Self-cleaving Intein Research Articles

A novel protein purification scheme based on salt inducible self-assembling peptides

BackgroundProtein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release.ResultsIn this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72–97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%.ConclusionOur results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.

Use Intein Cleavable Polyhydroxyalkanoate Synthase Fusions to Improve Protein Solubility.

Recombinant E. coli producing intein-cleavable polyhydroxyalkanoate synthase fusions mediates the intracellular formation of polyhydroxyalkanoate (PHA) particles densely coated with intein-cleavable target protein fusion. These PHA particles can be efficiently purified from lysed cells. The self-cleaving intein performs as a bio-linker between the PHA synthase and the target protein. The tagless target protein can be released as pure soluble protein from the PHA particles by a simple pH reduction to 6.0. Here we describe that PHA particles serve as bioseparation resin for purification of soluble target proteins with pharmaceutical grade purity, similar to commercial affinity separation technologies. This cost-effective technique does not involve multiple complicated protein purification procedures, and we have exploited this approach to purify six target proteins: green fluorescent protein (GFP) from A. victoria, antigen Rv1626 from M. tuberculosis, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).

Intein-mediated intracellular production of active microbial transglutaminase in Corynebacterium glutamicum

The microbial transglutaminase (mTGase) from Streptomyces mobaraense is widely used in the food industry. However, recombinant production of mTGase is challenging because the mTGase is synthesized as an inactive zymogen, and needs to be activated by proteolytic processing. In this study, self-cleaving intein Ssp DnaB was applied to activate the mTGase in Corynebacterium glutamicum. Premature cleavage of intein Ssp DnaB also occurred, but instead of suppressing premature cleavage, this phenomenon was used to produce active mTGase in C. glutamicum. Both SDS-PAGE analysis and mTGase activity assays indicated that the premature cleavage of intein Ssp DnaB activated the mTGase intracellularly in C. glutamicum. The subsequent N-terminal amino acid sequencing and site-directed mutagenesis studies further showed that the premature cleavage activated the mTGase intracellularly, in a highly specific manner. Moreover, the growth performance of C. glutamicum was not noticeably affected by the intracellular expression of active mTGase. Finally, the mTGase was produced in a 2 L bioreactor, with activity up to 49 U/mL, the highest intracellular mTGase activity ever reported. Using premature cleavage of intein Ssp DnaB to activate mTGase in C. glutamicum, we produced high levels of intracellular active mTGase. Moreover, this approach did not require any further processing steps, such as protease treatment or lengthy incubation, greatly simplifying the production of active mTGase. This efficient and simple approach has great potential for the large-scale industrial production of active mTGase.

Expression and one step intein-mediated purification of biologically active human G-CSF in Escherichia coli.

Recombinant form of granulocyte colony stimulating factor (G-CSF) was first approved by FDA in 1998 for chemotherapy induced neutropenia. However, despite production of its biosimilars, less expensive production of G-CSF could reduce the overall therapeutic cost. The aim of this study was to evaluate the possibility of producing biologically active recombinant G-CSF via a single step purification procedure mediated by a self-cleavable intein. G-CSF was expressed by E. coli BL21 (DE3) through IPTG induction, followed by its purification using pH optimization on a chitin column. Western blotting, ELISA, size exclusion chromatography, circular diachorism, peptide mapping, and in vitro assays were performed to compare the structural similarity and biological activity of the purified G-CSF with Neupogen™. Protein purification was confirmed by revealing a band of approximately 18.8kDa on SDS-PAGE. Bioactivity and physicochemical assays based on the US pharmacopeia showed almost identical or acceptable ranges of similarities between recombinant G-CSF and Neopogen™. this study, biologically active soluble recombinant G-CSF was successfully produced with high purity without using chaotropic solvents through a one-step procedure. This shorter and more efficient purification procedure can reduce the cost and time of G-CSF production which makes its industrial production more cost-effective and might be also applicable for production of other biopharmaceuticals.

