Studies on the Selective Trifluoroacetolytic Scission of Native Peptaibols and Model Peptides Using HPLC and ESI‐CID‐MS

Representative members of a group of linear, N‐acylated polypeptide antibiotics (peptaibols) containing α‐aminoisobutyric acid (Aib) and, in part, isovaline (Iva), as well as proteinogenic amino acids and a C‐terminal‐bonded 2‐amino alcohol, were treated with anhydrous trifluoroacetic acid (TFA) at 37° for 0.5–26 h. The resulting fragments were separated by HPLC and characterized by electrospray ionization collision‐induced dissociation mass spectrometry (ESI‐CID‐MS). The following 16–20‐residue peptaibols were investigated: natural, microheterogeneous mixtures of antiamoebins and alamethicin F50, uniform paracelsin A, and synthetic trichotoxin A50/E. In the natural peptides, bonds formed between Aib (Iva) and Pro (Hyp) were rapidly and selectively cleaved within 0.5 h. Furthermore, TFA esters of the C‐terminal amino alcohols were formed. Depending on time, release of C‐terminal tri‐ and tetrapeptides as well as amino acids from the major fragments was observed. Synthetic homooligopeptides, namely Z‐ and Ac‐(Aib)₁₀‐O^tBu and Z‐(Aib)₇‐O^tBu, were analyzed for comparison. On treatment with TFA, a regular series of Z‐(Aib)_10–5‐OH from Z‐(Aib)₁₀‐O^tBu were detected within 0.5 h, and, after 3 h, release of a regular series of Z‐(Aib)_7–3‐OH from Z‐(Aib)₇‐O^tBu were observed. Moreover, concomitant release of the series of H‐(Aib)_10–3‐OH from the decapeptide occurred. From these data, a repetitive cleavage mechanism via intermediate formation of C‐terminal oxazolones on trifluoroacetolysis is proposed. Furthermore, their formation and stability in native peptaibols are correlated with subtle structural differences in protein amino acids linked to Aib. From the conspicuous concordance of the formation and abundance of regular series of trifluoroacetolytic fragments and of positive ions of the b‐series in CID‐MS, the generation of intermediate oxazolonium ions in both gas and liquid phase is concluded.     

A rapid and easy protein N‐terminal profiling strategy using (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) labeling and StageTip

Protein N‐terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N‐terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N‐terminal peptide losses. In this study, a fast, one‐step approach utilizing (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N‐terminal peptide enrichment and analysis. Based on the characteristics of TMPP‐derivatized samples, such as a higher hydrophobicity and increased likelihood to produce a and b ions in collision‐induced dissociation or HCD fragmentation modes, first the SDS‐PAGE was optimized to increase protein loading and gel entry and to remove unbound TMPP. Then, this process was combined with a simplified StageTip separation and a new scoring criterion (considering a, b and y ions) to identify more TMPP‐modified N‐terminal spectra. When utilizing a low amount of starting material (～20 μg protein), a total of 581 yeast N‐terminal peptides were identified, with 485 of them being TMPP modified, in only about one third of the general experimental time. It is hoped that the workflow constructed herein will provide a fast and practical strategy for N‐terminomic studies.     

Development of novel mass spectrometric methods for identifying HOCl‐induced modifications to proteins

Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label‐free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl‐oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS² analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS³ fragment ions from the immonium ions and collisionally‐activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS³ fragment ions were also identified for 2‐hydroxytryptophan and 5‐hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.     

Comparative Analyses of Data Independent Acquisition Mass Spectrometric Approaches: DIA,WiSIM‐DIA,and Untargeted DIA

Data‐independent acquisition (DIA) is an emerging technology for quantitative proteomics. Current DIA focusses on the identification and quantitation of fragment ions that are generated from multiple peptides contained in the same selection window of several to tens of m/z. An alternative approach is WiSIM‐DIA, which combines conventional DIA with wide‐SIM (wide selected‐ion monitoring) windows to partition the precursor m/z space to produce high‐quality precursor ion chromatograms. However, WiSIM‐DIA has been underexplored; it remains unclear if it is a viable alternative to DIA. We demonstrate that WiSIM‐DIA quantified more than 24 000 unique peptides over five orders of magnitude in a single 2 h analysis of a neuronal synapse‐enriched fraction, compared to 31 000 in DIA. There is a strong correlation between abundance values of peptides quantified in both the DIA and WiSIM‐DIA datasets. Interestingly, the S/N ratio of these peptides is not correlated. We further show that peptide identification directly from DIA spectra identified >2000 proteins, which included unique peptides not found in spectral libraries generated by DDA.     

