pET22b Vector Research Articles

Cloning, expression, purification and characterisation of serine alkaline protease from Bacillus subtilis RD7

Abstract Proteases (E.C 3.4) are enzymes that break peptide bonds between amino acid groups of proteins. Bacillus subtilis RD7, an extracellular protease producer was genetically characterised, B. subtilis genomic DNA was isolated, oligonucleotide primers specific to serine alkaline protease gene of B. subtilis were designed and its PCR amplification was done. The purified PCR product and pET15b vector were subjected to restriction digestion with Nde1 and BamH1 and transformed into Escherichia coli DH5α competent cells. The recombinant expression of serine alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the serine alkaline protease protein had an estimated molecular size of 43 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1203bp gene encoding a protein of 400 amino acids. The sequence was blasted and aligned with known serine alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with closely related subsp. of B. subtilis. The insert also showed many substitutions (mutations with other sp. of Bacillus which established that serine alkaline protease of B. subtilis RD7 is a novel gene. The phylogenetic analysis of serine alkaline protease gene and its predicted amino acid sequences also validated that serine alkaline protease gene is a novel gene and has been accessioned in GenBank with accession number (MN097797). When expressed in E. coli, the recombinant enzyme was over expressed in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by PMSF. The enzyme showed maximum activity at pH 10 and 40 °C. It was stable at pH from 6 to 11 and below 70 °C. The aim of this study is to clone serine alkaline protease gene from B. subtilis RD7 (MG255317), its expression in mesophilic E. coli strain BL21, Purification and characterisation of the expressed protein.

Cloning and Expression of Mannheimia haemolytica PlpE Gene in Escherichia coli and its Immunogenicity Assessment.

Mannheimia haemolytica is responsible for considerable economic losses to cattle, sheep, and goat industries in many parts of the world. This bacterium isone of the causative agents of shipping fever in cattle. Current vaccines against M. haemolytica are moderately efficacious since they do not provide complete protection against the disease. Production of an economic vaccine for protecting farm animals against M. haemolytica has attracted the attention of many scientists. The outer membrane proteins (OMPs) play a major role in the pathogenesis and immunogenicity of M. haemolytica. Research on M. haemolytica OMPs has shown that antibodies to a particular OMP may be important in immune protection. In the current study, the gene for M. haemolytica OMP PlpE was cloned into the expression vector pET26-b, and then expressed in Escherichia coli BL21. The expression of the protein was carried out by the induction of cultured Escherichiacoli Bl21 cells with 1mM isopropyl-β-D-thiogalactopyranoside. The recombinant PlpE was purified using Ni-NTA agarose resin, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The identity of the expressed protein was analyzed by western blotting. It was revealed that rPlpE was expressed and produced properly. To assess the immunogenicity of the recombinant protein, the purified rPlpE was used as an antigen for antibody production in goats. The observations suggested that the produced recombinant protein can be used as a antigen for developing diagnostic tests and or as a vaccine candidate.

Cyanide-free synthesis of an aromatic nitrile from a biorenewable-based aldoxime: Development and application of a recombinant aldoxime dehydratase as a biocatalyst

An Escherichia coli host microorganism was used for the production of the aldoxime dehydratase from Rhodococcus sp. YH3-3 (OxdYH3-3), which showed good activity toward aromatic aldoximes. Biotransformation of aromatic aldoximes to nitriles was studied using this recombinant biocatalyst OxdYH3-3, exemplified in particular for the synthesis of biorenewable-based 2-furonitrile. The gene encoding for the enzyme OxdYH3-3 was cloned to a histidine tag-containing overexpression vector pET15b, pET22b and pET28b and transformed to E. coli BL21 (DE3) and BL21-Codonplus (DE3). The preferred overexpression was achieved when utilizing the strain BL21 (DE3)/pET28b. Apart from this expression system, however, an overexpression was not observed. The bioconversion transforming E-pyridine-3-aldoxime into 3-cyanopyridine was dependent on the overexpression level of OxdYH3-3. The initial activity for 3 h of BL21 (DE3)/pET28b-OxdYH3-3 toward E-2-furfurylaldoxime (75%) was 13% lower compared to the initial activity toward E-pyridine-3-aldoxime (88%). Even though the initial conversion was lower, BL21 (DE3)/pET28b-OxdYH3-3 showed full conversion of E-2-furfurylaldoxime under formation of 2-furonitrile after 9 h of reaction time. The new recombinant enzyme OxdYH3-3 was successfully overexpressed and used for the biocatalytic synthesis of the pharmaceutical intermediate 2-furonitrile. The enzyme OxdYH3-3 showed a high catalytic activity for aromatic aldoximes, which enables a perspective for an efficient production of aromatic nitriles.

