Advances in the Production and Batch Reformatting of Phage Antibody Libraries Abstract Phage display antibody libraries have proven an invaluable resource for the isolation of diagnostic and potentially therapeutic antibodies, the latter usually being antibody fragments converted into IgG formats. Recent advances in the production of highly diverse and functional antibody libraries are considered here, including for Fabs, scFvs and nanobodies. These advances include codon optimisation during generation of CDR diversity, improved display levels using novel signal sequences, molecular chaperones and isomerases and the use of highly stable scaffolds with relatively high expression levels. In addition, novel strategies for the batch reformatting of scFv and Fab phagemid libraries, derived from phage panning, into IgG formats are described. These strategies allow the screening of antibodies in the end-use format, facilitating more efficient selection of potential therapeutics.

Molecular Cloning, Expression and Biochemical Characterization of a Family 5 Glycoside Hydrolase First Endo-Mannanase ( Rf GH5_7) from Ruminococcus flavefaciens FD-1 v3 Abstract The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

Formation of Virus-Like Particles of the Dengue Virus Serotype 2 Expressed in Silkworm Larvae Abstract To explore virus-like particles formation of dengue virus serotype type 2 (DENV-2) structural proteins of, C, prM, E were expressed in silkworm larvae using recombinant Bombyx mori nucleopolyhedroviruses (BmNPV). Each recombinant BmNPV bacmid coding the 2C–prM–E polypeptide and E protein fused with the signal peptide of bombyxin from B. mori was injected into silkworm larvae. The expressed proteins were collected from hemolymph and fat body, and purified using affinity chromatography. E protein was observed at 55 kDa. The DENV virus-like particles (DENV-LPs) with a diameter approximately 35 nm was observed using transmission electron microscopy (TEM) and immunogold-labelling TEM analysis. The binding of each partially purified proteins to heparin, one of receptors for DENV was confirmed. DENV-LPs were secreted in silkworm larval hemolymph even still low amount, but the E protein and heparin binding function were confirmed.

Flavin-Based Fluorescent Protein EcFbFP Auto-Guided Surface Display of Methyl Parathion Hydrolase in Escherichia coli Abstract Methyl parathion hydrolase (MPH) plays an important role in degrading a range of organophosphorus compounds. In order to display MPH on the cell surface of Escherichia coli strain RosettaBlue™, the Flavin-based fluorescent protein EcFbFP was severed as an auto-anchoring matrix. With net negative charges of EcFbFP supplying the driving forces, fusion protein MPH-EcFbFP through a two-step auto-surface display process was finally verified by (a) inner membrane translocation and (b) anchoring at outer membrane. Cells with surface-displayed MPH obtained activity of 0.12 U/OD600 against substrate methyl parathion. MPH when fused with engineered EcFbFP containing 20 net negative charges exhibited fivefold higher anchoring efficiency and tenfold higher enzymatic catalytic activity of 1.10 U/OD600. The above result showed that MPH was successfully displayed on cell surface and can be used for biodegradation of methyl parathion.

Heterologous Expression of S fp -Type Phosphopantetheinyl Transferase is Indispensable in the Biosynthesis of Lipopeptide Biosurfactant Abstract Phosphopantetheinyl transferases are of tremendous enthusiasm inferable from their fundamental parts in activating polyketide, fatty acid, and non-ribosomal peptide synthetase enzymes and additionally an increasing number of biotechnological applications. The present study reports the identification of sfp gene from the Paenibacillus sp. D9, which encompasses 693 bp encoding a 230-amino acid protein with a molecular weight of 25.3 kDa. The amino acid sequence Paenibacillus sp. D9 Sfp revealed more than 90% sequence identity to other Sfp proteins from other Paenibacillus. The sfp gene was cloned and recovered efficiently using affinity chromatography with maximal specific phosphopantetheinyl transferase activity at an optimal pH of 8.0 and temperature of 30 °C. The enzyme also exhibited stability under a wide-ranging pH and temperature. The presence of Zn2+, Cu2+, and Fe2+ ions improved the enzymatic activity, while other metals such as Ni2+, Co2+, and Mg2+ had inhibitory effects. The introduction of EDTA also displayed no inhibition. Kinetic parameters were obtained having values of 4.52 mg/mL, 35.33 U/mg, 3.64 s−1, and 0.104 mM−1 s−1 for Km, Vmax, kcat, and kcat/Km, respectively. The biosurfactant synthesized by the recombinant BioSp was found to be surface active, reducing the surface tension to 33.7 mN/m on the glucose substrate after 5 days of incubation at 37 °C. The recombinant Escherichia coli strain also exhibited an improvement in biosurfactant yield (1.11 g/L) when contrasted with 0.52 g/L from Paenibacillus sp. D9. High esterase activity of 2.55 IU/mL using p-nitrophenyl acetate was observed on the recombinant strain, as the protein connected with the release of the biosurfactant was observed to be an esterase. The characteristics of improved biosurfactant and esterase synthesis by hyper-producing recombinant strain possess numerous values from biotechnology standpoint.