Designing an ELP-intein system: toward a more realistic outlook

Despite the ever-growing demand for proteins in pharmaceutical applications, downstream processing imposes many technical and economic limitations to recombinant technology. Elastin-like polypeptides tend to aggregate reversibly at a specific temperature. These biopolymers have been joined with self-cleaving inteins to develop a non-chromatographic platform for protein purification without the need for expensive enzymatic tag removal. Following the design and expression of an ELP-intein-tagged GFP, herein, we report certain complications and setbacks associated with this protein purification system, overlooked in previous studies. Based on our results, a recovery rate of 68% was achieved using inverse transition cycling. Fluorescence intensity analysis indicated a production yield of 11 mg GFP fusion protein per liter of bacterial culture. The low expression level is attributable to several factors, such as irreversible aggregation, slipped-strand mispairing or insufficiency of aminoacyl tRNAs during protein translation of the highly repetitive ELP tag. While the goals we set out to achieve were not entirely met, a number of useful tips could be gathered as a generic means for implementing ELP-intein protein purification. Overall, we believe that such reports help clarify the exact capacity of emerging techniques and build a fairly realistic prospect toward their application.

Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli

Uricase as an important healthcare-related protein is extensively used in the treatment of tumor lysis syndrome and in the manufacture of serum uric-acid diagnostic kits. In this study, a gene of a new thermostable uricase (KmUOX) was cloned from thermotolerant yeast Kluyveromyces marxianus. The uricase was fused with a self-cleaving intein and cellulose-binding affinity tag and expressed in Escherichia coli BL21 (DE3). Through the binding to inexpensive cellulose and in situ intein cleavage induced by a pH change, tag-free uricase (KmUOX) was efficiently purified with a 77.11% yield via a single-step column purification strategy. This tag-free uricase showed Km, Vmax, and Kcat values of 67.60µM, 56.35µM/(minmg), and 32.74S-1, respectively. Furthermore, this pure uricase was relatively thermostable and retained 79.75% of activity when incubated at 40°C for 90h. Thus, this pH-induced self-cleavable intein system combined with a cellulose matrix for affinity chromatography is proven here to be an effective and low-cost method for recombinant-uricase purification. Moreover, the stability of KmUOX makes it useful for clinical applications.

High efficient prokaryotic expression and purification of bioactive human growth hormone using a cleavable self-aggregating tag

Human growth hormone (hGH) is synthesized by the anterior pituitary gland and promotes cell proliferation and growth. This protein has been authorized to use for the treatment of various human growth disorders and until recently, substantial efforts have been made to upgrade the previous introduced strategies. Due to the small size of hGH and absence of posttranslational modifications, Escherichia coli is the ideal host for hGH production. In the present work, we employed a previously established cleavable self-aggregating tag (cSAT) for the expression and purification of hGH in BL21 (DE3) strain of E. coli to evaluate its effectiveness. The tag is composed of a self-cleavable intein and a self-assembling peptide ELK16 (Mxe GyrA intein-ELK16). At the first step, an active insoluble aggregate of the recombinant hGH-Mxe GyrA intein-ELK16 fusion protein was expressed through an efficient T7 based expression system and then purified with a simple centrifugation. The thiol reagent dithiothreitol (DTT) was then added to induce the intein-mediated cleavage and as a result the peptide released into the soluble fraction. Afterward, the hGH production was determined by SDS-PAGE and then the final concentration of released hGH was measured by the Bradford assay (4.96 mg/ml). Furthermore, the bioactivity of purified hGHs was confirmed by calculating its growth-stimulating effect using Nb2 cell line proliferation assay. All in all, the current study offers a straightforward and fast procedure for the production of pure and bioactive hGH in E. coli.

Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions

BackgroundRecombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction.ResultsBy translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).ConclusionsHere a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

Cell‐free production of a therapeutic protein: Expression, purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care.

High-throughput purification of recombinant proteins using self-cleaving intein tags

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.

Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme

Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers' attention. Self-cleaving intein mediated single column purification is one of these novel approaches. Self-cleaving inteins are modified forms of natural inteins that can excise and join only at one junction site. In this study, the synthetic gene of Aspergillus flavus uricase, a homotetrameric protein, was cloned into pTXB1 vector as a fusion with the N-terminal of Mxe GyrA intein and chitin-binding domain (CBD) for simple purification. Expression was confirmed by western blot analysis. The fusion protein containing uricase-intein-CBD was purified on a chitin column. The cleavage was induced by adding DTT,1 as a reducing agent to release uricase. The purity of uricase and complete excision of the intein and CBD were confirmed by SDS-PAGE2 while its proper folding was proved by circular dichroism and fluorescent emission studies. Isoelectric focusing further confirmed its homogeneity when a single protein band was observed at the predicted pI value. This is the first report of successful purification of a multimeric therapeutic enzyme by intein-mediated protein cleaving using a well-established and facile system.