Cloning, expression, and purification of HpaA-CagA fusion recombinant protein of Helicobacter pylori in E. coli BL 21 strain

Helicobacter pylori colonizes in the stomach of nearly half of the world's population. It is one of the most common causes of chronic bacterial infection and a well-known risk factor for gastric cancer in humans. This study aimed to evaluate the cloning, expression, and purification of the HpaA-CagA fusion recombinant protein of H. pylori in E. coli BL21 (DE3) strain.The HpaA-CagA construct was designed using bioinformatics tools. The construct was then sub-cloned to the pET21b prokaryotic expression vector. Then, E. coli BL21 (DE3) was transfected with the pET21b-HpaA-CagA. Then, E. coli BL21 (DE3) was transformed with pET21b-HpaA-CagA. The gene expression was induced by IPTG. The protein expression was verified using SDS page and western blot analysis. The recombinant protein was finally purified using nickel nitrilotriacetic acid (Ni-NTA) resin. The waste materials were removed by dialysis. The concentration of recombinant protein was determined by the Bicinchoninic Acid Protein Assay Kit (Parstoos).In this study, HpaA-CagA was sub-cloned into the pET21b vector. The expression of the recombinant protein was confirmed in the host. The recombinant protein with the weight of 69 kDa was successfully purified. We here presented an efficient method to construct, clone, express, and purify the HpaA-CagA recombinant protein. The recombinant HpaA-CagA protein showed immunogenicity feature with human antiserum.

Characterization of recombinant endo-1,4-β-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach.

Increased demand of enzymes for industrial use has led the scientists towards protein engineering techniques. In different protein engineering strategies, rational approach has emerged as the most efficient method utilizing bioinformatics tools to produce enzymes with desired reaction kinetics; physiochemical (temperature, pH, half life, etc) and biological (selectivity, specificity, etc.) characteristics. Xylanase is one of the widely used enzymes in paper and food industry to degrade xylan component present in plant pulp. In this study endo 1,4-β-xylanase (Xyl-11A) from Bacillus halodurans C-125 was cloned in pET-22b (+) vector and expressed in Escherichia coli BL21 (DE3) expression strain. The enzyme had Michaelis constant Km of 1.32mgml-1 birchwoodxylan (soluble form) and maximum reaction velocity (Vmax) 73.53mmolmin-1mg-1 with an optimum temperature of 75°C and pH 9.0. The thermostability analysis showed that enzyme retained more than 80% of its residual activity when incubated at 75°C for 2h. In addition, to increase Xyl-11A thermostability, an in-silico analysis was performedto identify the hot spot amino acid residues. Consensus-based amino acid substitution was applied to evaluate multiple sequence alignment of homologs and identified 20 amino acids positions by following Jensen-Shnnon Divergence method. 3D models of 20 selected mutants were analyzed for conformational transition in protein structures by using NMSim server. Two selected mutants T6K and I17M of Xyl-11A retained 40, 60% residual activity respectively, at 85°C for 120min as compared to wild type enzyme which retained 37% initial activity under same conditions, confirming the enhanced thermostability of mutants. The present study showed a good approach for the identification of promising amino acid residues responsible for enhancing the thermostability of enzymes of industrial importance.

