scholarly journals Optimization of the fusion protein rhIL7-BAPmut renaturation process from the Escherichia coli inclusion bodies and its practical application

2019 ◽  
Vol 17 (1) ◽  
pp. 38-44
Author(s):  
M. O. Usenko ◽  
O. V. Okunev ◽  
K. I. Bentsionova ◽  
O. B. Gorbatiuk ◽  
D. M. Irodov ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Inclusion Bodies ◽  
Active Form ◽  
Plasmid Vector ◽  
Coli Strain ◽  
Metal Affinity ◽  
Interleukin 7 ◽  
Protein Renaturation ◽  
Secondary Antibodies

Aim. The aim of our work was to optimize the renaturation method of the rhIL7-BAPmut fusion protein based on recombinant human interleukin-7 (rhIL7) and bacterial alkaline phosphatase with enhanced catalytic properties (BAPmut) for its obtaining in functionally active form. Methods. The cells of E. coli strain BL21(DE3) were transformed with pET24-IL7-BAPmut plasmid vector. Protein synthesis was induced by autoinduction protocol. Immobilized-metal affinity chromatography (IMAС) and slow dilution methods were applied for rhIL7-BAPmut fusion protein renaturation from bacterial inclusion bodies in vitro. Results. Combination of IMAС method and slow dilution at the presence of arginine, GSH/ GSSG and Mg2+ ions provided obtaining of rhIL7-BAPmut in pure and active form. Bifunctional activity of rhIL7-BAPmut after refolding is confirmed immunochemically by binding with specific antibodies. Conclusions. It was shown that application of rhIL7-BAPmut allows to reduce the time of the screening of immune combinatory libraries of variable genes of IgG and does not require specific primary and secondary antibodies. The rhIL7-BAPmut fusion protein also can be used for qualitative and quantitative analysis of IL-7 receptors.Keywords: IL-7, BAPmut, inclusion bodies, renaturation.

scholarly journals Obtaining of the recombinant Rhil7-Cbd fusion protein

2020 ◽  
Vol 26 ◽  
pp. 282-286
Author(s):  
M. O. Usenko ◽  
O. B. Gorbatiuk ◽  
O. V. Okunev ◽  
D. M. Irodov ◽  
M. V. Koval’chuk ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Fusion Protein ◽  
Dna Sequences ◽  
Inclusion Bodies ◽  
Plasmid Vector ◽  
Coli Strain ◽  
Iptg Induction ◽  
E Coli ◽  
Interleukin 7

Aim. The purpose was to obtain the recombinant fusion protein based on the human interleukin-7 (rhIL7) and cellulose binding domain (CBD). Methods. The DNA sequences encoding rhIL-7 and CBD were subcloned into the pET24a(+). Vector containing the 6His-tag sequence for further chromatographic purification of the target protein. The cells of E. coli strain BL21(DE3) were transformed with pET24-rhIL7-CBD plasmid vector. Protein synthesis was induced in clones by IPTG and by autoinduction. Results. An expression cassette of rhIL7-CBD protein has been designed. Producers of rhIL7-CBD protein were obtained. It was shown that the autoinduction protocol provides protein synthesis in E. coli (but IPTG induction doesn’t). Protein was obtained in the cytoplasmic fraction in form of inclusion bodies. In vitro method of purification of rhIL7-CBD from inclusion bodies has been worked out. Conclusions. The obtained rhIL7-CBD protein can be used for the microcrystalline cellulose immunosorbent construction for the purification of the specific polyclonal IL-7 antibodies and also for qualitative and quantitative analysis of IL-7 receptors. Keywords: IL-7, CBD, inclusion bodies, renaturation.

scholarly journals Obtaining of the recombinant rhIL7-BAPmut fusion protein and its functional characterization

2019 ◽  
Vol 25 ◽  
pp. 321-326 ◽  
Author(s):  
M. O. Usenko ◽  
O. V. Okunev ◽  
K. I. Bentsionova ◽  
O. B. Gorbatiuk ◽  
D. M. Irodov ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Fusion Protein ◽  
Dna Sequences ◽  
Inclusion Bodies ◽  
Functional Characterization ◽  
Active Form ◽  
Plasmid Vector ◽  
Cdna Libraries ◽  
Single Chain ◽  
E Coli

Aim. The purpose was to obtain a functionally active form of the recombinant fusion protein based on the human interleukin-7 (rhIL7) and bacterial alkaline phosphatase (BAPmut). Methods. The DNA sequences encoding rhIL-7 and BAPmut were subcloned into the pET24a(+) plasmid vector containing the 6His-tag sequence for further chromatographic purification of the target protein. The cells of E. coli strain BL21(DE3) were transformed with pET24-rhIL7-BAPmut plasmid vector. Protein synthesis was induced by IPTG and by autoinduction. Bifunctional activity of rhIL7-BAPmut after refolding via dilution is confirmed immunochemically by binding to specific antibodies. Results. RhIL7-BAPmut protein has been designed. It was shown that autoinduction protocol provides a significantly higher level of protein synthesis in E. coli compared with IPTG induction. In vitro method of purification and renaturation of rhIL7-BAPmut from inclusion bodies has been worked out. It has been shown that rhIL7-BAPmut has a specific biological activity after renaturation. Conclusions. The obtained rhIL7-BAPmut protein can be used for screening of immune combinatorial cDNA libraries of immunoglobulins variable genes, that are the sources of specific single chain antibodies. The protein also can be used for qualitative and quantitative analysis of IL-7 receptors. Keywords: IL-7, BAPmut, inclusion bodies, renaturation.

scholarly journals PhaC and PhaR Are Required for Polyhydroxyalkanoic Acid Synthase Activity in Bacillus megaterium