Recombinant expression of porcine lactoferrin peptide LF-6 with intein technology and its immunomodulatory function in ETEC K88-infected mice

LF-6 is a modified antibacterial peptide derived from LFP-20, a major active ingredient of porcine lactoferrin, whose antibacterial activity is 200 times higher than its native protein counterpart. Moreover, LF-6 displays even higher antibacterial activity than LFP-20 and negligible toxic adverse effects, make it a potential therapeutic agent for antibacterial purposes. Escherichia coli expression system has been a preferred choice and workhorse for most recombinant proteins. However, LF-6 must be coexpressed with a fusion partner to avoid its potentially fatal toxicity which would threat E. coli expression system. In this study, we successfully introduced intein system to solve this problem, which LF-6 was N-terminally fused to dithiothreitol (DTT)-induced self-cleavable intein, and it conduct cleavage when the intein-fusion peptide passing through a chromatography column filled with chitin, then the spliced peptide was purified with RP-HPLC and identified with mass spectroscopy. A bacteriostatic test showed that the recombinant LF-6 displayed nearly the same antibacterial activity as the chemically synthetized LF-6, and an in vivo immunoprotection analysis showed that the recombinant LF-6 exerted protective effects on Escherichia coli (ETEC)-K88-infected mice, which significantly reduced the pro-inflammatory cytokines level in plasma and intestine, and resistant to intestinal mucosal injury compared to the infective alone groups. Our study indicates that the intein system allows a safe and efficient method to produce recombinant LF-6, which not only has antibacterial activity, but more importantly, has an immunomodulatory function.

Spider silk-like proteins derived from transgenic Nicotiana tabacum.

The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids.

Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

BackgroundIn the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule.ResultsIn this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0–7.5 μg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme.ConclusionsThe all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a “pull-down” tag to produce purified soluble proteins with reasonable quantity and purity from active aggregates. Owing to the structural simplicity, strong hydrophobicity, and high aggregating efficiency, these peptides can be further explored for enzyme production and immobilization.

Protein Affinity Purification using Intein/Chitin Binding Protein Tags.

Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protein of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.

Ligation-independent cloning and self-cleaving intein as a tool for high-throughput protein purification

The rapid production of purified recombinant proteins has become increasingly important for countless applications. Many purification methods involve expression of target proteins in fusion to purification tags, which often must be removed from the target proteins after purification. Recently, engineered inteins have been used to create convenient self-cleaving tags for tag removal. Although intein methods can greatly simplify protein purification, commercially available expression vectors still rely on conventional restriction/ligation cloning methods for target gene insertion. We have streamlined this process by introducing Ligation-Independent Cloning (LIC) capability to our intein expression plasmids, which provides a simple method for constructing self-cleaving tag-target gene fusions. In this work, we demonstrate efficient gene insertion via this system, as well as target protein expression and purification consistent with previously reported results. Through this newly developed system, arbitrary protein genes can be rapidly incorporated into self-cleaving tag expression vectors, and their products purified using convenient platform methods.

