Machine Learning Integrating Protein Structure, Sequence, and Dynamics to Predict the Enzyme Activity of Bovine Enterokinase Variants.

Despite recent advances in computational protein science, the dynamic behavior of proteins, which directly governs their biological activity, cannot be gleaned from sequence information alone. To overcome this challenge, we propose a framework that integrates the peptide sequence, protein structure, and protein dynamics descriptors into machine learning algorithms to enhance their predictive capabilities and achieve improved prediction of the protein variant function. The resulting machine learning pipeline integrates traditional sequence and structure information with molecular dynamics simulation data to predict the effects of multiple point mutations on the fold improvement of the activity of bovine enterokinase variants. This study highlights how the combination of structural and dynamic data can provide predictive insights into protein functionality and address protein engineering challenges in industrial contexts.

Metformin inhibits digestive proteases and impairs protein digestion in mice.

Metformin is among the most prescribed medications worldwide and the first-line therapy for type 2 diabetes. However, gastrointestinal side effects are common and can be dose limiting. The total daily metformin dose frequently reaches several grams, and poor absorption results in high intestinal drug concentrations. Here, we report that metformin inhibits the activity of enteropeptidase and other digestive enzymes at drug concentrations predicted to occur in the human duodenum. Treatment of mouse gastrointestinal tissue with metformin reduces enteropeptidase activity; further, metformin-treated mice exhibit reduced enteropeptidase activity, reduced trypsin activity, and impaired protein digestion within the intestinal lumen. These results indicate that metformin-induced protein maldigestion could contribute to the gastrointestinal side effects and other impacts of this widely used drug.

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5.

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

Directed evolution for soluble and active periplasmic expression of bovine enterokinase in Escherichia coli.

Bovine enterokinase light chain (EKL) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EKL gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by kcat/Km, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased kcat and Tm values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site.

Design, Synthesis, and Biological Evaluation of a Novel Series of 4-Guanidinobenzoate Derivatives as Enteropeptidase Inhibitors with Low Systemic Exposure for the Treatment of Obesity.

To discover a novel series of potent inhibitors of enteropeptidase, a membrane-bound serine protease localized to the duodenal brush border, 4-guanidinobenzoate derivatives were evaluated with minimal systemic exposure. The 1c docking model enabled the installation of an additional carboxylic acid moiety to obtain an extra interaction with enteropeptidase, yielding 2a. The oral administration of 2a significantly elevated the fecal protein output, a pharmacodynamic marker, in diet-induced obese (DIO) mice, whereas subcutaneous administration did not change this parameter. Thus, systemic exposure of 2a was not required for its pharmacological effects. Further optimization focusing on the in vitro IC50 value and T1/2, an indicator of dissociation time, followed by enhanced in vivo pharmacological activity based on the ester stability of the compounds, revealed two series of potent enteropeptidase inhibitors, a dihydrobenzofuran analogue ((S)-5b, SCO-792) and phenylisoxazoline (6b), which exhibited potent anti-obesity effects despite their low systemic exposure following their oral administration to DIO rats.

Comparison of Periplasmic and Cytoplasmic Expression of Bovine Enterokinase Light Chain in E. coli.

Enterokinase enzyme is widely used in production of recombinant proteins. This enzyme is isolated from the intestine and recognizes a specific cleavage site (X↓LYS-ASP4). Several studies have been performed to produce recombinant active enterokinase. In this study, the coding sequence of bovine enteropeptidase light chain (bEKL) was isolated from Iranian Sarabi cattle and its expression was investigated in the periplasm and cytoplasm of E. coli by two different expression vectors, pET22 and pET32RH. RNA was extracted from the duodenum part of cattle, cDNA was amplified, the enterokinase light chain coding fragment was cloned and the expression was examined by SDS-PAGE analysis. The higher amounts of soluble enterokinase as a fusion with thioredoxin (Trx) were detected in cytoplasmic expression. The functional enterokinase was purified with a yield of 45 mg per litter by two-steps Ni2+ affinity chromatography. The effective activity of the enzyme implies that it can be produced in large scale for biotechnological applications.

A combination strategy of solubility enhancers for effective production of soluble and bioactive human enterokinase.

Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.

Recombinant Production of Bovine Enteropeptidase Light Chain in SHuffle® T7 Express and Optimization of Induction Parameters.