Chicken immunoglobulin Y based FRET assay for TSST-1 detection and its validation onto clinical isolates

Staphylococcus aureus (S. aureus) is commensal bacteria implicated in various opportunistic infections worldwide. Their numerous virulence factors along with mounting antibiotic resistance is an ominous problem associated with its clinical management. Among the numerous S. aureus virulence factors, toxic shock syndrome toxin 1 (TSST-1) is a superantigen that is pivotal in life-threatening multisystem disorder. The present study reports the development of a fluorescence resonance energy transfer (FRET) assay with hybrid gold nanoparticles (GNP) conjugated chicken antibodies (IgY) and Cadmium Telluride (CdTe) quantum dots labeled rabbit antibodies for TSST-1 detection. The TSST-1 gene cloned into pET22b (+) vector for recombinant protein production and employed for anti-TSST-1 antibody generation from chicken and rabbit systems. Thereafter, CdTe-QDs tagged anti-TSST-1 IgG was employed as the fluoroprobe and subsequently, GNP conjugated anti-TSST-1 IgY served as its quencher pair. Further analysis results showed that the assay was simple, sensitive and reliable with a limit of detection (LOD) of 0.0005 μgmL-1 and shows no cross-reactivity towards closely related toxins. The artificially spiked clinical sample analysis revealed insignificant matrix interference as well as considerable toxin recovery range (81.21%–99.3%) for the assay. Furthermore, its co-evaluation with commercially available TSST-1 detection kit demonstrated their feasibility for toxin screening with substantially less assay duration. Therefore, the assay may aid in better toxic shock management, apparently reducing empirical treatment, medical costs, and antimicrobial resistance.

Evaluation of Anticancer and Anti-hemolytic Activity of Azurin, a Novel Bacterial Protein from Pseudomonas aeruginosa SSj

Cancer is the second leading cause of death in the world. About 18 milllion people were affected with cancer in 2018. Research in novel anti-cancer agents of bacterial origin thus gain importance. Azurin is a novel bacterial protein that has unique ability to specifically target cancer cells without any harmful effect to normal cells. In this study, cytotoxic effect of azurin from Pseudomonas aeruginosa SSj expressed in pET22b (+) vector and standard azurin (Sigma) were studied on (MCF7), (HCT15), and (VERO) cell lines by MTT assay. Apoptosis assay was performed using Hoechst staining and confirmed by FRET based assay on colon cancer cell lines (SW480). In vivo ascites antitumor model assay was also done to detect the in vivo cytotoxicity of azurin. Anti-hemolytic potential of purified azurin on human erythrocytes was also evaluated. Azurin induced cytotoxicity in both HCT15 and MCF7 cell lines, without having any adverse effect on the normal cells. Cell cycle analysis by Hoechst staining and FRET analysis, both showed evidence of apoptosis induced by azurin. In vivo ascites antitumor model proved that azurin treatment can increase the lifespan of tumor-bearing mice. Anti-hemolytic assay showed a significant increase of inhibition of hemolysis.

The effects of overexpression of cytoplasmic chaperones on secretory production of hirudin-PA in E. coli

The secretory production of heterologous proteins in E. coli has revolutionized biotechnology. Efficient periplasmic production of foreign proteins in E. coli often requires a signal peptide to direct proteins to the periplasm. However, the presence of attached signal peptide does not guarantee periplasmic expression of target proteins. Overproduction of auxiliary proteins, such as chaperones can be a useful approach to enhance protein export. In the current study, three chaperone plasmid sets, including GroEL–GroES (GroELS), Dnak–Dnaj–GrpE (DnaKJE), and trigger factor (TF), were coexpressed in E. coli BL21 (DE3) in a pairwise manner with two pET22-b vectors carrying the recombinant hirudin-PA (Hir) gene and different signal sequences alkaline phosphatase (PhoA) and l-asparaginase II (l-ASP). Overexpression of cytoplasmic combinations of molecular chaperones containing GroELS and DnaKJE with PhoAHir increased the secretory production of PhoAHir by 2.6fold (p < 0.05) and 3.5fold (p < 0.01) compared with their controls, respectively. By contrast, secretory production of PhoAHir significantly reduced in the presence of overexpressed TF (p = 0.02). Further, periplasmic expression of l-ASP was significantly increased only in the presence of DnaKJE (p = 0.04). These findings suggest that using molecular chaperones can be helpful for improving periplasmic expression of Hir. However, tagged signal peptides may affect the physicochemical properties and secondary and tertiary structures of mature Hir, which may alter their interactions with chaperones. Hence, using overexpressed chaperones has various effects on secretory production of PhoAHir and l-ASPHir.