2001 ◽  
Vol 183 (14) ◽  
pp. 4235-4243 ◽  
Author(s):  
Gabriel J. McCool ◽  
Maura C. Cannon
Keyword(s):  
Bacillus Megaterium ◽  
Inclusion Bodies ◽  
Active Form ◽  
Cell Types ◽  
Pha Synthase ◽  
Class Iii ◽  
Polyhydroxyalkanoic Acid ◽  
Genes Encoding

ABSTRACT Polyhydroxyalkanoic acids (PHAs) are a class of polyesters stored in inclusion bodies and found in many bacteria and in some archaea. The terminal step in the synthesis of PHA is catalyzed by PHA synthase. Genes encoding this enzyme have been cloned, and the primary sequence of the protein, PhaC, is deduced from the nucleotide sequences of more than 30 organisms. PHA synthases are grouped into three classes based on substrate range, molecular mass, and whether or not there is a requirement for phaE in addition to thephaC gene product. Here we report the results of an analysis of a PHA synthase that does not fit any of the described classes. This novel PHA synthase from Bacillus megaterium required PhaC (PhaCBm) and PhaR (PhaRBm) for activity in vivo and in vitro. PhaCBm showed greatest similarity to the PhaCs of class III in both size and sequence. Unlike those in class III, the 40-kDa PhaE was not required, and furthermore, the 22-kDa PhaRBm had no obvious homology to PhaE. Previously we showed that PhaCBm, and here we show that PhaRBm, is localized to inclusion bodies in living cells. We show that two forms of PHA synthase exist, an active form in PHA-accumulating cells and an inactive form in nonaccumulating cells. PhaC was constitutively produced in both cell types but was more susceptible to protease degradation in the latter type. Our data show that the role of PhaR is posttranscriptional and that it functions directly or indirectly with PhaCBm to produce an active PHA synthase.

In silico approach for designing a novel recombinant fusion protein as a Candidate Vaccine against HPV

2020 ◽  
Vol 17 ◽  
Author(s):  
Mohsen Sisakht ◽  
Amir Mahmoodzadeh ◽  
Mohammadsaeid Zahedi ◽  
Davood Rostamzadeh ◽  
Amin Moradi Hasan-Abad ◽  
...  
Keyword(s):  
Immune Responses ◽  
Fusion Protein ◽  
In Silico ◽  
Precancerous Lesions ◽  
Candidate Vaccine ◽  
T Cell Epitopes ◽  
Binding Interaction ◽  
Hpv16 E6 ◽  
E7 Proteins

Background: Human papillomavirus (HPV) is the main biological agent causing sexually transmitted diseases (STDs), including precancerous lesions and several types of prevalent cancers. To date, numerous types of vaccines are designed to prevent high-risk HPV. However, their prophylactic effect is not the same and does not clear previous infections. Therefore, there is an urgent need for developing therapeutic vaccines that trigger cell-mediated immune responses for the treatment of HPV. The HPV16 E6 and E7 proteins are ideal targets for vaccine therapy against HPV. Fusion protein vaccines, which include both immunogenic interest protein and an adjuvant for augmenting the immunogenicity effects, are theoretically capable of guarantee the power of the immune system against HPV. Method: A vaccine construct, including HPV16 E6/E7 proteins along with a heat shock protein GP96 (E6/E7-NTGP96 construct), was designed using in silico methods. By the aid of the SWISS-MODEL server, the optimal 3D model of the designed vaccine was selected, followed by physicochemical and molecular parameters were performed using bioinformatics tools. Docking studies were done to evaluate the binding interaction of the vaccine. Allergenicity, immunogenicity, B, and T cell epitopes of the designed construct were predicted. Results: Immunological and structural computational results illustrated that our designed construct is potentially proper for stimulation of cellular and humoral immune responses against HPV. Conclusion: Computational studies showed that the E6/E7-NTGP96 construct is a promising candidate vaccine that needs further in vitro and in vivo evaluations.

scholarly journals Engineering subtilisin proteases that specifically degrade active RAS

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yingwei Chen ◽  
Eric A. Toth ◽  
Biao Ruan ◽  
Eun Jung Choi ◽  
Richard Simmerman ◽  
...  
Keyword(s):  
Active Form ◽  
High Specificity ◽  
Kinetic Properties ◽  
Target Sequence ◽  
Reporter System ◽  
Human Cell Culture ◽  
Conserved Sequence ◽  
Cofactor Binding ◽  
Selective Proteolysis

AbstractWe describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

scholarly journals DnaA, the Initiator of Escherichia coliChromosomal Replication, Is Located at the Cell Membrane

2000 ◽  
Vol 182 (9) ◽  
pp. 2604-2610 ◽  
Author(s):  
Gillian Newman ◽  
Elliott Crooke
Keyword(s):  
Cell Membrane ◽  
Spatial Organization ◽  
Active Form ◽  
Chromosomal Replication ◽  
E Coli ◽  
Dnaa Protein ◽  
Section Electron ◽  
Acidic Phospholipids

ABSTRACT Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.

scholarly journals Antioxidant Fusion Protein SOD1-Tat Increases the Engraftment Efficiency of Total Bone Marrow Cells in Irradiated Mice

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3395
Author(s):  
Ting Bei ◽  
Xusong Cao ◽  
Yun Liu ◽  
Jinmei Li ◽  
Haihua Luo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Bone Marrow Cells ◽  
Standard Procedure ◽  
Severe Infection ◽  
Copper Zinc Superoxide Dismutase ◽  
Donor Cells ◽  
Total Bone Marrow ◽  
Marrow Cells

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.

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