OmpA Signal Peptide Research Articles

Direct capture and selective elution of a secreted polyglutamate‐tagged nanobody using bare magnetic nanoparticles

The secretion and direct capture of proteins from the extracellular medium is a promising approach for purification, thus enabling integrated bioprocesses. We demonstrate the secretion of a nanobody (VHH) to the extracellular medium (EM) and its direct capture by bare, non-functionalized magnetic nanoparticles (MNPs). An ompA signal peptide for periplasmic localization, a polyglutamate-tag (E8 ) for selective MNP binding, and a factor Xa protease cleavage site were fused N-terminally to the nanobody. The extracellular production of the E8 -VHH (36mg L-1 ) was enabled using a growth-decoupled Escherichia coli-based expression system. The direct binding of E8 -VHH to the bare magnetic nanoparticles was possible and could be drastically improved up to a yield of 88% by adding polyethylene glycol (PEG). The selectivity of the polyglutamate-tag enabled a selective elution of the E8 -VHH from the bare MNPs while raising the concentration factor (5x) and purification factor (4x) significantly. Our studies clearly show that the unique combination of a growth-decoupled E. coli secretion system, the polyglutamate affinity tag, non-functionalized magnetic nanoparticles, and affinity magnetic precipitation is an innovative and novel way to capture and concentrate nanobodies.

Biocatalytic synthesis of lactosucrose using a recombinant thermostable β-fructofuranosidase from Arthrobacter sp. 10138

ABSTRACT As a prebiotics, lactosucrose plays an important role in maintaining human gastrointestinal homeostasis. In this study, a thermostable enzyme from Arthrobacter sp. 10138 was screened from six β-fructofuranosidase-producing strains for the lactosucrose production and the coding gene was heterologously expressed in Escherichia coli for efficient expression. Recombinant β-fructofuranosidase was purified and biochemically characterized by MALDI-TOFMS spectrometry. The transfructosylation product by this recombinant enzyme was determined to be lactosucrose rather than other oligosaccharides or polysaccharides by HPLC and LC-MS. Efficient extracellular secretion of β-fructofuranosidase was achieved by the optimization of signal peptide and induction conditions. It was found that with the signal peptide torT, the highest extracellular activity reached 111.01 U/mL, which was 38.4-fold higher than that with the OmpA signal peptide. Under the optimal conditions (pH 6.0, temperature 50°C, enzyme amount 40 μg/ml, sucrose 150 g/L and lactose 150 g/L), 109 g/L lactosucrose was produced with a molar conversion ratio of 49.3%. Here the thermostable β-fructofuranosidase from Arthrobacter sp. 10138 can be used for efficient synthesis of lactosucrose, and this provides a good startpoint for the industrial production of lactosucrose in the future.

Experimental Evaluation of In Silico Selected Signal Peptides for Secretory Expression of Erwinia Asparaginase in Escherichia coli

Erwinia chrysanthemi asparaginase is an important drug used in cancer treatment, especially in acute lymphoblastic leukemia (ALL). Escherichia coli periplasmic space is an ideal compartment for recombinant expression of certain proteins. To efficient secretion, an appropriate signal sequence should be chosen for each protein individually. In this study, the proper signal sequences for secretory production of Erwinia asparaginase were predicted by in silico methods. Consequently, two signal peptides, OmpA and DsbA, were selected for secretory expression of the enzyme in E. coli. Asparaginase was translocated through the cytoplasmic membrane with either DsbA signal peptide or OmpA signal peptide; Using DsbA signal peptide, 5.95 units of the enzyme was obtained per milliliter of culture media, whereas OmpA signal sequence led to some amount of periplasmic expression. Our study showed that the co-translational signal peptide, DsbA, targeted the asparaginase to cell membrane more efficiently in comparison to the post-translational signal peptide, OmpA. The combination of in silico and experimental approaches provides a way to test a wide variety of signal sequences for secretory production of the enzyme in a time- and cost-effective manner. It is a fundamental step for further studies in the enzyme production process.

