Immunodetection of recombinant proteins based on antibodies directed against a metal binding peptide engineered for purification by immobilized metal affinity chromatography

1992 ◽  
Vol 156 (2) ◽  
pp. 231-238 ◽  
Author(s):  
David B. Evans ◽  
Anne F. Vosters ◽  
Jane B. Carter ◽  
Satish K. Sharma
Keyword(s):  
Affinity Chromatography ◽  
Recombinant Proteins ◽  
Metal Binding ◽  
Immobilized Metal Affinity Chromatography ◽  
Binding Peptide ◽  
Metal Affinity

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.

scholarly journals Bifunctional nickel–iminodiacetic acid-core–shell silica nanoparticles for the exclusion of high molecular weight proteins and purification of His-tagged recombinant proteins

RSC Advances ◽  
2019 ◽  
Vol 9 (20) ◽  
pp. 11038-11045
Author(s):  
Sergio G. Hernandez-Leon ◽  
Jose A. Sarabia-Sainz ◽  
Gabriela Ramos-Clamont Montfort ◽  
José Ángel Huerta-Ocampo ◽  
Ana M. Guzman-Partida ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Silica Nanoparticles ◽  
Recombinant Proteins ◽  
Iminodiacetic Acid ◽  
Immobilized Metal Affinity Chromatography ◽  
Core Shell ◽  
Metal Affinity ◽  
Chemically Modified ◽  
High Molecular Weight Proteins

Silica nanoparticles were synthesized and chemically modified with iminodiacetic acid (IDA) and Ni2+ ions surrounded by a bis-acrylamide polymeric shell to obtain a new core–shell immobilized metal affinity chromatography (IMAC) based material.

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.

Immobilized metal-ion affinity systems for recovery and structure–function studies of proteins at molecular, supramolecular, and cellular levels

2010 ◽  
Vol 82 (1) ◽  
pp. 39-55 ◽  
Author(s):  
Rajasekar R. Prasanna ◽  
Mookambeswaran A. Vijayalakshmi
Keyword(s):  
Capillary Electrophoresis ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Metal Ion ◽  
Immobilized Metal Affinity Chromatography ◽  
Metal Affinity ◽  
Purification Technique ◽  
Affinity Gel Electrophoresis ◽  
Insight Into ◽  
Histidine Tag

Immobilized metal-ion affinity (IMA) adsorption is a collective term that is used to include all kinds of adsorptions where the metal ion serves as the characteristic and most essential part of adsorption center. Of all the IMA techniques, immobilized metal-affinity chromatography (IMAC) has been gaining popularity as the choice of purification technique for proteins. IMAC represents a separation technique that is primarily useful for proteins with natural surface exposed-histidine residues and for recombinant proteins with engineered histidine tag. This review also gives insight into other nonchromatographic applications of IMA adsorption such as immobilized metal-ion affinity gel electrophoresis (IMAGE), immobilized metal-ion affinity capillary electrophoresis (IMACE), and immobilized metal-ion affinity partitioning (IMAP).

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