Balancing robust quantification and identification for iTRAQ: Application of UHR‐ToF MS

iTRAQ reagents allow the simultaneous multiplex identification and quantification of a large number of proteins. Success depends on effective peptide fragmentation in order to generate both peptide sequence ions (higher mass region, 150–2200 m/z) and reporter ions (low mass region, 113–121 m/z) for protein identification and relative quantification, respectively. After collision‐induced dissociation, the key requirements to achieve a good balance between the high and low m/z ions are effective ion transmission and detection across the MS/MS mass range, since the ion transmission of the higher m/z range competes with that of the low m/z range. This study describes an analytical strategy for the implementation of iTRAQ on maXis UHR‐Qq‐ToF instruments, and discusses the impact of adjusting the MS/MS ion transmission parameters on the quality of the overall data sets. A technical discussion highlights a number of maXis‐specific parameters, their impact of quantification and identification, and their cross‐interactions.     

C-terminal sequencing by mass spectrometry: application to gelatine-derived proline-rich peptides

Protonated peptides derived from proline‐rich proteins (PRP) are often difficult to sequence by standard collision‐induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N‐terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al., Analyst 2004, 129, 111–115). Herein, we report the identification of these marker peptides using the strategy of C‐terminal sequencing by CID MS from their sodium and lithium adducts. Upon fragmentation a new cationized peptide was produced that is one C‐terminal amino acid shorter in length. This dissociation pathway allowed for the facile identification of the C‐terminal residue by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry. Each newly formed cationized peptide was further fragmented by up to seven stages of electrospray ionization ion trap MS. Proline‐rich C‐terminal sequence tags were established which permitted successful database identification of collagen alpha type I proteins.     

PAS‐cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry

Mass spectrometers equipped with matrix‐assisted laser desorption/ionization (MALDI‐MS) require frequent multipoint calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS‐cal tailored for MALDI‐MS based bottom‐up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C‐terminal arginine residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multipoint calibration of MS spectra using PAS‐cal peptides compares well to current commercial reagents for protein identification by PMF. Calibration of tandem mass spectra from LC‐MALDI experiments using the longest peptide, PAS‐cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS‐cal standard generically useful for bottom‐up proteomics.     

ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

Cn‐AMP2 from green coconut water is an anionic anticancer peptide

Globally, death due to cancers is likely to rise to over 20 million by 2030, which has created an urgent need for novel approaches to anticancer therapies such as the development of host defence peptides. Cn‐AMP2 (TESYFVFSVGM), an anionic host defence peptide from green coconut water of the plant Cocos nucifera, showed anti‐proliferative activity against the 1321N1 and U87MG human glioma cell lines with IC₅₀ values of 1.25 and 1.85 mM, respectively. The membrane interactive form of the peptide was found to be an extended conformation, which primarily included β‐type structures (levels > 45%) and random coil architecture (levels > 45%). On the basis of these and other data, it is suggested that the short anionic N‐terminal sequence (TES) of Cn‐AMP2 interacts with positively charged moieties in the cancer cell membrane. Concomitantly, the long hydrophobic C‐terminal sequence (YFVFSVGM) of the peptide penetrates the membrane core region, thereby driving the translocation of Cn‐AMP2 across the cancer cell membrane to attack intracellular targets and induce anti‐proliferative mechanisms. This work is the first to demonstrate that anionic host defence peptides have activity against human glioblastoma, which potentially provides an untapped source of lead compounds for development as novel agents in the treatment of these and other cancers. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Optimized Temperature Effect of Li‐Ion Diffusion with Layer Distance in Li(NixMnyCoz)O2 Cathode Materials for High Performance Li‐Ion Battery

Understanding and optimizing the temperature effects of Li‐ion diffusion by analyzing crystal structures of layered Li(Ni_xMn_yCo_z)O₂ (NMC) (x + y + z = 1) materials is important to develop advanced rechargeable Li‐ion batteries (LIBs) for multi‐temperature applications with high power density. Combined with experiments and ab initio calculations, the layer distances and kinetics of Li‐ion diffusion of LiNi_xMn_yCo_zO₂ (NMC) materials in different states of Li‐ion de‐intercalation and temperatures are investigated systematically. An improved model is also developed to reduce the system error of the “Galvanostatic Intermittent Titration Technique” with a correction of NMC particle size distribution. The Li‐ion diffusion coefficients of all the NMC materials are measured from ?25 to 50 °C. It is found that the Li‐ion diffusion coefficient of LiNi_0.6Mn_0.2Co_0.2O₂ is the largest with the minimum temperature effect. Ab initio calculations and XRD measurements indicate that the larger Li slab space benefits to Li‐ion diffusion with minimum temperature effect in layered NMC materials.     