Secretory expression of biologically active chondroitinase ABC I for production of chondroitin sulfate oligosaccharides

Chondroitin sulfate ABC lyases (csABCs) have attracted intensive attention because of their wide potential applications in promoting tissue regeneration and generating oligosaccharides. In the present study, three csABC I encoding sequences were analyzed and site-directed mutagenesis results demonstrate that residues Leu125 and Leu322 are essential to activity and mutation of each leucine residue to proline dramatically decreased enzymatic activity. Additionally, our results showed that mutation of I309 V significantly increased the catalytic efficiency. By recruiting OmpA signal peptide and engineering the permeability of cell membrane with deletion of a lipoprotein encoding gene lpp, all recombinant enzymes were secreted and the extracellular activity was finally increased to 2.99 ± 0.1 U/mL in batch fermentation. More importantly, the engineered csABC I with high activity can rapidly degrade chondroitin sulfate to the end tetrasaccharides and disaccharides, demonstrating its applicability for preparation of chondroitin sulfate oligosaccharides.

Optimization extracellular secretion of Recombinant Human Epidermal Growth Factor (hEGF) in Escherichia coli BL21 (DE3) pD881-OmpA-hEGF by using Response Surface Method (RSM)

Human Epidermal Growth Factor is techno-economically protein that has the function for stimulates a process of proliferation and cell differentiation. Because of that function, it can be a candidate for wound healing in ulcer diabetic. In the previous study, hEGF can extracellular secreted by OmpA signal peptide using recombinant DNA. The production method of recombinant hEGF will be extracellular secreted by Escherichia coli. However, the optimum secretion of OmpA has not been studied. Therefore the purpose of this study is to determine the optimum conditions using the RSM method. This research begins with a rejuvenation of culture, extracellular secretion, and optimization with the RSM method, followed by protein production at optimum condition. Secreted recombinant protein measured by Sandwich ELISA method and LOWRY. The result showed that the optimum condition was found in the medium concentration of 1.5x and induction time at 2 hours 10 minutes and the total protein yield of expression was 21.247 mg/mL with optimization percentage 2.96% and efficiency 50.56 % and recombinant protein hEGF concentration in fermentation media 416 μg/mL

Secretion of the recombination α-amylase in Escherichia coli and purification by the gram-positive enhancer matrix (GEM) particles

α-Amylases are important enzymes in industry. A recombinant α-amylase with a secretion signal peptide and an AcmA tag was expressed in Escherichia coli to improve the yield. The induction concentrations were optimized, and the temperature had a significant influence on soluble expression and secretion. A visible band could be obtained when the induction was conducted at 16 °C. The gram-positive enhancer matrix (GEM) particles could separate and purify the recombinant α-amylase with the AcmA tag, and no visible band could be seen in the culture even after the culture was concentrated ten times. The solution and concentration of the recombinant α-amylase could be adjusted by GEM particles. The recombinant untagged α-amylase was obtained after digestion. The α-amylase was characterized. The recombinant α-amylase was a thermophilic enzyme with a broad pH tolerance. In addition, the enzyme activity of the recombinant α-amylase was independent of Ca2+. The recombinant α-amylase contained the OmpA signal peptide and the AcmA tag and was expressed and purified quickly and easily.

Improving Fab\u2019 fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA

ObjectivesTo reduce unwanted Fab’ leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab’ fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives.ResultsWe attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab’ grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab’ coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab’ leakage relative to the original autonucleolytic Fab’ strain with OmpA-fused staphylococcal nuclease.ConclusionsWe successfully rescued Fab’ leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab’ fragment to the surrounding growth media.

