Secretion Vector Research Articles

Assessing the influence of the algebraic structure of q-ary lattices on the complexity of cryptanalysis of problems on lattices

The work is devoted to the influence of the algebraic structure of q-ary lattices on the complexity of cryptanalysis of cryptographic problems on lattices. The root-mean-square errors for existing lattice reduction models are obtained. It is shown that the deterministic Albrecht-Lee simulator shows the smallest mean square deviations on small lattices. Collapsibility estimates for nesting and decoding attacks on the algebraic structure of q-ary lattices have been found. A new method for selecting attack parameters has been proposed for the decoding attack. It is shown that a decoding attack with such a choice of parameters can be at the expense of nesting attacks. To distribute the power of the secret vector in decoding attacks, which differ from the normal one, the approximation method is used by the normal division, which minimizes the Kolmogorov-Smirnov equation and calculates the optimal values for some distributions. There are no parameters for any divisions. For the SIS problem, a new approach to safety assessment has been proposed, which takes into account the possibility of various Euclidean norms. Based on the new approach, estimates of the SIS problem for Crystals-Dilithium electronic signature schemes have been calculated.

Forging quantum data: classically defeating an IQP-based quantum test

Recently, quantum computing experiments have for the first time exceeded the capability of classical computers to perform certain computations – a milestone termed "quantum computational advantage." However, verifying the output of the quantum device in these experiments required extremely large classical computations. An exciting next step for demonstrating quantum capability would be to implement tests of quantum computational advantage with efficient classical verification, such that larger system sizes can be tested and verified. One of the first proposals for an efficiently-verifiable test of quantumness consists of hiding a secret classical bitstring inside a circuit of the class IQP, in such a way that samples from the circuit's output distribution are correlated with the secret. The classical hardness of this protocol has been supported by evidence that directly simulating IQP circuits is hard, but the security of the protocol against other (non-simulating) classical attacks has remained an open question. In this work we demonstrate that the protocol is not secure against classical forgery. We describe a classical algorithm that can not only convince the verifier that the (classical) prover is quantum, but can in fact can extract the secret key underlying a given protocol instance. Furthermore, we show that the key extraction algorithm is efficient in practice for problem sizes of hundreds of qubits. Finally, we provide an implementation of the algorithm, and give the secret vector underlying the "\$25 challenge" posted online by the authors of the original paper.

Biochemical Characterization of an Endoglucanase GH7 from Thermophile Thermothielavioides terrestris Expressed on Aspergillus nidulans

Endoglucanases (EC 3.2.1.4) are important enzymes involved in the hydrolysis of cellulose, acting randomly in the β-1,4-glycosidic bonds present in the amorphous regions of the polysaccharide chain. These biocatalysts have been classified into 14 glycosyl hydrolase (GH) families. The GH7 family is of particular interest since it may act on a broad range of substrates, including cellulose, β-glucan, and xylan, an attractive feature for biotechnological applications, especially in the renewable energy field. In the current work, a gene from the thermophilic fungus Thermothielavioides terrestris, encoding an endoglucanase GH7 (TtCel7B), was cloned in the secretion vector pEXPYR and transformed into the high-protein-producing strain Aspergillus nidulans A773. Purified TtCel7B has a molecular weight of approximately 66 kDa, evidenced by SDS-PAGE. Circular dichroism confirmed the high β-strand content consistent with the canonical GH7 family β-jellyroll fold, also observed in the 3D homology model of TtCel7B. Biochemical characterization assays showed that TtCel7B was active over a wide range of pH values (3.5–7.0) and temperatures (45–70 °C), with the highest activity at pH 4.0 and 65 °C. TtCel7B also was stable over a wide range of pH values (3.5–9.0), maintaining more than 80% of its activity after 24 h. The KM and Vmax values in low-viscosity carboxymethylcellulose were 9.3 mg mL−1 and 2.5 × 104 U mg−1, respectively. The results obtained in this work provide a basis for the development of applications of recombinant TtCel7B in the renewable energy field.