BioSpotlight

BioTechniquesVol. 54, No. 4 BioSpotlightOpen AccessBioSpotlightPatrick Lo & Nathan BlowPatrick LoSearch for more papers by this author & Nathan BlowSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114004AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Playing tag with proteinsNon-chromatographic methods for protein purification are attractive in light of the expense and time required for chromatography. Perhaps the most promising of these approaches has been the use of protein or peptide stimulus-responsive tags that allow the rapid and reversible precipitation of target proteins from complex solutions. After isolation of the fusion protein, the tag can be removed by inducing a coupled self-cleaving intein domain or through treatment with a site-specific protease. A number of these tags, however, require heat or high salt treatment to induce their precipitation, which can be potentially deleterious for some target proteins. In this month's issue of BioTechniques, S. Banta and colleagues at Columbia University (New York, NY) describe a gentler approach using a synthetic peptide tag they developed that can be reversibly precipitated in response to calcium. The new tag came about as a serendipitous discovery during the authors’ research into repeat scaffolds for stimulus-responsive protein engineering applications. The calcium-responsive repeat-in-toxin (RTX) domain is found in various bacterial proteins secreted through the type I system and consists of repeats of a nine-amino-acid sequence. After designing a consensus RTX repeat sequence, the authors found that constructs containing multiple repeats of the consensus unit fused to the C-terminus of maltose-binding protein (MBP) caused its precipitation in the presence of calcium. Precipitation was most efficient for constructs with 13 or 17 consensus repeats and was easily reversed upon the addition of EGTA. Of several cations tested, only calcium could induce precipitation when the 17-repeat tag was fused to various target proteins. High yields of all the fusion proteins were obtained, and the functionality of the fused target proteins was also retained. To increase the utility of this tag, an enterokinase cleavage site was engineered between the tag and the target protein. Cleavage of the isolated fusion protein with enterokinase, followed by calcium-induced precipitation of the tag, left the purified target protein in solution. This new calcium-precipitable tag should prove to be a welcome and valuable new method for the rapid and selective purification of recombinant proteins.See “A designed, phase-changing RTX-based peptide for efficient bioseparations”Biochemistry's 15-minute workoutAffinity isolation of protein complexes can be greatly enhanced by speed–the faster the time between isolation and a downstream assay, the better the chance everything is completely recovered in a native state. One widely used affinity isolation system is based on the interaction between S. aureus Protein-A (SpA) and immunoglobulin G (IgG). Here, a bait protein is tagged with SpA and interacting partners are isolated using either competitive elution or cleavage. When it comes to cleavage, the reaction may not be uniform and also tends to be rather slow. On the other hand, while competitive elution is more uniform, it also requires several hours of incubation. In this issue of BioTechniques, John LaCava and his colleagues from Rockefeller University (New York, NY) describe the design of an improved reagent for the competitive elution of SpA tagged native complexes that works within 15 minutes under very mild conditions. Previously, the authors described a modified peptide generated from the Fc fragment of IgG called Bio-Ox that requires a 2-hour or longer incubation for competitive elution. They reasoned that by increasing the solubility of this peptide, it might be possible to reduce the elution time. The original Bio-Ox peptide was modified at the N terminus to achieve increased solubility, so LaCava and his team tested alternative substitutions at that same N-terminal portion of the peptide, identifying an additional polyethylene glycol (PEG) moiety of four unit lengths that was most effective in increasing solubility. Comparisons between Bio-Ox and the newly generated peptide PEGylOx showed that PEGylOx could effectively release 60%-85% of two test protein complexes within 15 minutes while Bio-Ox was unable to release either of these complexes within that time period. The authors also demonstrated that, similar to the original Bio-Ox, PEGylOx could be removed from a sample using a simple spin column with a 40kDa molecular weight cutoff for downstream assays. This newly improved peptide should prove highly advantageous to the large number of researchers taking advantage of SpA-tags in their affinity isolation workflows.PEGylOx structure and effectiveness as an elution reagentSee “Improved native isolation of endogenous protein-A tagged protein complexes”FiguresReferencesRelatedDetails Vol. 54, No. 4 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print April 2013 Information© 2013 Author(s)PDF download

Facile expression and purification of the antimicrobial peptide histatin 1 with a cleavable self-aggregating tag (cSAT) in Escherichia coli

Human histatin 1 (Hst1), a member of the histatin family, possesses antimicrobial properties. In this study, we applied a previously developed cleavable self-aggregating tag (cSAT) for the expression and purification of histatin 1 to demonstrate its utility for peptide expression and purification. The tag consists of a self-cleavable intein and a self-assembling peptide ELK16 (I-ELK16). First, an active insoluble aggregate of the recombinant histatin 1-Mxe GyrA intein-ELK16 (Hst1-I-ELK16) fusion protein was produced with a yield of 28.9μg/mg wet cell pellet. The thiol reagent dithiothreitol (DTT) was then used to induce the intein-mediated cleavage and peptide release into the soluble fraction with a yield of 2.06μg/mg wet cell pellet and a purity of 70%. The peptide was further purified by high performance liquid chromatography. These results were comparable to the yield and purity achieved when the more conventional glutathione transferase (GST) tag was used. The antimicrobial activities of this recombinant histatin 1 were confirmed against three Candida strains. This cSAT technique offers considerable advantages in terms of its simplicity and speed, eliminating the need for an exogenous protease, and reducing the number of chromatography purification steps. This technique should also be useful for the expression and purification of other AMPs.