Enhanced Solubility of Anti-HER2 scFv Using Bacterial Pelb Leader Sequence

Single chain Fragment variable (scFv) is an antibody fragment consisting variable regions of heavy and light chains. scFvs enhance their penetrability into tissues while maintaining specific affinity and having low immunogenicity. Insoluble inclusion bodies are formed when scFvs are expressed in reducing bacterial cytoplasm. One strategy for obtaining functionally active scFv is to translocate the scFv into the oxidized environment of the periplasm where the possibility for disulfide bond formation is increased. This can be achieved by cloning the gene in a vector containing N-terminal pelB leader peptide that export foreign proteins to the periplasmic space. The aim of this study is to evaluate the influence of periplasmic localization using pelB leader peptide on the solubility of anti HER2-scFv. Herein, anti HER2-scFv gene was cloned between NcoI and XhoI sites of pET22-b (+) containing pelB leader peptide and in same sites of pET28-b (+) (without pelB). The expression in BL21 (DE3) was induced using IPTG and was analyzed using SDS-PAGE and Western blot experiment. Then, the solubility of anti HER2-scFvin BL21 (DE3) containing both pET22- and pET28-(anti HER2-scFv) was determined. The results of the present study demonstrated that anti HER2-scFv was expressed by both pET22-b (+) and pET28-b (+) vectors in BL21 (DE3). The proper expression of anti-HER2 scFv was confirmed by appearance of a  28 kDa band in Western blot analysis. The most anti HER2-scFv expression from BL21 containing pET28-(anti HER2-scFv) was achieved when it was induced by 0.25 mM IPTG at 37 C, 24 h post-induction. The ratio of soluble/insoluble anti HER2-scFv was significantly higher in BL21 containing pET22-(anti HER2-scFv) than in that containing pET28-(anti HER2-scFv). Totally, fusion of pelB signal sequence to anti HER2-scFv resulted in solubility enhancement. Therefore, production of functional anti HER2-scFv with proper disulfide bond can be achieved by directing the recombinant protein to periplasmic space using pelB signal peptide in pET22 (+) vector.

High expression level of human epidermal growth factor (hEGF) using a well-designed fusion protein-tagged construct in E. coli.

The study was aimed at design a good fusion construct that would successfully express the recombinant proteins and produce peptides inEscherichia coli.Two different constructs including human epidermal growth factor (hEGF) gene were designed to obtain an efficient expression level of hEGF. The hEGF sequence was inserted in pET32a vector containing thioredoxin (Trx) sequence and modified pET15b vector containing intein and elastin-like polypeptide (ELP). The vectors were transformed into E. coli TOP10F' for multiplication and further into E. coli BL21 (DE3) to express protein. The hEGF expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) while the expression levels were evaluated by SDS-PAGE and western blotting and compared by ImageJ analysis, BCA and Elisa assays. The expression level after 2 hours of IPTG induction was significantly higher than after other induction times. ImageJ, BCA and Elisa analyses demonstrated that the Trx presence enhanced protein expression significantly when compared to ELP-intein-based construct. The pET32a-Trx-hEGF construct had a higher expression than pET15b-ELP-intein-hEGF. Overall, considering Trx, the fusion protein in construct design can make it suitable to significantly express hEGF compared to ELP-intein while its combination with ELP-intein may improve the expression of the ELP-intein construct (Tab. 2, Fig. 7, Ref. 34).

