools for a variety of applications in enzyme industry, vaccines, biomaterials, and cellular applications.

Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk Proteins.

A type of protein/peptide pair known as Catcher/Tag pair spontaneously forms an intermolecular isopeptide bond which can be applied for biomolecular click reactions. Covalent protein conjugation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines, but it is essential to increase the current toolbox of orthogonal Catcher/Tag pairs to expand the range of applications further, for example, for controlled multiple-fragment ligation. We report here the engineering of novel Catcher/Tag pairs for protein ligation, aided by a crystal structure of a minimal CnaB domain from Lactobacillus plantarum.

Liquid-Liquid Phase Separation and Assembly of Silk-like Proteins is Dependent on the Polymer Length.

Phase transitions have an essential role in the assembly of nature's protein-based materials into hierarchically organized structures, yet many of the underlying mechanisms and interactions remain to be resolved. A central question for designing proteins for materials is how the protein architecture and sequence affects the nature of the phase transitions and resulting assembly. In this work, we produced 82 kDa (1×), 143 kDa (2×), and 204 kDa (3×) silk-mimicking proteins by taking advantage of protein ligation by SpyCatcher/Tag protein-peptide pair.

The NMR structure of the engineered halophilic DnaE intein for segmental isotopic labeling using conditional protein splicing.

Protein trans-splicing catalyzed by split inteins has been used for segmental isotopic labeling of proteins for alleviating the complexity of NMR signals. Whereas inteins spontaneously trigger protein splicing upon protein folding, inteins from extremely halophilic organisms require a high salinity condition to induce protein splicing. We designed and created a salt-inducible intein from the widely used DnaE intein from Nostoc punctiforme by introducing 29 mutations, which required a lower salt concentration than naturally occurring halo-obligate inteins.

Psychosocial difficulties and treatment retention in inpatient detoxification programmes.

Aims: Treatment retention is associated with addiction treatment outcomes. Research regarding predictors of retention at inpatient detoxification treatment is limited. The aim of this study was to investigate whether psychosocial difficulties (PSDs) are associated with treatment retention among Finnish inpatients undergoing detoxification treatment.

Molecular mechanisms mediating stiffening in the mechanically adaptable connective tissues of sea cucumbers.

Mutable collagenous tissues (MCTs) from echinoderms (e.g., sea stars, sea urchins) possess the remarkable ability to change their mechanical properties rapidly and reversibly thanks to the release of effector molecules regulating the number of cross-links between collagen fibrils.

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins.

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields.

The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms.

Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins.

Substrate specificities of inteins investigated by QuickDrop-cassette mutagenesis.

Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the -1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins.

Biomimetic composites with enhanced toughening using silk-inspired triblock proteins and aligned nanocellulose reinforcements.

Silk and cellulose are biopolymers that show strong potential as future sustainable materials. They also have complementary properties, suitable for combination in composite materials where cellulose would form the reinforcing component and silk the tough matrix. A major challenge concerns balancing structure and functional properties in the assembly process.

Phase transitions as intermediate steps in the formation of molecularly engineered protein fibers.

A central concept in molecular bioscience is how structure formation at different length scales is achieved. Here we use spider silk protein as a model to design new recombinant proteins that assemble into fibers. We made proteins with a three-block architecture with folded globular domains at each terminus of a truncated repetitive silk sequence.

Salt-inducible Protein Splicing in cis and trans by Inteins from Extremely Halophilic Archaea as a Novel Protein-Engineering Tool.

Intervening protein sequences (inteins) from extremely halophilic haloarchaea can be inactive under low salinity but could be activated by increasing the salt content to a specific concentration for each intein. The halo-obligatory inteins confer high solubility under both low and high salinity conditions. We showed the broad utility of salt-dependent protein splicing in cis and trans by demonstrating backbone cyclization, self-cleavage for purification, and scarless protein ligation for segmental isotopic labeling.

Nature's recipe for splitting inteins.

Protein splicing in trans by split inteins has increasingly become a powerful protein-engineering tool for protein ligation, both in vivo and in vitro. Over 100 naturally occurring and artificially engineered split inteins have been reported for protein ligation using protein trans-splicing. Here, we review the current status of the reported split inteins in order to delineate an empirical or rational strategy for constructing new split inteins suitable for various applications in biotechnology and chemical biology.

Structure-based engineering and comparison of novel split inteins for protein ligation.

Protein splicing is an autocatalytic process involving self-excision of an internal protein domain, the intein, and concomitant ligation of the two flanking sequences, the exteins, with a peptide bond. Protein splicing can also take place in trans by naturally split inteins or artificially split inteins, ligating the exteins on two different polypeptide chains into one polypeptide chain. Protein trans-splicing could work in foreign contexts by replacing the native extein sequences with other protein sequences.

Intermolecular domain swapping induces intein-mediated protein alternative splicing.

Protein sequences are diversified on the DNA level by recombination and mutation and can be further increased on the RNA level by alternative RNA splicing, involving introns that have important roles in many biological processes. The protein version of introns (inteins), which catalyze protein splicing, were first reported in the 1990s. The biological roles of protein splicing still remain elusive because inteins neither provide any clear benefits nor have an essential role in their host organisms.

Structural basis for protein trans-splicing by a bacterial intein-like domain--protein ligation without nucleophilic side chains.

Unlabelled: Protein splicing in trans by split inteins has become a useful tool for protein engineering in vivo and in vitro. Inteins require Cys, Ser or Thr at the first residue of the C-terminal flanking sequence because a thiol or hydroxyl group in the side chains is a nucleophile indispensable for the trans-esterification step during protein splicing. Newly-identified distinct sequences with homology to the hedgehog/intein superfamily, called bacterial intein-like (BIL) domains, often do not have Cys, Ser, or Thr as the obligatory nucleophilic residue found in inteins.

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.

Use of protein trans-splicing to produce active and segmentally (2)H, (15)N labeled mannuronan C5-epimerase AlgE4.

Alginate epimerases are large multidomain proteins capable of epimerising C5 on beta-D-mannuronic acid (M) turning it into alpha-L-guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers.

Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro.

Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.

In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins.

Background: Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized.

Principal Findings: We report the identification of new functional split sites in DnaE inteins from Synechocystis sp.

Solution structure of DnaE intein from Nostoc punctiforme: structural basis for the design of a new split intein suitable for site-specific chemical modification.

Naturally split DnaE intein from Nostoc punctiforme (Npu) has robust protein trans-splicing activity and high tolerance of sequence variations at the splicing junctions. We determined the solution structure of a single chain variant of NpuDnaE intein by NMR spectroscopy. Based on the NMR structure and the backbone dynamics of the single chain NpuDnaE intein, we designed a functional split variant of the NpuDnaE intein having a short C-terminal half (C-intein) composed of six residues.