Expression and production optimization of sucrose isomerase from Pantoea dispersa in Escherichia coli

To improve the yield of sucrose isomerase from Pantoea dispersa UQ68J, we studied the effect of different signal peptides and fermentation conditions on sucrose isomerase expression in Escherichia coli. The gene of sucrose isomerase was optimized and expressed in E. coli BL21 (DE3) with native signal peptide which was named as ORI strain. The total and extracellular enzyme activity was 85 and 65 U/mL in the flask, respectively. The mature protein, which started from the 22th amino acid, was connected with the PelB and OmpA signal peptide to construct P22 and O22 strain, respectively. The total activity of P22 reached 138 U/mL, which was 1.6 times of ORI strain. The total activity of O22 strain was similar to that of ORI strain. Induced by 3.0 g/L lactose, the total activity of P22 strain increased to 168 U/mL. In 3 L fermentor, the effects of glycine concentration and induction time were studied. Induction when the DCW reached 18 g/L (OD₆₀₀=30), with 0.5% glycine, the extracellular enzyme activity reached 1 981 U/mL, and the total enzyme activity reached 2 640 U/mL, which is the highest activity of sucrose isomerase that was expressed in recombinant E. coli.

OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system.

The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2893-y) contains supplementary material, which is available to authorized users.

Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.

BackgroundHeterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. Lactic acid bacteria are interesting cell factories for the expression of hydrolytic enzymes as many of them are generally recognized as safe and require only a simple cultivation process. We are studying a potentially food-grade expression system for secretion of hydrolytic enzymes into the culture medium, since this enables easy harvesting and purification, while allowing direct use of the enzymes in food applications.ResultsWe studied overexpression of a chitosanase (CsnA) and a β-mannanase (ManB), from Bacillus licheniformis and Bacillus subtilis, respectively, in Lactobacillus plantarum, using the pSIP system for inducible expression. The enzymes were over-expressed in three forms: without a signal peptide, with their natural signal peptide and with the well-known OmpA signal peptide from Escherichia coli. The total production levels and secretion efficiencies of CsnA and ManB were highest when using the native signal peptides, and both were reduced considerably when using the OmpA signal. At 20 h after induction with 12.5 ng/mL of inducing peptide in MRS media containing 20 g/L glucose, the yields and secretion efficiencies of the proteins with their native signal peptides were 50 kU/L and 84 % for ManB, and 79 kU/L and 56 % for CsnA, respectively. In addition, to avoid using antibiotics, the erythromycin resistance gene was replaced on the expression plasmid with the alanine racemase (alr) gene, which led to comparable levels of protein production and secretion efficiency in a suitable, alr-deficient L. plantarum host.ConclusionsManB and CsnA were efficiently produced and secreted in L. plantarum using pSIP-based expression vectors containing either an erythromycin resistance or the alr gene as selection marker.

Transesterification of plant oils using Staphylococcus haemolyticus L62 lipase displayed on Escherichia coli cell surface using the OmpA signal peptide and EstAβ8 anchoring motif

Staphylococcus haemolyticus L62 (SHL62) lipase was displayed on the outer membrane of Escherichia coli using the OmpA signal peptide and the autotransporter EstAβ8 protein. Localization of SHL62 lipase on the outer membrane of E. coli was confirmed using immunofluorescence microscopy and flow cytometry analysis. Lipase activity of the displayed SHL62 lipase was also measured using spectrophotometry and pH titration. SHL62 lipase activity of whole cells reached 2.0U/ml culture (OD600nm of 10) when it was measured by the p-nitrophenyl caprylate assay after being induced with 1mM IPTG for 24h. The optimum temperature and pH for the lipase was 45°C and 10, respectively. Furthermore, it maintained more than 90% of maximum lipase activity at up to 50°C and in a pH range of 5–9. The hydrolytic activity assay conduted with various substrates confirmed that p-nitrophenyl caprylate and corn oil were preferred substrates among various synthetic and natural substrates, respectively. The displayed SHL62 lipase produced fatty acid esters from various alcohols and plant oils through transesterification.