Time-Memory Trade-Offs for Saber+ on Memory-Constrained RISC-V Platform

Saber is a module-lattice-based key encapsulation scheme that has been selected as a finalist in the NIST Post-Quantum Cryptography standardization project. As Saber computes on considerably large matrices and vectors of polynomials, its efficient implementation on memory-constrained IoT devices is very challenging. In this paper, we present an implementation of Saber with a minor tweak to the original Saber protocol for achieving reduced memory consumption and better performance. We call this tweaked implementation ‘Saber+’, and the difference compared to Saber is that we use different generation methods of public matrix <inline-formula><tex-math notation="LaTeX">$\boldsymbol{A}$</tex-math></inline-formula> and secret vector <inline-formula><tex-math notation="LaTeX">$\boldsymbol{s}$</tex-math></inline-formula> for memory optimization. Our highly optimized software implementation of Saber+ on a memory-constrained RISC-V platform achieves 48% performance improvement compared with the best state-of-the-art memory-optimized implementation of original Saber. Specifically, we present various memory and performance optimizations for Saber+ on a memory-constrained RISC-V microcontroller, with merely 16KB of memory available. We utilize the Number Theoretic Transform (NTT) to speed up the polynomial multiplication in Saber+. For optimizing cycle counts and memory consumption during NTT, we carefully compare the efficiency of the complete and incomplete-NTTs, with platform-specific optimization. We implement 4-layers merging in the complete-NTT and 3-layers merging in the 6-layer incomplete-NTT. An improved on-the-fly generation strategy of the public matrix and secret vector in Saber+ results in low memory footprint. Furthermore, by combining different optimization strategies, various time-memory trade-offs are explored. Our software implementation for Saber+ on selected RISC-V core takes just 3,809K, 3,594K, and 3,193K clock cycles for key generation, encapsulation, and decapsulation, respectively, while consuming only 4.8KB of stack at most.

Adaptive visual cryptography scheme design based on QR codes.

QR code based visual encryption schemes are still relatively rare, and the existing schemes are mainly implemented by averaging grayscale maps. They become distorted when recovering complex secret images. In this paper, we propose a visual cryptography scheme based on QR codes. We use two adaptive schemes to improve the distortion problem in secret image recovery. The algorithm for generating the QR code shared matrix is improved, and the secret image can be restored quickly. This scheme could ensure the uniform distribution of secret vectors. Meanwhile individual shared QR codes would never reveal any secret information. The proposed algorithm reduces the space consumed by the secret vector and the probability of falling prey to illegal attacks. Compared with other schemes, the secret image recovered via the proposed method is clearer and the scheme is suitable for scenarios in which the secret images are more complex, as it yields better security and practicality.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain.

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

SlpB Protein Enhances the Probiotic Potential of L. lactis NCDO 2118 in Colitis Mice Model.

Bacteria used in the production of fermented food products have been investigated for their potential role as modulators of inflammation in gastrointestinal tract disorders such as inflammatory bowel diseases (IBD) that cause irreversible changes in the structure and function of gut tissues. Ulcerative colitis (UC) is the most prevalent IBD in the population of Western countries, and it is marked by symptoms such as weight loss, rectal bleeding, diarrhea, shortening of the colon, and destruction of the epithelial layer. The strain Propionibacterium freudenreichii CIRM-BIA 129 recently revealed promising immunomodulatory properties that greatly rely on surface-layer proteins (Slp), notably SlpB. We, thus, cloned the sequence encoding the SlpB protein into the pXIES-SEC expression and secretion vector, and expressed the propionibacterial protein in the lactic acid bacterium Lactococcus lactis NCDO 2118. The probiotic potential of L. lactis NCDO 2118 harboring pXIES-SEC:slpB (L. lactis-SlpB) was evaluated in a UC-mice model induced by Dextran Sulfate Sodium (DSS). During colitis induction, mice receiving L. lactis-SlpB exhibited reduced severity of colitis, with lower weight loss, lower disease activity index, limited shortening of the colon length, and reduced histopathological score, with significant differences, compared with the DSS group and the group treated with L. lactis NCDO 2118 wild-type strain. Moreover, L. lactis-SlpB administration increased the expression of genes encoding tight junction proteins zo-1, cln-1, cln-5, ocln, and muc-2 in the colon, increased IL-10 and TGF-β, and decreased IL-17, TNF-α, and IL-12 cytokines in the colon. Therefore, this work demonstrates that SlpB recombinant protein is able to increase the probiotic potential of the L. lactis strain to alleviate DSS-induced colitis in mice. This opens perspectives for the development of new approaches to enhance the probiotic potential of strains by the addition of SlpB protein.

