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.

Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides.

AIMS: The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. METHODS AND RESULTS: To improve the pMAL expression vector, we introduced the His6 tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His6 -Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His6 tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His6 and His6 -enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human ß-defensin. CONCLUSIONS: We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory.

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 .

[Optimization of enterokinase secretion in Pichia pastoris].

Enterokinase is a class of serine proteases that specifically recognize the cleavage DDDDK sequences. Therefore, enterokinase has been widely used as a tool enzyme in the field of biomedicine. Currently, the expression level of enterokinase in Pichia pastoris is low, which hinders related practical applications. In this study, the effects of six different signal peptides SP1, SP2, SP3, SP4, SP7 and SP8 on the secretory expression of enterokinase in Pichia pastoris were studied. Compared with α-factor, SP1 significantly increased the secretory expression of enterokinase (from 6.8 mg/L to 14.3 mg/L), and the enterokinase activity increased from (2 390±212) U/mL to (4 995±378) U/mL in shaking flask cultures. On this basis, the enterokinase activity was further enhanced to (7 219±489) U/mL by co-expressing the endogenous protein Kex2. Moreover, the activity that the mutant strain with N-terminal fusion of three amino acids of WLR was increased to (15 145±920) U/mL with a high specific activity of (1 174 600±53 100) U/mg. The efficient secretory expression of enterokinase laid a foundation for its applications in near future.

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.

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.

Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris.

Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.

Antithrombin controls tumor migration, invasion and angiogenesis by inhibition of enteropeptidase.

Antithrombin is a key inhibitor of the coagulation cascade, but it may also function as an anti-inflammatory, anti-angiogenic, anti-viral and anti-apoptotic protein. Here, we report a novel function of antithrombin as a modulator of tumor cell migration and invasion. Antithrombin inhibited enteropeptidase on the membrane surface of HT-29, A549 and U-87 MG cells. The inhibitory process required the activation of antithrombin by heparin, and the reactive center loop and the heparin binding domain were essential. Surprisingly, antithrombin non-covalently inhibited enteropeptidase, revealing a novel mechanism of inhibition for this serpin. Moreover, as a consequence of this inhibition, antithrombin was cleaved, resulting in a molecule with anti-angiogenic properties that reduced vessel-like formation of endothelial cells. The addition of antithrombin and heparin to U-87 MG and A549 cells reduced motility in wound healing assays, inhibited the invasion in transwell assays and the degradation of a gelatin matrix mediated by invadopodia. These processes were controlled by enteropeptidase, as demonstrated by RNA interference experiments. Carcinoma cell xenografts in nude mice showed in vivo co-localization of enteropeptidase and antithrombin. Finally, treatment with heparin reduced experimental metastasis induced by HT29 cells in vivo. In conclusion, the inhibition of enteropeptidase by antithrombin may have a double anti-tumor effect through inhibiting a protease involved in metastasis and generating an anti-angiogenic molecule.

Production and purification of recombinant enteropeptidase expressed in an insect-baculovirus cell system.

Enteropeptidase (EC 3.4.21.9) is the glycoprotein enzyme in the small intestine that triggers the activation of the zymogens in pancreatic juice by converting trypsinogen into trypsin. Because of its physiological significance, there have been many studies on the expression, purification, and characterization of enteropeptidase from different species. The baculovirus expression system has been commonly used in research communities and scientific industries for the production of high levels of recombinant proteins, which require posttranslational modifications for functional activity. In the present study, we isolated bovine enteropeptidase catalytic subunit gene from Bos taurus indicus (GenBank accession no. KC756844), and cloned it in pFast Bac HT "A" baculovirus expression donor vector, under the polyhedrin promoter. Recombinant bovine enteropeptidase was expressed in SF-9 insect cells with high expression levels. Recombinant enteropeptidase was purified using Ni-NTA affinity chromatography. A 6-mg quantity of pure active protein was obtained from 100 mL culture using this approach. Its activity and kinetic parameters were determined by cleavage of its fluorogenic substrate Gly-(Asp) 4-Lys-ß-naphthylamide. The recombinant bovine enteropeptidase showed a K m value of 0.75 ± 0.02 mM with K cat 25 ± 1 s.

Characterization and expression of trypsinogen and trypsin in medaka testis.

Previously, we reported that the medaka testis abundantly expresses the mRNA for trypsinogen, which is a well-known pancreatic proenzyme that is secreted into and activated in the intestine. Currently, we report our characterization of the medaka trypsin using a recombinant enzyme and show that this protein is a serine protease that shares properties with trypsins from other species. Two polypeptides (28- and 26-kDa) were detected in the testis extracts by Western blot analysis using antibodies that are specific for medaka trypsinogen. The 28-kDa polypeptide was shown to be trypsinogen (inactive precursor), and the 26-kDa polypeptide was shown to be trypsin (active protease). We did not detect enteropeptidase, which is the specific activator of trypsinogen, in the testis extract. Immunohistochemical analyses using the same trypsinogen-specific antibody produced a strong signal in the spermatogonia and spermatozoa of the mature medaka testis. Substantial staining was found with spermatocytes, whereas extremely weak signals were observed with spermatids. In vitro incubation of testis fragments with the trypsinogen antibody strongly inhibited the release of sperm from the testis into the medium. Trypsin activity was detected in sperm extracts using gelatin zymographic analysis. Immunocytochemistry showed that trypsinogen and trypsin were localized to the cell membranes surrounding the sperm head. Collectively, these results suggest that trypsin plays an important role in the testis function of the medaka.