Enteropeptidase is a duodenum serine protease that triggers the activation of pancreatic enzymes by remarkably specific cleavages after lysine residues of peptidyl substrate (Asp)4-Lys. This high specific cleavage makes the enzyme a widely used biotechnological tool in laboratory researches and industrial scale. Previous studies both in small and large scales were showed low expression and miss-folding of the expressed protein. In this study, the DNA sequence encoding the light chain (catalytic subunit) of bovine enteropeptidase (EPL) was subcloned into plasmid pET-32b, downstream to the DNA encoding the fusion partner thioredoxin immediately after the EPL cleavage site. SHuffle® T7 Express was selected as an expression host due to the ability to promote proper folding and correction of the mis-oxidized bonds. Expression and purification of protein was performed, and the result of biological activity confirmed that the active EPL was obtained. Optimization of protein expression conditions was accomplished by response surface methodology for significant factors including induction temperature, duration of induction, inducer concentration and OD600 of induction. The best conditions were achieved in 1.05 mM IPTG at OD600 of 0.6 for seven h incubation at 26.5 °C, and a high level of protein expression was obtained in the optimized condition.

Impact of media components from different suppliers on enterokinase productivity in Pichia pastoris.

BACKGROUND: The aim of this study was to provide an information about the homogeneity on the level of enterokinase productivity in P. pastoris depending on different suppliers of the media components. RESULTS: In previous studies, we performed the optimisation process for the production of enterokinase by improving the fermentation process. Enterokinase is the ideal enzyme for removing fusion partners from target recombinant proteins. In this study, we focused our optimization efforts on the sources of cultivation media components. YPD media components were chosen as variables for these experiments. Several suppliers for particular components were combined and the optimisation procedure was performed in 24-well plates. Peptone had the highest impact on enterokinase production, where the difference between the best and worst results was threefold. The least effect on the production level was recorded for yeast extract with a 1.5 fold difference. The worst combination of media components had a activity of only 0.15 U/ml and the best combination had the activity of 0.88 U/ml, i.e., a 5.87 fold difference. A substantially higher impact on the production level of enterokinase was observed during fermentation in two selected media combinations, where the difference was almost 21-fold. CONCLUSIONS: Results demonstrated in the present study show that the media components from different suppliers have high impact on enterokinase productivity and also provide the hypothesis that the optimization process should be multidimensional and for achieving best results it is important to perform massive process also in terms of the particular media component supplier .

Analysis of reflux as the aetiology of laryngeal dysplasia progression through a matched case-control study.

OBJECTIVES: Laryngeal dysplasia (LD) is a precancerous lesion of the larynx. In this study, the laryngeal tissue of patients with laryngeal dysplasia was taken as the research object, and the aetiology of reflux was analysed. METHOD: Patients with laryngeal dysplasia after surgery were selected as our subjects. The levels of pepsin, enterokinase and bilirubin in laryngeal tissue samples of the two groups were detected by immunohistochemical method. RESULTS: The OR values (95% CI) of pepsin, enterokinase and bilirubin were 0.67 (0.19-2.36), 0.80 (0.22-2.98) and 1.33 (0.30-5.96), respectively, in the univariate analysis. Besides, in the multivariate analysis, the OR values (95% CI) of pepsin, enterokinase and bilirubin were 0.57 (0.14-2.30), 0.73 (0.18-2.92) and 1.40 (0.30-6.53), respectively. CONCLUSION: Larger sample size should be applied to prospective studies on whether reflux is a risk factor for laryngeal cancer.

Destabilizing single chain major histocompatibility complex class I protein for repurposed enterokinase proteolysis.

The lack of a high throughput assay for screening stabilizing peptides prior to building a library of peptide-major histocompatibility complex class I (pMHC-I) molecules has motivated the continual use of in silico tools without biophysical characterization. Here, based on de novo protein fragmentation, the EASY MHC-I (EZ MHC-I) assay favors peptide antigen screening to an unheralded hands-on time of seconds per peptide due to the empty single chain MHC-I protein instability. Unlike tedious traditional labeling- and antibody-based MHC-I assays, repurposed enterokinase directly fragments the unstable single MHC-I chain protein unless rescued by a stabilizing peptide under luminal condition. Herein, the principle behind EZ MHC-I assay not only characterizes the overlooked stability as a known better indicator of immunogenicity than classical affinity but also the novel use of enterokinase from the duodenum to target destabilized MHC-I protein not bearing the standard Asp-Asp-Asp-Asp-Lys motif, which may protend to other protein instability-based assays.

Efficient Soluble Expression and Purification of Recombinant Human Acidic Fibroblast Growth Factor from Escherichia coli via Fusion with a Novel Collagen-like Protein Scl2.