Collision‐induced thermochemistry of reactions of dissociation of glycyl–homopeptides—An experimental and theoretical analysis

The research draws on experimental and theoretical data about energetics and kinetics of mass spectrometric (MS) reactions of glycyl homopenta– ( G5 ) and glycyl homohexapeptides ( G6 ). It shows the great applicability of the methods of quantum chemistry to predict MS profile of peptides using energetics of collision induced dissociation (CID) fragment species. Mass spectrometry is among irreplaceable methods, providing unambiguous qualitative, quantitative and structural information about analytes, applicable to many scientific areas like environmental chemistry; food chemistry; medicinal chemistry; and more. Our study could be considered of substantial interdisciplinary significance, where MS proteomics is widely used. The experimental design involves electrospray ionization (ESI) and CID MS/MS. Theoretical design is based on ab initio and density functional theory (DFT) methods. Experimental MS and theoretical free Gibbs energies as well as rate constants of fragment reactions are compared. The thermodynamic encompasses gas–phase and polar continuum analysis, including polar protic and aprotic solvents within temperature T = 10–500 K; dielectric constant ε = 0–78, pH, and ionic strengths μ = 0.001–1.0 mol dm^?1. There are computed and discussed 39 protonated forms of peptides at amide N– and –(NHC)= O centers; corresponding fragment ions studying their thermodynamic stability depending on experimental conditions. A correlation analysis between molecular conformations of parent ions and fragment species; their proton accepting ability and internal energy distribution is carried out. Data about ionization potentials (IPs) and electron affinities (EAs) are discussed, as well.     

Mass spectrometric analysis of rabbit and bovine trypsin-solubilized cytochrome b5

The sequence and blocking group of the amino-terminal 15 amino acids of rabbit trypsin-solubilized cytochrome b₅ were determined by liquid secondary ion mass spectrometry (LSIMS) and tandem mass spectrometry (MS/MS). The molecular weights of peptides generated from aStaphylococcus aureus V8 protease digest of this protein were determined by LSIMS analysis and the two peptides containing the blocked amino-terminus were sequenced by tandem mass spectrometry to yield the sequence; N-acetyl-Ala-Ala-Glu-Ser-Asp-Lys-Asp-Val-Lys-Tyr-Tyr-Thr-Leu-Glu-Glu. Comparison of this sequence with a recently reported cDNA sequence (Dariushet al., 1988) indicates that Gln at position 3 is selectively deamidated, although no other discrepancies were found. Intact rabbit and bovine trypsin-solubilized cytochrome b₅ were also analyzed by LSIMS on a high-field mass spectrometer equipped with a diode array detector. Mass measurement of the unresolved protonated molecular ion peak tops gave average molecular weights of 9462.2±2 and 9502.3±2 for bovine and rabbit trypsin-solubilized cytochrome b₅, respectively. In both cases, these molecular weights correspond to a cytochrome b₅ fragment consisting of amino acids Asp(7)-Arg(88). The average molecular weight for the rabbit amino-terminal-blocked form of trypsin-solubilized cytochrome b₅ was found to be 10,144.5±2, which was consistent with the molecular weight predicted for the extended N-acetylated form (residues 1–88) of M_r 10,146.1.     

N‐terminal diproline and charge group effects on the stabilization of helical conformation in alanine‐based short peptides: CD studies with water and methanol as solvent

