The two faces of His‐tag: Immune response versus ease of protein purification

Randolph, Theodore W.

doi:10.1002/biot.201100459

Cited by 20 publications

(7 citation statements)

References 11 publications

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“…5C and D indicate that all vaccines elicited a significantly higher Ab titer to the Histidine tag than those vaccines with a foreign epitope insertion N-terminal to the Histidine tag (HBcAg + SSM and mHBcAg + SSM), following the same pattern as the Ab titers to the whole DHBcAg protein. Together these data demonstrate that the histidine tag located at the N-terminus of the vaccine candidates may play a large role in Ab recognition to each protein, as has been seen previously by others, and the placement of an epitope N-terminal to the tag may reduce its immune interaction [40].…”

Section: Vector Protein Antibody Generationsupporting

confidence: 80%

Epitope selection and their placement for increased virus neutralization in a novel vaccination strategy for porcine epidemic diarrhea virus utilizing the Hepatitis B virus core antigen

Gillam

Zhang

2018

Vaccine

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Porcine epidemic diarrhea virus (PEDV) is a member of the Alphacoronaviridae genus within the Coronaviridae family. It is the causative agent of porcine epidemic diarrhea, a disease that can have mortality rates as high as 100% in suckling piglets. PEDV causes severe economic loss, and has been in existence for decades. A panzootic starting in 2010 renewed interest in the development of a universal vaccine toward PEDV. This report details several design changes made to a Hepatitis B virus core antigen (HBcAg)-based recombinant vaccine strategy, and their effect in vivo. Initially, several multi-antigen vaccine candidates were able to elicit antibodies specific to three out of four B-cell epitopes inserted into the chimeric proteins. However, a lack of virus neutralization led to a redesign of the vaccines. The focus of the newly redesigned vaccines was to elicit a strong immune response to the YSNIGVCK amino acid motif from PEDV. Genetically modified new vaccine candidates were able to elicit a strong antibody (Ab) response to the YSNIGVCK epitope, which correlated with an increased ability to neutralize the CO strain of PEDV. Additionally, the location of the inserted PEDV epitopes within the vector protein was shown to affect the immune recognition toward the native HBcAg during vaccination.

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Section: Vector Protein Antibody Generationsupporting

confidence: 80%

Epitope selection and their placement for increased virus neutralization in a novel vaccination strategy for porcine epidemic diarrhea virus utilizing the Hepatitis B virus core antigen

Gillam

Zhang

2018

Vaccine

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“…The inclusion of a His‐tag downstream of the sortag allowed purification of the sdAb via IMAC during sdAb production, while it resulted in the loss of the His‐tag upon conjugation. The latter is a great advantage for clinical translation where the use of His‐tagged proteins is avoided since they increase the possible risk of immune responses . Of note, the safety profile of the use of SrtA‐mediated conjugation in clinical tracer production, and in particular the non‐immunogenic character of the sortag and absence of metal contaminants coming from the IMAC step in purification, still needs to be verified.…”

Section: Discussionmentioning

confidence: 99%

Sortase A‐mediated site‐specific labeling of camelid single‐domain antibody‐fragments: a versatile strategy for multiple molecular imaging modalities

Massa

Vikani

Betti

et al. 2016

Contrast Media & Molecular

108

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A generic site-specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein-ligation capacity of the enzyme Sortase A to label camelid single-domain antibody-fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX-A"-DTPA and NOTA for single-photon emission computed tomography (SPECT) with indium-111 and positron emission tomography (PET) with gallium-68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single-conjugated tracer populations were obtained in high yield (30-50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1-tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A-mediated conjugation as a valuable strategy for the development of site-specifically labeled camelid single-domain antibody-fragments for use in multiple molecular imaging modalities.

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“…Interestingly, these studies have revealed that the tacn‐based IMAC resins exhibit significantly increased association constants and higher kinetic binding rates with recombinant proteins containing N‐terminal metal binding tags specifically designed for this purpose, such as the NT1A tag, compared with the [His]6 tag, whilst the efficiency of removal of tags specifically selected to be good substrates for DAP‐1 could be improved compared with [His]6 as a result of greater control over enzyme activation by attaining the optimal pH window for the cleavage conditions. Moreover, recent studies with recombinant malarial antigens have demonstrated (Khan et al ., ; Randolph, ) that a [His]6 tag can have significant humoral and cellular immunological consequences and have thus provided an additional driver for the development of alternative tags for use in IMAC purification of recombinant proteins.…”

Section: Discussionmentioning

confidence: 99%

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures

et al. 2015

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The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes.

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The two faces of His‐tag: Immune response versus ease of protein purification

Cited by 20 publications

References 11 publications

Epitope selection and their placement for increased virus neutralization in a novel vaccination strategy for porcine epidemic diarrhea virus utilizing the Hepatitis B virus core antigen

Epitope selection and their placement for increased virus neutralization in a novel vaccination strategy for porcine epidemic diarrhea virus utilizing the Hepatitis B virus core antigen

Sortase A‐mediated site‐specific labeling of camelid single‐domain antibody‐fragments: a versatile strategy for multiple molecular imaging modalities

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures

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