Human acidic fibroblast growth factor (haFGF) is a multifunctional protein involved in regulating a wide range of cellular processes. As a potent therapeutic agent, it is highly desirable to produce recombinant haFGF (r-haFGF) at low cost. However, the complex structure and formation of aggregation confines its high-level soluble expression and functional form. Herein, to produce r-haFGF efficiently in E. coli, we devised a novel soluble expression and cost-effective purification approach based on fusion with Scl2-M (a novel modified collagen-like protein) for the first time. By using this strategy, more than 95% of the Scl2-M-haFGF fusion protein was highly expressed in soluble form and the expression level of targeted fusion protein in shake flasks and 5-L fermenter was 0.42 g/L and 2.28 g/L, respectively. Subsequently, the recombinant Scl2-M-haFGF was readily purified through a facile process of acid precipitation and subjected to enterokinase (EK) cleavage. After Scl2-M cleavage, tag-free r-haFGF was further purified using ion-exchange chromatography. The recovery rate of the whole purification process attained 34.2%. Furthermore, the resulting high-purity (96.0%) r-haFGF was prepared by freeze-drying as a final product, and its bioactivity was confirmed to potentiate the proliferation of L929 and BALB-3T3 fibroblasts. Overall, our developed method has the potential for the massive production of the r-haFGF in the future.

Effects of His-tag on Catalytic Activity and Enantioselectivity of Recombinant Transaminases.

Recombinant proteins were often expressed with His-tag to simplify the purification process. Among them, transaminase was mostly expressed with fusion tags and widely used in the production of numerous amino moieties. However, the existence of the His-tag has been reported to affect various properties of different recombinant enzymes, while the effect on transaminase was rarely studied. In this paper, we investigated the effect of His-tag on transaminase based on the various activities of 4-aminobutyrate-2-oxoglutarate transaminase (GabT) when it was expressed in vector pETDuet-1. We found that His-tag did not affect the enantioselectivity, but decreased the catalytic activity to different extents according to its existence and location. Native GabT maintained the highest catalytic activity; GabT with C-terminal His-tag showed slightly lower activity than native GabT but about 2.2-fold higher than GabT with N-terminal His-tag. Besides, other fusion tags like T7-tag and S-tag inserted between N-His-tag and GabT can relieve the decreasing effect of His-tag on GabT activity. Furthermore, whole cell catalytic activity of several transaminases was improved by deleting the N-terminal His-tag. This study provided a strategy for the efficient expression of recombinant transaminase with improved catalytic activity and might attract attention to the effect of His-tag on other enzymatic properties.

Truncated Thioredoxin Peptides Serves as an Efficient Fusion Tag for Production of Proinsulin.

BACKGROUND: Insulin is a peptide hormone used for regulating blood glucose levels. Human insulin market is projected to grow at a rate of 12.5% annually. To meet the needs of patients, a cost effective insulin manufacturing strategy has to be developed. This can be achieved by selecting a competent host, ideal fusion tag and streamlined downstream process. OBJECTIVE: In this article, we have demonstrated that selecting a right fusion partner for expression of toxic proteins like insulin, plays a major role in increasing the recombinant protein yield. METHODS: In this article, we have focused on identifying a peptide tag fusion partner for expressing proinsulin by truncating thioredoxin tag. Truncations were carried out from both Amino and Carboxy terminus of the protein and efficiency of truncated sequences was evaluated by expressing it with proinsulin gene. FCTRX (1-15) sequence fused to proinsulin was processed further to establish downstream protocol for purification. RESULTS: Thioredoxin tag was truncated appropriately by considering the fusion tag: protein ratio. A couple of sequences ranging 10 - 15 amino acids were identified based on its in silico properties. Of these FCTRX (1-15) showed increased expression and stability of fusion protein. 156 mg of purified insulin was generated from 1g of inclusion body after enzymatic conversion and chromatographic steps. CONCLUSION: As a result of the current study, it was concluded that FCTRX (1-15) peptide has advantageous attributes to be considered as an ideal fusion tag for expression of proinsulin. This can be further explored by expressing it with other proteins.

Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminase-catalyzed bioconjugation.

Enterokinase (EK) is one of the most popular enzymes for the in vitro cleavage of fusion proteins due to its high degree of specificity for the amino-acid sequence (Asp)4-Lys. Enzyme reusability is desirable for reducing operating costs and facilitating the industrial application of EK. In this work, we report the controlled, site-specific and covalent cross-linking of an engineered EKLC on amine-modified magnetic nanoparticles (NH2-MNPs) via microbial transglutaminase-catalyzed bioconjugation for the development of the oriented-immobilized enzyme, namely, EKLC@NH2-MNP biocatalyst. Upon the site-specific immobilization, approximately 90% EKLC enzymatic activity was retained, and the biocatalyst exhibited more than 85% of initial enzymatic activity regardless of storage or reusable stability over a month. The EKLC@NH2-MNP biocatalyst was further applied to remove the His tag-(Asp)4-Lys fusion partner from the His tag-(Asp)4-Lys-(GLP-1)3 substrate fusion protein, result suggested the EKLC@NH2-MNP possessed remarkable reusability, without a significant decrease of enzymatic activity over 10 cycles (P > 0.05). Supported by the unique properties of MNPs, the proposed EKLC@NH2-MNP biocatalyst is expected to promote the economical utilization of enterokinase in fusion protein cleavage.