Facilitated Substrate Channeling in a Self‐Assembled Trifunctional Enzyme Complex

Most cascade enzymes in metabolic pathways are spatially held together by noncovalent protein–protein interactions. The formation of a cascade enzyme complex often allows the product of one enzyme to be transferred to an adjacent enzyme where it acts as the substrate, thereby resulting in an enhanced reaction rate, because reaching equilibrium in the cytoplasm is not required; this mechanism is called substrate channeling. In nature, most intracellular enzyme complexes are dynamic so that they may be dissociated or associated, thereby resulting in forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of metabolite inhibition, and circumvention of unfavorable equilibrium and kinetics. The simplest way to facilitate substrate channeling between cascade enzymes is the construction of fusion proteins, but substrate channeling in fusion proteins might not take place. The assembly of numerous enzymes and/or co-enzymes in vitro is called cascade enzyme biocatalysis and has been proposed for the implementation of complicated bioconversion that microbes and chemical catalysts cannot do, such as hydrogen production from cellulosic materials and water with high yield. Inspired by natural enzyme complexes (e.g., metabolons, which are complexes of sequential enzymes of a metabolic pathway), the construction of static rather than dynamic enzyme complexes could be an important approach to accelerating reaction rates among cascade enzymes and to avoiding the regulation of enzyme–enzyme interactions. For example, Wilner et al. linked glucose oxidase and horseradish peroxidase by DNA scaffolds of different lengths, resulting in reaction rates that were enhanced by 20–30-fold. However, DNA scaffolds may be too costly for scale-up as compared to protein scaffolds. Minteer and co-workers demonstrated that chemical cross-linking of proteins within the mitochondria of Saccharomyces cerevisiae resulted in significant increases of the power output in enzymatic fuel cells. But chemical covalent linking often impairs enzyme activity so that it may not be applied to most intracellular enzymes. Herein we demonstrate a general approach for constructing a static self-assembled enzyme complex by using the highaffinity interaction between cohesin and dockerin modules, which occur in natural extracellular complexed cellulase systems, called cellulosomes. Cohesin domains are part of the natural scaffoldin protein of the cellulosome, which is crucial to the construction of the cellulase complex by binding to enzymes carrying dockerin domains. Bayer et al. proposed to construct designed enzyme complexes by utilizing speciesspecific dockerins and cohesins, which can bind tightly in these complexes at a molar ratio of 1:1. Later, several synthetic mini-cellulosomes containing various extracellular glycoside hydrolases were constructed. However, no one attempted to construct an enzyme complex containing cascade enzymes from a metabolic pathway by using dockerins and cohesins and investigated its potential applications in cascade enzyme biocatalysis. Triosephosphate isomerase (TIM, EC 5.3.1.1), aldolase (ALD, EC 4.1.2.13), and fructose 1,6-bisphosphatase (FBP, EC3.1.3.11) are cascade enzymes in the glycolysis and gluconeogenesis pathways. TIM catalyzes the reversible conversion of glycer-aldehyde-3-phosphate (G3P) to dihydroxy-acetone phosphate (DHAP). ALD catalyzes the reversible aldol condensation of G3P and DHAP to fructose-1,6bisphosphate (F16P). FBP catalyzes the irreversible conversion of F16P to fructose-6-phosphate (F6P; Scheme 1). Previous studies reported that substrate channeling existed in dynamic metabolons of enzymes such as TIM, ALD, or FBP. Three dockerin-free proteins: Thermus thermophilus HB27 TIM (TTC0581) as well as the Thermotoga maritima ALD (TM0273) and FBP (TM1415) were expressed in E. coli and purified to homogeneity by using nickel–nitrilotriacetate (Ni–NTA) resin or a self-cleaving intein. However, a mixture of these three enzymes did not form a putative enzyme complex, as examined by affinity electrophoresis (data not shown). The synthetic static three-enzyme complex was assembled in vitro through a synthetic trifunctional scaffoldin containing a family 3 cellulose-binding module (CBM3) at the N terminus followed by three different types of cohesins from the Clostridium thermocellum ATCC 27405 CipA, Clostridium cellulovorans ATCC 35296 CbpA, and Ruminococcus flavefaciens ScaB (cohesins CTCoh, CCCoh, and RFCoh, [*] Dr. C. You, S. Myung, Y.-H. P. Zhang Biological Systems Engineering Department Virginia Tech, 304 Seitz Hall Blacksburg, VA 24061 (USA) E-mail: ypzhang@vt.edu Homepage: http://www.sugarcar.com