Expression and purification of truncated recombinant B8/1 protein of Echinococcus granulosus for diagnosis of hydatid infection in human

Hydatidosis is one of the most important diseases common between animals and human beings. Caused by Echinococcus granulosus tapeworm, the disease has a global epidemic. The serological diagnostic tests that are now utilized to confirm the imaging approaches have some drawbacks such as low sensitivity and cross-reaction with the serum of the patients infected with other parasites. The application of recombinant and synthetic antigens has proven improvement in the functionality of serological diagnostic tests. The purpose of this study was to demonstrate the expression and purification of truncated recombinant B8/1 (trB8/1) antigen and its application in ELISA for diagnosis of hydatid infection in human. The tEgB8/1 was colonized in the expression vector pET28b (+) and expressed in different strains of E. coli. This protein was purified by Ni2+-NTA chromatography. The antigenicity of the protein was evaluated by Western blotting and ELISA. In the test, 50 positive serum samples from hydatid infected patients, 50 samples from healthy people, and 30 serum samples from patients with other parasitic diseases were used to determine the sensitivity and the specificity of this antigen. The measured sensitivity and specificity of this antigen were identified to be 75.75% and 96.38% respectively. The P value of <0.0001 by using ROC curve, confirmed that this antigen is able to differentiate between healthy and hydatid-infected individuals. Considering the excellent specificity of this antigen and in order to enhance the sensitivity, it is recommended to use a combination of this antigen with other antigens (e.g., EgB8/2-8/5).

Extracellular Production of a Potent and Chemically Resistant Nattokinase in Immobilized Escherichia coli Using Response Surface Methodology

Nattokinase is a potent fibrinolytic protease, which is used as a drug for treatment or a supplement for preventing thrombosis besides various industrial applications. The present study aimed to produce a soluble nattokinase in low cost media, which has with high activity and resistance to metal ions, detergents, and organic solvents, and can be easily used in medicines or as detergents. Generally, most of the native extracellular proteins, such as nattokinase from Bacillus subtilis, are lysed by secretory proteases. One way for solving this problem is to employ other hosts for nattokinase production. For producing secretory form of nattokinase from Bacillus subtilis, different factors such as a suitable host, optimized media for the maximum enzyme activity and easy purification are important. These factors are studied in this investigation. Escherichia coli BL21 (DE3), as a reliable host was selected for a high yield production of an extracellular recombinant nattokinase. A mature nattokinase gene from Bacillus subtilis 1023, was cloned in the expression vector pET22b by which the host was transformed. The recombinant nattokinase was expressed through induction with IPTG. The expressed protein was confirmed by SDS-PAGE, and its fibrinolytic activity was assayed on the fibrin plates. Afterwards, the enzyme was purified by Ni-NTA native affinity column. Different media components were evaluated for maximum nattokinase production and activity. The highest enzyme activity of 883.107 U/ml was obtained, when a medium approximately consisting of yeast extract (4.38 g/L), tryptone (4 g/L), K2HPO4 (1.61 g/L) and CaCl2 (0.01 g/L) was used. Entrapping the transformed host in calcium alginate could lead to more enzyme activity and decrease media cost.

Enhanced biosynthesis of CdS nanoparticles through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli with fluorescence effect in detection of pyrogallol and gallic acid

In this work, CdS nanoparticles (CdS NPs) biosynthesized through Arabidopsis thaliana phytochelatin synthase-modified Escherichia coli (CdS/AtPCS1-E. coli) with fluorescence (FL) performance for detection of pyrogallol and gallic acid was investigated. Through expression of the AtPCS1 gene inside E. coli cells by pET28b vector, biosynthesis of CdS NPs was greatly enhanced due to generation of phytochelatins (PCs, (γ-Glu-Cys)n-Gly, n ≥ 2) for efficient capture of Cd2+. The expression of AtPCS1 and concentration of glutathione (GSH) and PCs were detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography (HPLC), respectively. The morphology and component were checked through scanning electron microscope (SEM), transmission electron microscopy (TEM) and energy dispersive spectrometer (EDS). FL effect with different experimental conditions were examined. In addition, it is also applied to determination of pyrogallol and gallic acid. These results revealed that multifunctional PCs could effectively facilitate biosynthesis of CdS NPs with higher yield, better distribution and lower cost while stronger FL intensity could be obtained for quantitative analysis.

