Bacterial Protein Overexpression Systems and Strategies

Kinsland, Cynthia

doi:10.1016/b978-008045382-8.00199-4

Cited by 3 publications

(2 citation statements)

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“…They concluded that the presence or absence of a His-tag should be taken into consideration when investigating protein dynamics and other properties [ 45 ]. This is contrary to many published studies and protocols that state that the His-tag, due to having a small size of ~2.5 kDa, is assumed to have no significant impact or interference on a protein's structure, function, or folding within a cell [ [46] , [47] , [48] , [49] , [50] , [51] ].…”

Section: Discussionmentioning

confidence: 63%

Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity

Turkewitz

Moghaddasi

Alghalayini

et al. 2021

Biochemistry and Biophysics Reports

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The chloride intracellular ion channel protein (CLIC) family are a unique set of ion channels that can exist as soluble and integral membrane proteins. New evidence has emerged that demonstrates CLICs' possess oxidoreductase enzymatic activity and may function as either membrane-spanning ion channels or as globular enzymes. To further characterize the enzymatic profile of members of the CLIC family and to expand our understanding of their functions, we expressed and purified recombinant CLIC1, CLIC3, and a non-functional CLIC1-Cys24A mutant using a Histidine tag, bacterial protein expression system. We demonstrate that the presence of the six-polyhistidine tag at the amino terminus of the proteins led to a decrease in their oxidoreductase enzymatic activity compared to their non-His-tagged counterparts, when assessed using 2-hydroxyethyl disulfide as a substrate. These results strongly suggest the six-polyhistidine tag alters CLIC's structure at the N-terminus, which also contains the enzyme active site. It also raises the need for caution in use of His-tagged proteins when assessing oxidoreductase protein enzymatic function.

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Section: Discussionmentioning

confidence: 63%

Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity

Turkewitz

Moghaddasi

Alghalayini

et al. 2021

Biochemistry and Biophysics Reports

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“…An antigen is a key molecule that stimulates an immune response during immunization for antibody discovery. Normally, antigens are expressed and purified, with a 6× Histidine tag 1 for ease of purification. However, some antigens have issues with aggregation, precipitation, and protein misfolding, which all lead to poor expression and purification yields.…”

Section: Introductionmentioning

confidence: 99%

Difficult‐to‐express antigen generation through a co‐expression and disassociation methodology

Lieu,

Chao,

Kennedy

et al. 2023

Biotechnology Progress

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Extracellular domain (ECD) antigens are crucial components for antibody discovery, in vitro assays, and epitope mapping during therapeutical antibody development. Oftentimes, those antigens are difficult to produce while retaining the biologic function/activity upon extracellular secretion in commonly used expression systems. We have developed an effective method to cope with the challenge of generating quality antigen ECDs. In this method, a monoclonal antibody (Mab) or antibody fragment antigen‐binding (Fab) region acts as a “chaperone” to stabilize the antigen ECD through forming an antibody:antigen complex. This methodology includes transient co‐expression of the complex in Chinese hamster ovary cells and then dissociation of the purified complex into individual components by low pH treatment in the presence of arginine. The antigen is then separated from the chaperone on a preparative size exclusion chromatography (pSEC) followed by an optional affinity chromatography process to remove residual Mab or Fab. We demonstrate this co‐expression/disassociation methodology on two difficult‐to‐express antigen ECDs from cluster‐of‐differentiation/cytokine family and were successful in producing stable, biologically active antigens when the common methods using Histidine‐tagged and/or Fc‐fused protein failed. This can be applied as a general approach for antigen production if a Mab or binding partner is available.

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Functional mononuclear molybdenum enzymes: challenges and triumphs in molecular cloning, expression, and isolation

et al. 2020

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Bacterial Protein Overexpression Systems and Strategies

Cited by 3 publications

References 298 publications

Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity

Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity

Difficult‐to‐express antigen generation through a co‐expression and disassociation methodology

Functional mononuclear molybdenum enzymes: challenges and triumphs in molecular cloning, expression, and isolation

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