An Approach for Recombinant Epidermal Growth Factor Purification Using an Elastin-Like Protein Tag

Introduction: Wide applications in research, clinical and cosmetic industry of human epidermal growth factor (hEGF) made it a research interest target. Its production in different expression systems has shown several limitations. Recombinant expression of hEGF in E. coli is always accompanied by inclusion body formation. The object of this study is to the evaluation of a chromatography-independent approach for the production of EGF in E. coli as soluble form. Materials and Methods: In order to evaluate a chromatogram independent purification approach for recombinant hEGF production in a soluble form, the hEGF gene was fused to an elastin-like protein (ELP) and expressed in E. coli BL21 (DE3) using pET26b expression vector for secretion the product into periplasmic space. Results: Periplasmic protein content analysis confirmed that the recombinant protein is secreted into the periplasm. The purification process was done by using 0.4 M ammonium sulfate in two cycles of inverse phase transition (ITC). After two cycles of purification, purity reached more than 95%. Western blotting analysis with the monoclonal anti-EGF antibody has confirmed the accuracy of EGF. Biological activity of the purified protein was investigated on NIH-3T3 cell line and results indicated EGF-induced proliferation in treated cells. Our results showed periplasmic expression is the proper approach to the production of soluble recombinant hEGF. By using ELP fused to EGF, the purification process was established without applying chromatography which will result in decreasing in final costs. Conclusions: This study introduced a new economic and efficient approach to the production and purification of recombinant hEGF.

Fragile Watermarking of 3D Models in a Transformed Domain

This paper presents an algorithm aimed at the integrity protection of 3D models represented as a set of vertices and polygons. The proposed method defines a procedure to perform a fragile watermarking of the vertices’ data, namely 3D coordinates and polygons, introducing a very small error in the vertices’ coordinates. The watermark bit string is embedded into a secret vector space defined by the Karhunen–Loève transform derived from a key image. Experimental results show the good performance of the method and its security.

Impact ofthe modulus switching technique on some attacks against learning problems

The modulus switching technique has been used in some cryptographic applications as well as in cryptanalysis. For cryptanalysis against the learning with errors (LWE) problem and the learning with rounding (LWR) problem, it seems that one does not know whether the technique is really useful or not. This work supplies a complete view of the impact of this technique on the decoding attack, the dual attack and the primal attack against both LWE and LWR. For each attack, the authors give the optimal formula for the switching modulus. The formulas get involved the number of LWE/LWR samples, which differs from the known formula in the literature. They also attain the corresponding sufficient conditions saying when one should utilise the technique. Surprisingly, restricted to the LWE/LWR problem that the secret vector is much shorter than the error vector, they also show that performing the modulus switching before using the so-called rescaling technique in the dual attack and the primal attack make these attacks worse than only exploiting the rescaling technique as reported by Bai and Galbraith at the Australasian conference on information security and privacy (ACISP) 2014 conference. As an application, they theoretically assess the influence of the modulus switching on the LWE/LWR-based second round NIST PQC submissions.

Molecular cloning, heterological expression and production of the human growth hormone

Growth hormone (GH) is a polypeptide produced in the anterior pituitary lobe that triggers different biochemical pathways and increases cell proliferation and growth. In this work, the pNic based vector for periplasmatic secretion was developed. Recombinant human growth hormone was produced via periplasmatic secretion (42h, 30 °С) followed by several steps of purification. GH obtained does not contain His-tag. Expression level of 2,85 mg per1 literof bacterial culture was achieved.

Functional Secretion of Granulocyte Colony Stimulating Factor in Bacillus subtilis and Its Thermogenic Activity in Brown Adipocytes

Granulocyte colony-stimulating factor (G-CSF) is involved in generation of colonies hematopoietic cells by stimulation of bone marrow cells, resulting in the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. To express and secrete G-CSF in Bacillus system, the G-CSF gene was amplified using gene-specific primers and template DNA (pUNO1-hGCSFb). As a result, the PCR product of 530 bp was obtained, sequenced, and analyzed using the BLAST program. The open reading frame encoded a 204-amino acid polypeptide precursor consisting of the 30-residue signal sequence and a 174-residue mature polypeptide. Recombinant secretion vector (pRBAS-CSF3, 6.96 kb) was constructed using pRBAS vector which contains promoter (0.45 kb) and signal sequence (87 bp) of alkaline protease gene (aprE), ribosome binding site (GGAGAGGG) and transferred to Bacillus subtilis LKS. G-CSF protein was secreted and optimized to increase the productivity (19% of total secreted proteins, 0.12 mg/ml). The culture broth was harvested and followed by metal chelating, ethanol precipitation, and gel chromatography. The purified G-CSF was tested for thermogenic activity in cultured brown adipocytes. G-CSF activated HIB1B brown adipocytes by increasing expression levels of brown fat-specific marker proteins (UCP1 and PRDM16) and genes (Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. This result suggests that G-CSF per se or G-CSF-containing foodstuff can be used to treat obesity.