A Novel Signal Peptide Derived from Bacillus Licheniformis α-Amylase Efficiently Targets Recombinant Human Activin A to the Periplasm of Escherichia coli

Human activin A is a member of the transforming growth factor-β superfamily consists of two similar beta subunits. Activin A is expressed by different cells and displays numerous biological activities such as control of neuronal cell proliferation and differentiation, promotion of neuronal survival in the body. Therefore, recombinant production of activin A is beneficial because it can be used to treat many neurodegenerative diseases such as Alzheimer's and Parkinson diseases. In this study E. coli as a cheap and fast-growing host was selected to produce recombinant human activin A. As cytoplasmic expression of human activin A with complex structure and disulfide bonds produces inclusion bodies, so periplasmic expression of it can be beneficial. Therefore, we used modified Iranian B. licheniformis α-amylase signal peptide as a new signal peptide in order to translocate the recombinant activin A through the inner membrane. In this study human pro-activin A cDNA and signal sequence were cloned in pET21b vector and resulting vector transformed into the two strains of E. coli BL21. SDS-PAGE and western blot techniques were used to confirm recombinant activin A expression. Finally, our results indicated that the signal peptide used in this study was effective for secretion of activin A into the periplasmic space of E. coli.

Design and Production of a Novel Recombinant Chimeric IL2-Omp31 Antigen against Brucella Infection.

Brucellosis is a zoonotic disease in human and animals. Brucella melitensis is one of the most pathogenic species of Brucella in goat and sheep. Omp31 is an outer membrane protein of Brucella that acts as an immunogenic protein. Cytokines are glycoproteins with low molecular weight that play the role of an immune adjuvant and regulate immune responses. Interleukin-2 is one of the most important cytokines, which are secreted by the white blood cells and involved in T cell immune responses. In the present study, a chimeric Omp31-Interleukin2 recombinant protein was generated by means of genetic engineering techniques. This chimeric coding sequence was amplified by using specific primers and using Splicing Overlap Extension (SOE) PCR technique. The fusion of the two mentioned proteins was accomplished using a rigid linker. The generated chimeric IL2-Omp31 fragment was TA cloned, and then subcloned into pEt22b vector as an expression vector. The chimeric protein was successfully expressed in E. coli BL21 (DE3) and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and also Western-blotting analysis. Finally, in order to assess the antigenic features of the recombinant chimeric IL2-Opm31 protein, its secondary structure and antigenicity were predicted in silico.

Engineering, Cloning and Expression of DNA Sequence Coding of OMP31 Epitope of Brucella melitensis linked to IL-2 in Escherichia coli

: Brucellosis is a zoonetic disease that is caused by Brucella melitensis, which affects human health and animal husbandry industry. Omp31 is the most exposed outer membrane proteins (OMPs) and is a dominant antigen in smooth strains of B. melitensis, thus it is considered a desirable protein in subunit vaccine against brucellosis disease. Besides, IL-2 acts as a growth and differentiation stimulator of T-cells in cellular immune responses, therefore, it can serve as an adjuvant in subunit vaccines against intracellular pathogens. It seems that chimera scaffold, consisting of some copies of OMP31 experimental epitope region and IL-2, can provide a good immunization. In this regard, these tow fragments are linked together through using rigid linker and splicing overlap extension (SOE) PCR technique. Successful PCR products were TA cloned and then sub-cloned into pET-22b (+) vector as an expression vector. Recombinant protein was expressed in Escherichia coli BL21 (DE3) as an expressing lineage bacterium and then confirmed by SDS-PAGE and Western-blotting analysis. Secondary and tertiary structure prediction of recombinant protein and its immunity characterization were evaluated using in silico process. Further studies on the immunogenicity properties of this protein are expected to be carried out in the future to confirm the results in vivo.