Generation of Highly Specific Proteolytic Biocatalysts by Screening Technologies.

We propose a yeast display-based system for screening of proteolytic enzyme libraries that utilizes substrate protein adsorbed on the yeast cell surface and containing a desired cleavage sequence. Specific cleavage of the substrate protein releases its biotin-binding center. The cells carrying the target proteinase can be selected by cytofluorometry due to interaction with biotinylated fluorescent protein. Using human enterokinase light chain as the model proteinase we showed that the proposed screening system highly effectively selects the proteolytic enzymes with preset specificity.

Enterokinase monolithic bioreactor as an efficient tool for biopharmaceuticals preparation: on-line cleavage of fusion proteins and analytical characterization of released products.

One of the most popular enzymes used for the in vitro cleavage of fusion proteins is enterokinase (EK, E.C. 3.4.21.9). EK cleaves with high specificity after the sequence Asp4-Lys (DDDDK), which allows the fusion protein to preserve its native amino acid terminus without any additional unwanted cleavage residue from the recognition sequence. However, the complete removal of EK after protein cleavage is a critical step to ensure protein identity and stability. As enzyme immobilization increases stability and reusability of the biocatalyst while reducing operating costs and sample contamination, in this work we report the covalent immobilization of recombinant EK (rEK) on monolithic chromatographic supports with different binding chemistries for the development of a rEK-chromatographic-bioreactor. An on-line assay for the determination of the activity of the immobilized rEK was set up using a synthetic substrate (Gly-Asp4-Lys-ß-naphthylamide, GD4K-NA). The assay was used to study the improvement of the operational conditions (temperature and flow rate) on hydrolytic activity of the bioreactor. The immobilization yields, as well as the cleavage activity of immobilized rEK on GD4K-NA, were highly satisfactory when the immobilized enzyme reactor was used in recirculation. The ability of the immobilized rEK to cleave fusion proteins was tested by recirculation of thioredoxin (Trx)-TB10.4 and Trx-Ag85B His-tagged proteins yielding the mature antigens TB10.4 and Ag85B, to be used in the preparation of potential novel glycovaccines against tuberculosis. The prepared rEK-based immobilized enzyme reactors proved to efficiently cleave the considered fusion proteins even if the cleavage specificity at the canonical site was not fully achieved. The immobilized rEK showed very good stability and reusability.

Effects of N-glycosylation on the biochemical properties of recombinant bEKL expressed in Pichia pastoris.

Enterokinase is an ideal tool protease for cleaving fusion proteins in genetic engineering. The bovine enterokinase light chain (bEKL) produced in Pichia pastoris was shown to be a glycoprotein. To study the effects of N-glycosylation on the biochemical properties of bEKL, the enzyme was deglycosylated via site-directed mutagenesis. The results showed that elimination of the N-glycosylation sites of bEKL (N64, N103 and N165) did not significantly affect the protein secretion level in P. pastoris, but it does greatly influence its enzymatic activity. The N64Q increased the specific activity of the enzyme for GD4K-ß-naphthylamide and improved its catalytic efficiency. Moreover, the glycosylated bEKL is more thermostable than its deglycosylated counterparts. Structural analysis of glycosylated and deglycosylated bEKL revealed that the removal of N-glycosylation did not have pronounced changes on the secondary structure but there was a significant difference in the tertiary structure. In conclusion, this study demonstrated that the effects of glycosylation at different degrees and sites in bEKL were diverse. Moreover, this work will provide theoretical support for designing enzymes on the basis of N-glycosylation to meet the demands of the biochemical industry.

Enzymatic Cleavage of Branched Peptides for Targeting Mitochondria.

Most of the reported mitochondria-targeting molecules are lipophilic and cationic, and thus they may become cytotoxic with accumulation. Here we show enzymatic cleavage of branched peptides that carry negative charges for targeting mitochondria. Conjugating a well-established protein tag (i.e., FLAG-tag) to self-assembling motifs affords the precursors that form micelles. Enzymatic cleavage of the hydrophilic FLAG motif (DDDDK) by enterokinase (ENTK) turns the micelles to nanofibers. After being taken up by cells, the micelles, upon the action of intracellular ENTK, turn into nanofibers to locate mainly at mitochondria. The micelles of the precursors are able to deliver cargos (either small molecules or proteins) into cells, largely to mitochondria and within 2 h. Preventing ENTK proteolysis diminishes mitochondria targeting. As the first report of using enzymatic self-assembly for targeting mitochondria and delivery cargos to mitochondria, this work illustrates a fundamentally new way to target subcellular organelles for biomedicine.