Improvement of an antibody-enzyme coupling yield by enzyme surface supercharging.

BACKGROUND: Protein cross-coupling reactions demand high yields, especially if the products are intended for bioanalytics, like enzyme-linked immunosorbent assays. Amongst other factors, the coupling yield depends on the concentration of the proteins being used for coupling. Protein supercharging of enzymes can increase the solubility dramatically, which could promote enzyme-antibody coupling reactions. A highly soluble, supercharged variant of the enzyme human enteropeptidase light chain was created by a site-directed mutagenesis of surface amino acids, used for the production of an antibody-enzyme conjugate and compared to the wild type enzyme. RESULTS: Wild type and mutant enzyme could successfully be cross-coupled to an antibody to give antibody-enzyme conjugates suitable for ELISA. Their assay performances and the analysis of the enzyme activities in solution demonstrate that the supercharged version could be coupled to a higher extent, which resulted in better assay sensitivities. The generated conjugate, based on the supercharged enzyme, was feasible as a reporter molecule in a sandwich ELISA and allowed the detection of epidermal growth factor with a detection limit of 15.63 pg (25 pmol/L). CONCLUSION: The highly soluble, surface supercharged, human enteropeptidase light chain mutant provided better yields in coupling the enzyme to an antibody than the wild type. This is most likely related to the higher protein concentration during the coupling. The data suggest that supercharging can be applied favourably to other proteins which have to be covalently linked to other polymers or surfaces with high yields without losses in enzyme activity or specificity.

Rare genetic diseases with human lean and/or starvation phenotype open new avenues for obesity and type II diabetes treatment.

Treatments of obesity and type II diabetes target often gene functions involved in appetite-satiety, fat and carbohydrate metabolism or thermogenesis. None of these, have provided efficient drug therapy due to a large number of genes involved in weight and energy management, the redundancy of biochemical pathways and the environmental factors. Here I discuss a new approach based on studies of genes/proteins that are associated with human "lean or starvation" phenotype that became very rare in the course of evolution. This approach has led to the identification of the congenital enteropeptidase deficiency gene and the Anderson's Disease gene as a potential targets for obesity and type II diabetes treatment. The advantages of these targets are: 1) they are expressed exclusively in the intestine; 2) they are peripheral targets as opposed to systemic targets; 3) they are not redundant targets. These targets open new hopes for the development of novel drugs for the treatment of common metabolic syndrome.

Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin)-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6) M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

Surface display of human growth hormone on Bacillus subtilis spores for oral administration.

Human growth hormone (hGH) is the major and important hormone component of human being. At present, hGH for clinical uses is mostly produced in Escherichia coli, which requires costly denaturation and refolding to recover functionality. To obtain long-term bioactive hormone, we used hGH as a foreign gene and constructed a recombinant plasmid pJS700-hGH which carries a recombinant gene cotC-hgh with an enterokinase site under the control of cotC promoter. Plasmid pJS700-hGH was transformed into Bacillus subtilis by double crossover and an amylase-inactivated mutant was produced. After spore formation, Western blot and fluorescence immunoassay were used to monitor hGH surface expression on spores. Oral administration to silkworm with spores displaying hGH further showed that the recombinant spores may have potential ability to be digested and absorbed into the silkworm's hemolymph due to both the resistant characters of spores and the addition of enterokinase site.

Do-it-yourself histidine-tagged bovine enterokinase: a handy member of the protein engineer's toolbox.

Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme.

Inhibition effect of enteropeptidase on RANKL-RANK signalling by cleavage of RANK.

Enteropeptidase can cleave trypsinogen on the sequence of Asp-Asp-Asp-Asp-Lys and plays an important role in food digestion. The RANKL-RANK signalling pathway plays a pivotal role in bone remodelling. In this study, we reported that enteropeptidase can inhibit the RANKL-RANK signalling pathway through the cleavage of RANK. A surrogate peptide blocking assay indicated that enteropeptidase could specifically cleave RANK on the sequence NEEDK. Osteoclast differentiation assay and NF-κB activity assay confirmed that enteropeptidase could inhibit osteoclastogenesis in vitro through the cleavage of RANK. This is the first study to prove that the RANKL-RANK signalling pathway can be inhibited by cleavage of RANK instead of targeting RANKL.

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-ß-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 µM and 20 µM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.