scholarly journals Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography

2011 ◽  
Vol 77 (13) ◽  
pp. 4634-4646 ◽  
Author(s):  
Carine Robichon ◽  
Jianying Luo ◽  
Thomas B. Causey ◽  
Jack S. Benner ◽  
James C. Samuelson
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Metal Binding ◽  
Divalent Cations ◽  
Immobilized Metal Affinity Chromatography ◽  
Content Type ◽  
Cobalt Ions ◽  
Chitin Binding Domain ◽  
E Coli ◽  
Metal Affinity

ABSTRACTRecombinant His-tagged proteins expressed inEscherichia coliand purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with nativeE. coliproteins, especially if the recombinant protein is expressed at a low level. TheE. colicontaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineeredE. coliBL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of twoE. coliBL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that eachE. coliCBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

scholarly journals ThechbGGene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

2012 ◽  
Vol 194 (18) ◽  
pp. 4959-4971 ◽  
Author(s):  
Subhash Chandra Verma ◽  
Subramony Mahadevan
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Inflammatory Diseases ◽  
Regulatory Protein ◽  
Loss Of Function ◽  
Content Type ◽  
Reading Frame ◽  
E Coli ◽  
Evolutionarily Conserved

ABSTRACTThechboperon ofEscherichia coliis involved in the utilization of the β-glucosides chitobiose and cellobiose. The function ofchbG(ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show thatchbGencodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of thechbpromoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations inchbRconferring the ability to grow on both cellobiose and chitobiose are independent ofchbGfunction for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-β-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence ofchbG, suggesting an additional role ofchbGin the hydrolysis of chitooligosaccharides. The homologs ofchbGin metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function ofE. colichbGhas a broader significance.

scholarly journals Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography

2004 ◽  
Vol 78 (18) ◽  
pp. 9820-9827 ◽  
Author(s):  
Kaiming Ye ◽  
Sha Jin ◽  
Mohammad M. Ataai ◽  
Jerome S. Schultz ◽  
Jeanette Ibeh
Keyword(s):  
Affinity Chromatography ◽  
Metal Binding ◽  
Viral Vectors ◽  
Retroviral Vectors ◽  
Immobilized Metal Affinity Chromatography ◽  
Binding Peptide ◽  
Metal Affinity ◽  
Contaminant Level ◽  
Fold Reduction ◽  
One Step

ABSTRACT Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6 peptide. Display of the His6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 μg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.

OPTIMIZACIÓN DE LA EXTRACCIÓN Y PURIFICACIÓN DE UNA β-CAROTENO 15,15’-MONOOXIGENASA RECOMBINANTE A PARTIR DE CUERPOS DE INCLUSIÓN

Agrociencia ◽  
2021 ◽  
Vol 55 (4) ◽  
pp. 317-329
Author(s):  
Martín Barbosa Amezcua ◽  
Luz Vázquez Moreno ◽  
Laura González Dávalos ◽  
Armando Shimada ◽  
Ofelia Mora
Keyword(s):  
Escherichia Coli ◽  
Liquid Chromatography ◽  
Affinity Chromatography ◽  
Gallus Gallus ◽  
Fast Protein Liquid Chromatography ◽  
Immobilized Metal Affinity Chromatography ◽  
Gen Y ◽  
Metal Affinity ◽  
High Polarity

Los forrajes utilizados en la producción de ganado bovino en el trópico tienen altos niveles de β-caroteno, que produce canales con grasa de color amarillo y demerita su valor económico. La pigmentación amarilla se debe a la actividad baja de la enzima β -caroteno 15,15’-monooxigenasa (BCMO1) en el intestino delgado e hígado. Un uso biotecnológico de esta enzima podría escindir al β -caroteno en dos moléculas de retinal, eliminar la fuente de coloración y optimizar el valor comercial de la carne del ganado bovino alimentado en pastoreo. El objetivo de este estudio fue obtener una enzima BCMO1 recombinante con actividad similar a las enzimas nativas, a partir de bacterias transformadas con el gen que codifica la b-caroteno 15,15’-monooxigenasa de Gallus gallus (gBCMO1). La enzima se obtuvo por sobre expresión a partir de una Escherichia coli XL1-Blue transformada con dicho gen, y se purificó por Cromatografía rápida de proteína líquida (Fast Protein Liquid Chromatography, FPLC); se midió la actividad in vitro del proceso por Cromatografía de afinidad por metales inmovilizados (Immobilized Metal Affinity Chromatography, IMAC) y el producto final se detectó por Cromatografía de líquidos de polaridad alta (High Polarity Liquid Chromatography, HPLC). Una proteína de aproximadamente 63 kDa se obtuvo, la cual presentó una actividad enzimática de 2993 (± 108.2) pmol mg-1 de proteína h-1 (n=3). La proteína aislada se puede evaluar como aditivo en estudios in vitro con el fin de disminuir la coloración amarilla de las canales de bovinos.

scholarly journals Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

2012 ◽  
Vol 78 (7) ◽  
pp. 2289-2296 ◽  
Author(s):  
Ruth Hall Sedlak ◽  
Marketa Hnilova ◽  
Carolynn Grosh ◽  
Hanson Fong ◽  
Francois Baneyx ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Metal Ion ◽  
Binding Protein ◽  
Metal Resistance ◽  
Transmission Electron Microscopy Analysis ◽  
Binding Peptide ◽  
Content Type ◽  
E Coli

ABSTRACTSilver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerantEscherichia colistrain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of theE. colimaltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strongin vivophenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as thisE. colistrain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactionsin vivo.

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