Engineering of CHO cells for the production of vertebrate recombinant sialyltransferases.

BackgroundSialyltransferases (SIATs) are a family of enzymes that transfer sialic acid (Sia) to glycan chains on glycoproteins, glycolipids, and oligosaccharides. They play key roles in determining cell–cell and cell-matrix interactions and are important in neuronal development, immune regulation, protein stability and clearance. Most fully characterized SIATs are of mammalian origin and these have been used for in vitro and in vivo modification of glycans. Additional versatility could be achieved by the use of animal SIATs from other species that live in much more variable environments. Our aim was to generate a panel of stable CHO cell lines expressing a range of vertebrate SIATs with different physicochemical and functional properties.MethodsThe soluble forms of various animal ST6Gal and ST3Gal enzymes were stably expressed from a Gateway-modified secretion vector in CHO cells. The secreted proteins were IMAC-purified from serum-free media. Functionality of the protein was initially assessed by lectin binding to the host CHO cells. Activity of purified proteins was determined by a number of approaches that included a phosphate-linked sialyltransferase assay, HILIC-HPLC identification of sialyllactose products and enzyme-linked lectin assay (ELLA).ResultsA range of sialyltransferase from mammals, birds and fish were stably expressed in CHO Flp-In cells. The stable cell lines expressing ST6Gal1 modify the glycans on the surface of the CHO cells as detected by fluorescently labelled lectin microscopy. The catalytic domains, as isolated by Ni Sepharose from culture media, have enzymatic activities comparable to commercial enzymes. Sialyllactoses were identified by HILIC-HPLC on incubation of the enzymes from lactose or whey permeate. The enzymes also increased SNA-I labelling of asialofetuin when incubated in a plate format.ConclusionStable cell lines are available that may provide options for the in vivo sialylation of glycoproteins. Proteins are active and should display a variety of biological and physicochemical properties based on the animal source of the enzyme.

Enhanced antifungal activity of bovine lactoferrin-producing probiotic Lactobacillus casei in the murine model of vulvovaginal candidiasis

BackgroundVulvovaginal candidiasis (VVC) is a common vaginitis caused by Candida species,a frequently recurring condition. Fungal azole-resistant strains with azole-resistance have developed for long and wide explosion to the first-line antifungal azole agent. Bovine lactoferrin (BLF) is a protein from transferrin family secreted by the bovine mammary tissue. Its various biological functions are well known, especially the pronounced antifungal activity.ResultsIn the current study, we constructed a Lactobacillus casei strain (L.casei/pPG612.1-BLF), which secreted BLF encoded by a mature secretion vector plasmid pPG612.1, and evaluated its antifungal activity in vitro and in vivo. In a two-layer agar plate in vitro assay, the number of C. albicans CFUs decreased and the average colony size shrunk upon exposure to L. casei/pPG612.1-BLF. In a murine VVC model, the infection burden of mice intra-vaginally pre-inoculated with L. casei/pPG612.1-BLF was lower than in control groups. Furthermore, the infection burden in mice with VVC was reduced when the animals were continually given L. casei/pPG612.1-BLF as a topical treatment for 5 days.ConclusionCombined, these results suggested that the L. casei/pPG612.1-BLF strain is a promising preventative and therapeutic anti-VVC agent, highlighting the possibility of employing the probiotic L. casei as a vehicle for biotherapy in the female genital tract and exploiting the natural antibiotic antimicrobial peptides for other applications.

Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions

BackgroundTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro.ResultsThe human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells.ConclusionsWe designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.

Abstract 2781: "Wheelz": A novel engineered human antibody for possible CAR T-cell therapy