Glycine and Triton X-100 enhanced secretion of recombinant α-CGTase mediated by OmpA signal peptide in Escherichia coli

OmpA signal peptide mediated cgt gene from Paenibacillus macerans JFB05-01 was cloned and expressed in E. coli BL21 (DE3). The effects of glycine and Triton X-100 on extracellular production of α-cyclodextrin glycosyltransferase (α-CGTase) were investigated. When supplemented with Gly or Triton X-100 to the culture media individually, the secreted extracellular enzyme reached 32 or 33 U/mL at 48 h of cultivation, respectively. When supplemented with Gly and Triton X-100 together, the extracellular α-CGTase activity reached 48 U/mL after 48 h cultivation, which was 20-fold of the control group without any additives. Analysis of membrane permeability demonstrated that addition of glycine and Triton X-100 enhanced the permeability of both outer and inner membrane. The potential mechanism of the enhanced protein secretion was discussed.

Production of recombinant Bacillus subtilis chitosanase, suitable for biosynthesis of chitosan-oligosaccharides

Chitosanases are enzymes that catalyse the hydrolysis of the β-1,4 glycosidic bond of chitosan. One of the most promising applications of this enzyme is for the bioconversion of chitosan into value-added chitosan-oligosaccharides (COS). GH46 chitosanase (Csn) from Bacillus subtilis 168 was expressed in Escherichia coli by fusing the gene encoding mature Csn to the E. coli OmpA signal peptide sequence. The recombinant enzyme was secreted into the culture supernatant. The recombinant Csn showed high specific activity and stability over a wide range of pH. The enzyme was >100 times more thermostable in the presence of the substrate, with a half-life time of activity (τ1/2) of approximately 20h at 50°C and pH 5.5. Efficient bioconversion of chitosan into different mixtures of COS, using crude culture supernatant containing secreted enzyme was demonstrated.

Efficient E. coli expression systems for the production of recombinant β-mannanases and other bacterial extracellular enzymes

Two Escherichia coli expression systems based on T7 RNA polymerase promoter (pET system) and tac promoter (pFLAG system) have been used for the production and secretion of recombinant b-mannanases from Bacillus sp. Both E. coli OmpA signal peptide and native Bacillus signal peptide could be used efficiently for the secretion of recombinant enzymes into periplasmic space and culture media. The genes could be induced for over-expression with 0.1 - 1 mM isopropyl-β-D- 1-thiogalactopyranoside (IPTG) when the OD600 of the culture broth reached 0.6-1.5. The recombinant enzymes could be harvested from whole cell lysate, perimplasmic extract, or culture broth after induction for 4 - 20 hours. Since the enzyme is C-terminally tagged with hexahistidine, the recombinant enzymes could be conveniently purified to apparent homogeneity by one-step immobilized-metal affinity chromatography (IMAC) using Ni-NTA resins. The characteristics of purified recombinant b-mannanases from B. licheniformis and B. subtilis, which share 78% amino acid identity, are slightly different. These systems should be applicable for the production of various recombinant bacterial extracellular enzymes.

Secretion of human superoxide dismutase in Escherichia coli using the condensed single‐protein‐production system

A secretion vector, pColdV for the Single-Protein-Production (SPP) system was constructed using the E. coli OmpA signal peptide. Using this vector, human superoxide dismutase (hSOD) was co-expressed with MazF, an ACA-specific mRNA interferase, allowing E. coli cells to produce only hSOD, which was secreted into the periplasmic space with a yield of ∼20% of total cellular proteins. The signal peptide was properly cleaved. Using cells overproducing DsbA protein, two S-S bridges were also properly formed to yield enzymatically active SOD. A well resolved heteronuclear single quantum coherence (HSQC) spectrum of hSOD isotope-labeled in the condensed SPP (cSPP) system was obtained by simply isolating the periplasmic fraction. These results indicate that human secretory proteins can be expressed well in the cSPP system using pColdV.

Constructing Modified Protein-producing Escherichia coli Capable of Autohydrolysing Host Nucleic Acid During Cell Lysis

We used Red recombination system to construct a modified E. coli protein-producing strain capable of autohydrolysing host nucleic acid. E. coli BL21 (DE3), a common protein-producing strain, was used as starting material. The modified E. coli expression host had a staphylococcal nuclease expression cassette within the host chromosome lpxM locus. The Staphylococcus aureus nuclease was expressed as a fusion to the ompA signal peptide, and was translocated to the periplasm of the cell, protecting the host nucleic acid from the toxic activity during growth. The nuclease was released during cell lysis and subsequently hydrolyzed host nucleic acid in the lysate. Results show that the modified strain had sufficient nuclease activity to completely autohydrolyze the host chromosomal DNA and produced same amount of recombinant proteins as the original strain.