Preparation and identification of the nano antibodies of anti-programmed death receptor-1

Objective To prepare camelid-derived nano antibodies with high affinity binding to programmed death receptor-1 (PD-1) antigen, and to provide experimental basis for subsequent functional studies. Methods The PD-1-Fc recombinant protein expressed in eukaryotic expression was used to immunize Xinjiang Bactrian camel 6 times. The peripheral blood was collected and the lymphocytes were isolated. Nested PCR amplification was performed to obtain the genes in variable region of camelid heavy chain antibody (VHH), and to construct a phage display library. The phage display library was screened by solid phase enzyme-linked immunosorbent assay (ELISA). The PD-1 antigen, which was sequentially reduced in mass concentration (5.00、2.50、1.00 μg/ml), was coated in an ELISA plate, and the phage display library was subjected to 3 rounds of affinity selection. Individual clones that bind to PD-1 were further screened by soluble monoclonal ELISA. According to the results of DNA sequencing, three VHH monoclonals with multiple repeats were selected and ligated into pET22b vector, and transformed into E. coli BL21(DE3) competent cells, and then induced by isopropyl-β-D-thiogalactoside. The recombinant VHH antibody protein was purified by nickel column affinity chromatography, and its binding activity and affinity to PD-1 antigen were detected by Western Blot and ELISA. Results After immunization of Bactrian camel 6 times with recombinant protein PD-1-Fc, high titer specific antibody was stimulated, and the immune serum titer reached 1∶32 000. A VHH phage display library with a reservoir size of 2.6×108 cfu/ml was constructed from the immunized camel lymphocytes. After 3 rounds of affinity selection, 46 VHH monoclonals with absorbance (A600) values above 0.6 were obtained by soluble monoclonal ELISA. Among them, three clones of VHH-B7, VHH-H5 and VHH-H12 had higher repeats, indicating that significant enrichment was obtained. The results of Western Blot and ELISA showed that the purified B7, H5 and H12 nanobodies had good binding activity to PD-1 antigen and had high affinity. Their affinity constants were 1.19×1011 and 1.63×1011, 1.59×1011 L/mol, respectively. Conclusion The anti-PD-1 camelid-derived nanobodies were obtained by affinity selection of VHH phage display library, which can bind to the PD-1 antigen with high affinity. This study can provide an experimental basis for subsequent functional studies. Key words: PD-1; Nano antibodies; Heavy chain antibody variable region; Identification; Phage display

Cloning, expression and purification of m-cell specific binding peptide (CO1) fused with GFP

Despite many advantages over injection vaccines such as cost effectiveness, safety and easy to use, and so on, oral vaccines are negligibly concerned. This is mostly because of the availability of vast surface in the gastro-intestinal tract, thereby requiring lot of antigens which could hamper their potential. To circumvent this issue, a novel strategy for targeting antigens to M cells (microfold cells), a minority of cells located in the small intestine for antigen transportation, is utilized by making a fusion protein comprised of an antigen with an M cell specific ligand. Discovered via biopanning, Co1 peptide is a potential ligand because of its small size (12 amino acids) and having an adjuvant capacity. To develop a monitoring model, we fused GFP (green fluorescent protein) as a monitoring marker with Co1 peptide. Initially, co1-gfp fusion gene was created by overlap extension PCR on GFP-encoded vector backbone, then it was incorporated into an expression vector pET22b before transforming into E. coli DH5α. The recombinant vector was screened by PCR method, sequenced and aligned with designed sequences. The in-frame vector was then introduced into E. coli BL21(DE3) for expression by inducing with 0.5mM IPTG. Fusion protein was purified using Ni-affinity chromatography. The results showed that the fused genes were in-frame cloned and completely matched with the designed sequences. SDS-PAGE and Western blot analyses showed Co1-GFP protein expressed in soluble form and could be purified at one-step elution of 500mM imidazole. The purified fusion protein could emit fluorescent light under UV excitation. Collectively, recombinant Co1-GFP fusion protein was successfully produced and its potential applications need to be warranted.