Biochemical and Conformational Characterization of Recombinant VEGFR2 Domain 7 Abstract Angiogenesis is a biological process finely tuned by a plethora of pro- and anti-angiogenic molecules, among which vascular endothelial growth factors (VEGFs). Their biological activity is expressed through the interaction with three cognate receptor tyrosine kinases, VEGFR1, 2, and 3. VEGFR2 is the primary regulator of angiogenesis. Ligand-induced VEGFR2 dimerization and activation depend on direct ligand binding to extracellular domains 2 and 3 of receptor and in the establishment of interactions between proximal membrane domains. VEGFR2 domain 7 has been shown to play a crucial role in receptor dimerization and regulation, therefore, representing a convenient target for the allosteric modulation of VEGFR2 activity. The ability to prepare a functional VEGFR2D7 domain represents the starting point to the development of novel VEGFR2 binders acting as allosteric inhibitors of receptor activity. Here, we describe a robust and efficient procedure for the preparation in E. coli of the VEGFR2 domain 7. The protein was obtained with a good yield and was properly folded. It was investigated in a biochemical and structural study, providing information on its conformational arrangement and in solution properties.

Recent Trends in Enhancing the Resistance of Cultivated Plants to Heavy Metal Stress by Transgenesis and Transcriptional Programming Abstract Normal growth and development of high plants strongly depends on the concentration of microelements, including essential heavy metals, in the substrate. However, an excess of those elements may become harmful. Therefore, micronutrient concentrations in plant tissue should be well-balanced and controlled by homeostatic mechanisms. The advancement of knowledge on the regulation of metal homeostasis in plants is important for phytoremediation of metal-contaminated soil and for micronutrient malnutrition control. Experimental data from loss-of-function and gain-of-function studies, including functional descriptions and classifications have presented new opportunities for multiplex CRISPR/dCas9-driven control of gene expression and have opened up new prospects for the goal-seeking regulation of metal homeostasis in plants. The aim of this review is to help for multiplex transcriptional programming targets search by summarizing and analyzing data on possible ways to handle a plant’s ability to maintain metal homeostasis.

Expression of Breast Cancer-Related Epitopes Targeting the IGF-1 Receptor in Chimeric Human Parvovirus B19 Virus-Like Particles Abstract Breast cancer is a worldwide health problem, and the complexity of the disease, as well as the lack of treatment specificity, generates an urgent need for developing prophylactic and therapeutic measures. Searching for novel epitope-based approaches able to induce tumour immunity, we designed virus-like particles (VLPs) derived from Human parvovirus B19 assembled of chimeric VP2 proteins displaying two epitopes from the insulin-like growth factor-1 receptor (IGF-1R). Here, we present the generation of two chimeric VP2s that retain the stability, solubility and conditions of purification and assembly of the native VP2. We generated versatile chimeric multiepitope anti-cancer vaccine candidates, which prevented and delayed tumour growth when used in a prophylactic scheme of 4 weekly immunizations prior to 4T1 cell inoculation in female BALB/c mice. The presence of specific antibodies against the displayed epitopes suggests their participation in the protective effect; in contrast, no significant proliferative T-cell responses were recorded following stimulation by specific epitopes. The results comprise an approach whereby fusing desired epitopes from cancer to the N-terminus of B19 VP2 protein can generate a library of chimeric VP2-desired epitopes for further assembly in a designed and personalized epitope delivery system.

Recent Developments of Reverse Micellar Techniques for Lysozyme, Bovine Serum Albumin, and Bromelain Extraction Abstract Biomolecules produced by living organisms can perform vast array of functions and play an important role in the cell. Important biomolecules such as lysozyme, bovine serum albumin (BSA), and bromelain are often studied by researchers due to their beneficial properties. The application of reverse micelles is an effective tool for protein separation from their sources due to the special system structure. Mechanisms of transferring biomolecules and factors that influence the extraction of biomolecules are reviewed in this paper. The enhancement of biomolecule extraction could be achieved depending on the properties of reverse micelles. This paper provides an overall review on lysozyme, BSA, and bromelain extraction by reverse micelle for various applications.

Production of Lysostaphin by Nonproprietary Method Utilizing a Promoter from Toxin–Antitoxin System Abstract Lysostaphin is a staphylolytic protein of growing interest from biotechnological and pharmaceutical industry due to its potential use in preventing and combating staphylococcal infections. Here, we describe an optimized method for production of lysostaphin in an inductionless system utilizing constitutive promoter from staphylococcal toxin–antitoxin system PemIK-Sa1. We investigated the influence of ribosome-binding site sequence, Escherichia coli producer strain and growth media on yield and kinetics of recombinant protein production. Lysostaphin was purified in its native active form using one-step cation-exchange chromatography. The system provides a method for cost-efficient and scalable protein production, and can be applied to produce other biotechnologically significant proteins.