Protein folding problem remains a formidable challenge as main chain, side chain and solvent interactions remain entangled and have been difficult to resolve. Alanine‐based short peptides are promising models to dissect protein folding initiation and propagation structurally as well as energetically. The effect of N‐terminal diproline and charged side chains is assessed on the stabilization of helical conformation in alanine‐based short peptides using circular dichroism (CD) with water and methanol as solvent. A1 (Ac–Pro–Pro–Ala–Lys–Ala–Lys–Ala–Lys–Ala–NH₂) is designed to assess the effect of N‐terminal homochiral diproline and lysine side chains to induce helical conformation. A2 (Ac–Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) and A3 (Ac–d Pro–Pro–Glu–Glu–Ala–Ala–Lys–Lys–Ala–NH₂) with N‐terminal homochiral and heterochiral diproline, respectively, are designed to assess the effect of Glu...Lys (i , i  + 4) salt bridge interactions on the stabilization of helical conformation. The CD spectra of A1 , A2 and A3 in water manifest different amplitudes of the observed polyproline II (PPII) signals, which indicate different conformational distributions of the polypeptide structure. The strong effect of solvent substitution from water to methanol is observed for the peptides, and CD spectra in methanol evidence A2 and A3 as helical folds. Temperature‐dependent CD spectra of A1 and A2 in water depict an isodichroic point reflecting coexistence of two conformations, PPII and β‐strand conformation, which is consistent with the previous studies. The results illuminate the effect of N‐terminal diproline and charged side chains in dictating the preferences for extended‐β, semi‐extended PPII and helical conformation in alanine‐based short peptides. The results of the present study will enhance our understanding on stabilization of helical conformation in short peptides and hence aid in the design of novel peptides with helical structures. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Identifying N‐terminal peptides by a combination of the edman procedures with a bromine isotope tag: Application to the silicateins

Silicateins are proteins found within spicules of siliceous sponges. They are analogs of proteinases cathepsins; they catalyze the transformation of silicic acid esters into biogenic silica (SiO₂·nH₂O), and are believed to take part in the processes of silicification in marine and freshwater sponges. Earlier studies by Kalyuzhnaya et al. revealed that the Baikal Sponge Lubomirskia baicalensis Pallas, 1773 (L. baicalensis) contains a gene 1988 bp long, which hosts four sequences that encode four mRNAs giving rise to silicateins α1, α2, α3 and α4 (SILα1, SILα2, SILα3, SILα4) whose predicted amino acid sequences are similar to those of the predicted sequences of marine sponge silicateins. However, the sequences of mature silicateins of L. baicalensis remained unknown, since their N‐terminal peptides were not identified. We found the sequences of these N‐terminal peptides using a combination of the Edman procedure, which involved reaction with phenylisothiocyanate, treatment with trifluoroacetic acid and trypsinolysis followed by treatment with 4‐bromine‐phenylisothiocyanate performed directly within polyacrylamide gel bands, and subsequent mass spectrometry. The N‐terminal peptides are YAESIDWR (SILα1), YVDSIDWR (SILα2 and α4), and YADSLDWR (SILα3). All mature silicateins of L. baicalensis had a length 217 amino acid residues.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Lipopeptides Produced by a Soil <Emphasis Type="Italic">Bacillus Megaterium</Emphasis> Strain

A soil microorganism identified as Bacillum megaterium was found to produce several antibiotics substances after growth for 20 h at 37°C in a mineral culture medium. Analysis both by electron spray ionization (ESI) and matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry (MS) identified these substances as lipopeptides. Predominant peaks at m/z 1,041 and m/z 1,065 revealed ions which are compatible with surfactins and lichenysins, respectively. Two other ions m/z 1,057 and m/z 1,464 were further studied by collision-induced dissociation (CID) unveiling an iturin A at the first and fengycins A and B at the second m/z peaks. The CID spectrum of the m/z 1,464 ion also suggests the existence of fengycins A and B variants in which Ile was changed to Val in the position 10 of the peptide moiety. Raw mixtures of all these compounds were also assayed for antibiotic features. The data enlighten the unusual diversity of the lipopeptide mixture produced by a sole Bacillus species.     

Distinct functions of serial metal‐binding domains in the Escherichia coli P1B‐ATPase CopA

P_1B‐ATPases are among the most common resistance factors to metal‐induced stress. Belonging to the superfamily of P‐type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P_1B‐ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N‐terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal‐binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein‐internal pathway of copper and demonstrate the distal N‐terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane‐integral ion‐binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P_1B‐ATPases, which is governed by the length of the inter‐domain linker.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self‐assembling peptides and a C‐terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self‐assembling peptide. Upon simple separation, the target protein or peptide with an authentic N‐terminus is then released in the solution by intein‐mediated cleavage. Different combinations of four self‐assembling peptides (ELK16, L₆KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI‐CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI‐CM, which has pH‐shift inducible cleavage, was found to work well with three self‐assembling peptides (L₆KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx‐strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N‐terminus, which is especially effective for peptides of 30‐100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.