Cloning, Expression, and Purification of Recombinant CEL

Cloning, Expression, and Purification of Recombinant CEL I Endonuclease in HEK293T Cell Line [1] Mutation detection using a novel plant ... [2] Purification, cloning, and characterization of the CEL I ... [3] sunTILL: A TILLING resource for gene function analysis in ... [4] High-throughput screening

Recombinant nucleases CEL I from celery and SP I

2007-6-1  In the present study, we reported recombinant expression of plant mismatch endonuclease CEL I and a newly cloned CEL I ortholog, SP I, in a baculovirus system. Active enzymes were expressed as C-terminal hexahistidine fusions, purified from cell culture media using metal affinity chromatography and used for detection of mutations in BRCA1 gene of patient-derived DNA.

Evaluation Recombinant YeastCell EstrogenScreeningAssay

Transformedyeast cells. Recombinant estrogen sensitiveyeast cells (14) werestored in 30%glycerol at -80°C and grown as required at 30°C on selective media agar plates containing 0.17%yeast ...

Production and characterization of the celery

2012-11-25  Here, we demonstrate high-level expression of CEL II using silkworm-baculovirus system. The recombinant CEL II secreted in silkworm hemolymph was glycosylated and susceptible to N-glycosidase F. Additionally, larger metal ions such as Ca 2+ and Sr 2+ were able to replace Mg 2+ and enhanced mismatch cleavage activity of CEL II. These results indicate that the silkworm-baculovirus platform is a good alternative system to obtain the functional CEL II.

Cell Line Development, Single Cell Sorting Molecular

A small population of cells with the foreign DNA integrated into their genome that maintain their ability to express recombinant protein for long periods of time are referred to as stably transfected cells. Antibodies screening/titer ranking – Discovery and selection of high-value clones from a transfected pool of cells. To screen large populations by quantifying cell surface expression of protein-of-interest or secreting

Stabilizing Additives Added during Cell Lysis Aid in the ...

2012-12-20  We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo.

Expression of Recombinant PfCelTOS Antigen in the ...

2018-11-30  PCR Screening of Transformants. Transformants were subcultured on HSM agar and screened for the presence of gene of interest using gene-specific primers. Cells were resuspended in 10 × TE (Tris-EDTA) buffer and heated to 95 °C for 10 min. The cell lysate was then used as a template for PCR under standard conditions .

Improving the efficiency of CHO cell line generation

Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process.

Mapping and regulation of the cel genes in Erwinia ...

Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the ...

Mannose-recognition mutant of the galactose/N ...

2015-7-1  Because the recombinant proteins were obtained as inclusion bodies after disrupting the cells by sonication, they were solubilized in the solubilization buffer [50 mM Tris–HCl, pH 8.0; 0.2 M NaCl; 1 mM ethylenediamine tetraacetate (EDTA); and 6 M guanidine hydrochloride] and the protein was refolded in the refolding buffer (0.1 M Tris–HCl, pH 8.0; 0.4 M L-arginine; 2 mM EDTA; 5 mM reduced

Evaluation of a Recombinant Yeast Cell Estrogen

Exquisiie sensivty, a e of test compound bioans io, ese of use, and the possibili measurin nistogencactivityareimportant attr tt s c fiote suitand the environmen t.Rky antiestrogen, metabolism, py potenc, receptor, recombinant cel bioayuteorophic. Envxiron

Recombinant nucleases CEL I from celery and SP I

Recombinant expression of CEL I and SP I. Infection of a Sf9 cell culture with recombinant viruses containing the CEL I or SP I genes under control of a constitutive promoter resulted in accumulation of a single-strand specific nuclease activity both in the culture media and cell extract (Figure (Figure1C). 1C).This activity adhered to a Ni 2+ affinity column and was eluted with 150 mM imidazole.

Recombinant nucleases CEL I from celery and SP I

2007-6-1  Recombinant expression of CEL I and SP I. Infection of a Sf9 cell culture with recombinant viruses containing the CEL I or SP I genes under control of a constitutive promoter resulted in accumulation of a single-strand specific nuclease activity both in the culture media and cell extract (Figure 1C).This activity adhered to a Ni 2+ affinity column and was eluted with 150 mM imidazole.

High-throughput recombinant protein expression in ...

2016-8-1  Fusion tags can also be used in protein expression screening, which is essential for obtaining well-expressed and functional proteins. If a His-tag is attached to a target protein, an anti-His antibody can be used to detect the expression and solubility of the recombinant

Cell Line Development, Single Cell Sorting Molecular

Stable cell lines are widely used in a number of important applications including biologics (e.g. recombinant protein and monoclonal antibody) production, drug screening, and gene functional studies.The process of developing stable cell lines often starts with transfecting selected host cells, typically CHO or HEK 293 cells, with desired plasmids.

A recombined allele of the lipase gene CEL and its ...