Expression and Purification of Hydrophobic PEP‐Inhibiting Peptides from Bovine αS1 Casein

According to previous studies, there is a prolyl endopeptidase‐inhibiting sequence (LNENLLRFFVAPFPEVFG) within a relatively hydrophobic region of αS1 casein (Juillerat‐Jeanneret et al., 2011). Recently, sixteen recombinant peptides were designed in assorted lengths, each containing the inhibitory sequence at varied positions within the αS1 casein sequence. The corresponding cDNA sequences were originally ligated into the pET15b vector, which provides an N‐terminal poly‐histidine tag for purification. The vector was in turn transformed into a BL21(DE3)pLysS E. coli expression host (Vishram et al., 2016). However, the system proved unsuccessful in adequate expression of the hydrophobic peptide. The intention of this study was to assemble a new pET28a expression system, kindly provided by Dr. X. Cheng (MD Anderson in Houston, TX) that utilizes a SUMO (small ubiquitin‐like modifier) fusion tag. The SUMO tag is known for enhancing expression levels of potentially toxic or insoluble recombinant peptides (Bommarius et al., 2010). The expressed peptides were purified via Ni‐NTA column chromatography, with subsequent UlpI cleavage. This was followed by reversed‐phase high performance liquid chromatography (RP‐HPLC) to further purify the peptides. Tricine‐SDS‐PAGE, a useful screening tool for hydrophobic peptides smaller than 30kDa, was used to analyze expression and purification. A comparison was made between SUMO‐fused expression and non‐fused expression of the hydrophobic casein peptides.Support or Funding InformationOffice of Research and Sponsored Projects, SFASU and Ed &amp; Gwen ColeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prokaryotic Expression and Purification of the Notch Ligand and Its Effect on Hematopoiesis after Carbon Tetrachloride Damage

To express and purify the mouse endothelial cell-targeted recombinant Notch ligand protein mD1R, and to investigate its effect on hematopoiesis after carbon tetrachloride damage. PCR was performed to clone and construct the expression vector pET22b(+)-mD1R. The mD1R successfully transformed into E. coli was induced by IPTG, and purified with Ni2+-beads affinity chromatography. The target protein was detected by SDS-PAGE. The fluorescence-activated cell sorting analysis (FACS), cell adhesion test, immunofluorescence staining and quantitative real-time PCR were employed to detect the endothelial cell-targeted and Notch signaling-activated biological characteristics of mD1R. The carbon tetrachloride mouse model was established to observe the effects of mD1R on the hematopoietic stem cell (HSC), myeloid cells and lymphoid cells by flow cytometry. The Lin-Scal-1+c-Kit+ cells were sorted by magnetic bead, FACS was performed to analyze the cell cycle, and RT-PCR was employed to observe the expression of interleukin (IL)-10. The prokaryotic expression vector was successfully cloned and constructed. The purity and the activity were confirmed in mD1R recombinant protein. The purified mD1R activated the Notch signaling pathway of hematopoietic stem cells in carbon tetrachloride damaged mouse, and internally elevated the number of HSC and long-term HSC to 2.96-fold and 6.18-fold. In addition, mD1R improved the amplification of the myeloid progenitor cells and the myeloid-derived suppressor cells, particularly the granulocyte/monocyte into blood. Mechanistically, the further analyses suggested that Notch pathway could increase the proliferation of HSC and enhance expression of IL-10 after stress injury. A new and activated recombinant Notch ligand protein has been obtained successfully to communicate hematopoietic stem cells and hematopoietic microenvironment. The Notch- mediated intrinsic hematopoiesis has been regulated by the anti-inflammatory factor after stress injury.