Improved periplasmic production of biologically active murine interleukin-2 in Escherichia coli through a single amino acid change at the cleavage site

We fused the mature murine Interleukin-2 (mIL-2) gene to the signal peptide of the Outer membrane protein A (OmpA). We generated mutants mimicking different cleavage sites. A hybrid protein consisting of the OmpA signal peptide fused precisely to mature mIL-2, thereby mimicking the cleavage site of the OmpA native protein, was very poorly secreted into the periplasm of Escherichia coli (200 U/ml). Insertion of a serine residue between the OmpA signal peptide and the mIL-2 mature sequence, thus mimicking the mIL-2 natural cleavage site, increased the secretion by a factor of 40,000 (8 × l0 6 U/ml). The specific biological activity of secreted mIL-2 equaled that of natural mIL-2 and was about five times higher than that of mIL-2 refolded from inclusion bodies. We also show that the temperature at which the culture is grown has a major impact on the secretion level.

Extracellular production of Streptomyces lividans acetyl xylan esterase A in Escherichia coli for rapid detection of activity

Acetyl xylan esterase A (AxeA) from Streptomyces lividans belongs to a large family of industrially relevant polysaccharide esterases. AxeA and its truncated form containing only the catalytically competent domain, AxeA tr, catalyze both the deacetylation of xylan and the N-deacetylation of chitosan. This broad substrate specificity lends additional interest to their characterization and production. Here, we report three systems for extracellular production of AxeA tr: secretion from the native host S. lividans with the native signal peptide, extracellular production in Escherichia coli with the native signal peptide, and in E. coli with the OmpA signal peptide. Over five to seven days of a shake flask culture, the native host S. lividans with the native signal peptide secreted AxeA tr into the extracellular medium in high yield (388 mg/L) with specific activity of 19 U/mg corresponding to a total of 7000 U/L. Over one day of shake flask culture, E. coli with the native secretion signal peptide produced 84-fold less in the extracellular medium (4.6 mg/L), but the specific activity was higher (100 U/mg) corresponding to a total of 460 U/L. A similar E. coli culture using the OmpA signal peptide, produced 10 mg/L with a specific activity of 68 U/mg, corresponding to a total of 680 U/L. In 96-well microtiter plates, extracellular production with E. coli gave ∼30 and ∼86 μg/mL in S. lividans. Expression in S. lividans with the native signal peptide is best for high level production, while expression in E. coli using the OmpA secretion signal peptide is best for high-throughput expression and screening of variants in microtiter plate format.

Function of the signal peptide and N- and C-terminal propeptides in the leucine aminopeptidase from Aeromonas proteolytica

The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP's native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type AAP to be proposed. The role of the A. proteolytica signal peptide in protein secretion as well as comparison to other known signal peptides reveals a close resemblance of the A. proteolytica signal peptide to the outer membrane protein (OmpA) signal peptide. Over-expression of the full 54 kDa AAP enzyme provides an enzyme that is significantly less active, due to a cooperative inhibitory interaction between both propeptides. Over-expression of AAP lacking its C-terminal propeptide provided an enzyme with an identical k cat value to wild-type AAP but exhibited a larger K m value, suggesting competitive inhibition of AAP by the N-terminal propeptide ( K i∼0.13 nM). The recombinant 32 kDa form of AAP was characterized by kinetic and spectroscopic methods and was shown to be identical to mature, wild-type AAP. Therefore, the ease of purification and processing of rAAP along with the fact that large quantities can be obtained now allow new detailed mechanistic studies to be performed on AAP through site-directed mutagenesis.

Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C: use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C.

Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins. This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils. One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life. The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors. The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C. In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers. The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C. In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C. It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.