2015-3-16  CEL protein concentration was determined by SDS-PAGE and immunoblotting using titration of recombinant mouse CEL (5658-CE, RD Systems) as a standard. Treatments of stably transfected cells HEK293 cells were seeded in triplicate in 6-well plates (3 × 10 5 cells per well), coated with poly- L -lysine (Sigma), and cultured for 48 hours before ...

Mannose-recognition mutant of the galactose/N ...

2015-7-1  EPNH-CEL-I was crystallized by the hanging-drop and sitting-drop vapor diffusion methods. The initial screening was performed using the screening kits Crystal Screen 1 and 2 (Hampton Research, Aliso Viejo, CA, USA) and Wizard I and II (Emerald BioSystems, Inc., Bedford, MA, USA).

Determination of bioethanol production potential

2020-6-15  Cel-5M gene sequence was cloned and expressed in E. coli and the subsequent recombinant enzyme was purified and run on 12% SDS-PAGE . The SDS-PAGE analysis of the purified Cel-5M showed a single band with the molecular mass of approximately 33 kDa on the gel which is according to protein molecular weight obtained theoretically.

Recombinant DNA Technology - an overview

Recombinant DNA has been effectively used to produce various human proteins in microorganisms, such as insulin and growth hormone, used in the treatment of diseases (see Chapter 4: Recombinant DNA Technology and Genetically Modified Organisms).Unlike chemically synthesized drugs, these are biomacromolecules—primarily endogenous proteins, and present a variety of special ...

Mapping and regulation of the cel genes in Erwinia ...

Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the ...

Recombinant nucleases CEL I from celery and SP I

Recombinant expression of CEL I and SP I. Infection of a Sf9 cell culture with recombinant viruses containing the CEL I or SP I genes under control of a constitutive promoter resulted in accumulation of a single-strand specific nuclease activity both in the culture media and cell extract (Figure (Figure1C). 1C).This activity adhered to a Ni 2+ affinity column and was eluted with 150 mM imidazole.

Recombinant SETD7 protein for use in enzyme kinetics ...

Recombinant SETD7 is suitable for use in the study of enzyme kinetics, inhibitor screening, and selectivity profiling. HMT Assay Conditions: 1 μM H3K4me0 (1-21aa) peptide was incubated with different concentrations of recombinant SETD7 protein in reaction buffer containing 50 mM TrisCl pH 8.6, 0.02% Triton X-100, 2 mM MgCl2, 1 mM TCEP, 100 μM ...

A recombined allele of the lipase gene CEL and its ...

2015-3-16  CEL protein concentration was determined by SDS-PAGE and immunoblotting using titration of recombinant mouse CEL (5658-CE, RD Systems) as a standard. Treatments of stably transfected cells HEK293 cells were seeded in triplicate in 6-well plates (3 × 10 5 cells per well), coated with poly- L -lysine (Sigma), and cultured for 48 hours before ...

Determination of bioethanol production potential

2020-6-15  Cel-5M gene sequence was cloned and expressed in E. coli and the subsequent recombinant enzyme was purified and run on 12% SDS-PAGE . The SDS-PAGE analysis of the purified Cel-5M showed a single band with the molecular mass of approximately 33 kDa on the gel which is according to protein molecular weight obtained theoretically.

Recombinant DNA Technology - an overview

Recombinant DNA has been effectively used to produce various human proteins in microorganisms, such as insulin and growth hormone, used in the treatment of diseases (see Chapter 4: Recombinant DNA Technology and Genetically Modified Organisms).Unlike chemically synthesized drugs, these are biomacromolecules—primarily endogenous proteins, and present a variety of special ...

Competent Cell - an overview ScienceDirect Topics

These colonies are subjected to screening with phenol–chloroform to confirm the presence of the plasmid. After screening by electrophoresis (Fig. 18.4), four colonies (P1, P2, P3, and P4) have the same running pattern as the negative control samples. P5 has a

Mapping and regulation of the cel genes in Erwinia ...

Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the ...

Cell-Free Protein Expression Thermo Fisher Scientific

In vitro protein expression (also known as in vitro translation, cell-free protein expression, cell-free translation, or cell-free protein synthesis) is a technique that enables researchers to rapidly express and manufacture small amounts of functional proteins.Compared to in vivo techniques based on bacterial or tissue culture cells, in vitro protein expression is considerably faster because ...

Microbial cell-surface display - PubMed

Cell-surface display allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs. The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-terminal fusion, C-terminal fusion or sa

pnpla3 - Recombinant Protein, Native Protein, GMP

PNPLA3. Official Full Name. patatin-like phospholipase domain containing 3. Background. The protein encoded by this gene is a triacylglycerol lipase that mediates triacylglycerol hydrolysis in adipocytes. The encoded protein, which appears to be membrane bound, may be involved in the balance of energy usage/storage in adipocytes. Synonyms.



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