Abstract The purpose of this study is to present a novel engineered human antibody specific to thymidine kinase 1 (TK1). Recent developments in chimeric antigen receptor (CAR) T-cell therapeutic approaches have rocketed into the spotlight in the field of cancer immunotherapy. One of the limitations to CAR T-cell therapy is the need for novel targets on cancer cells. The upregulation of serum TK1 has been shown to be an important biomarker in cancer detection and prognosis. Furthermore, we have previously reported the cell surface expression of TK1 on multiple cancer cell lines and in clinical samples. This data merits an investigation of the use of TK1 as a target for CAR T-cells. We have created an engineered human antibody against TK1. We present “Wheelz,” a single chain fragment variable monoclonal antibody with a constant region (scFv-Fc) to potentially target cancer cells expressing surface TK1. Our engineered antibody was isolated from a yeast display library (donated by the Wittrup lab at MIT) using magnetic sorting and flow cytometry to find the top 0.1% of TK1-binding single chain fragments. We fused the resulting scFv region to a constant region (Fc) fragment through restriction digestion and ligation. We then transformed the construct with a secretion vector into yeast and induced the transformed yeast to produce the antibody, which we called “Wheelz.” We purified the antibody using His-tag. In order to confirm specificity to TK1, we used dot blotting and saw specific binding to purified TK1. We performed a western blot using chemiluminescence imaging. This indicated that the engineered human scFv-Fc binds to TK1 present in normal serum, cancer serum, and cancer cell extract in TK1's monomeric, dimeric, and tetrameric forms and did not bind to any other human cellular proteins. In the past, anti-TK1 scFv antibodies of murine origin were used in CAR experiments with some cytotoxic effect. Because “Wheelz” is an engineered human antibody, it could improve previous models by minimizing immune rejection in the patient. Future research will explore the cytotoxic-inducing effects of “Wheelz” before inserting the antibody into a CAR. We will also explore the specificity and cytotoxicity of eight other scFv regions isolated from yeast display that preliminarily appear to be specific for TK1. The specific binding of the engineered scFv-Fc antibody to cell-surface biomarker TK1 makes it a promising approach in cancer immunotherapy. “Wheelz” has the potential to expand the repertoire of tumor targets in CAR T-cell therapy. Citation Format: Kiara V. Whitley, Edwin J. Velazquez, Kelsey B. Bennion, Brianne M. Kingery, Scott K. Weber, Kim L. O'Neill. "Wheelz": A novel engineered human antibody for possible CAR T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2781.

Efficient Extracellular Expression of Phospholipase D in Escherichia Coli with an Optimized Signal Peptide

New secretion vectors containing the synthetic signal sequence (OmpA’) was constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli Phospholipase D structural gene (Accession number:NC_018658) fused to various signal sequence were expressed from the Lac promoter in E. coli Rosetta strains by induction with 0.4mM IPTG at 28°C for 48h. SDS-PaGe analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of Phospholipase D (PLD) not only to the medium but also remained in periplasm of E. coli with OmpA’ signal sequence at the N-terminus of PLD. Thus the study on the effects of various surfactants on PLD extracellular production in Escherichia coli in shake flasks revealed that optimal PLD extracellular production could be achieved by adding 0.4% Triton X-100 into the medium. The maximal extracellular PLD production and extracellular enzyme activity were 0.23mg ml-1 and 16U ml-1, respectively. These results demonstrate the possibility of efficient secretory production of recombinant PLD in E. coli should be a potential industrial applications.

고초균에서 His-Pro 반복서열을 갖는 Oligopeptide의 분비 최적화 및 항당뇨 효과

To verify anti-diabetic effect of oligopeptide with His-Pro repeats (mHP peptide), the oligopeptide was first secreted and optimized using the secretion vector, pRBAS with alkaline protease gene promoter and the signal sequence in Bacillus subtilis and directly the anti-diabetic effect of the mHP peptide was investigated in insulinoma cell, RINm5F cell line. The oligopeptide gene was obtained by annealing oligonucleotides with repeated His-Pro sequence and finally was constructed as 18 dipeptides (108 bp and 4.0 kDa) coding gene, named oligopeptide with His-Pro repeats (mHP peptide) to make cyclo(His-Pro) known to be anti-diabetic effects. The region encoding the oligopeptide gene was subcloned into the pRBAS secretion vector (E.coli-Bacillus shuttle vector) after PCR amplification using the designed primers including initiation and termination codons and His tag, named pRBAS-mHP (6.56 kb). To optimize secretion of the oligopeptide, various culture conditions were investigated in Bacillus subtilis LKS. As a result, the secreted oligopeptide was maximally measured (approximately 59.6 μg/mL) in 3 L batch culture and the highest secretion was achieved at 30°C, PY medium, and carbon sources (particularly barley and glycerol). In the RINm5F cells treated with 2 mM STZ, the oligopeptide treatment (0.1 mg/mL) restored the cell viability (10%) and reduced the nitric oxide (NO) generation (35%) and DNA fragmentation (90%). And also, insulin secretion level was increased to 17% higher than in STZ-treated RINm5F cells. These results suggest that the oligopeptide with His-Pro repeats could be a candidate material for anti-diabetic agent against STZ-induced diabetes.

Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli

ABSTRACTFusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380 mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86 mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.

Wireless Packet Communications Protected by Secret Sharing and Vector Coding

Wireless Packet Communications Protected by Secret Sharing and Vector Coding