Purification-independent immunoreagents obtained by displaying nanobodies on bacteria surface

Oloketuyi, Sandra Folarin; Dilkaute, Carina; Mazzega, Elisa; Jose, Joachim; Marco, Ario de

doi:10.1007/s00253-019-09823-x

Cited by 5 publications

(3 citation statements)

References 43 publications

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“…The first is due to the presence of incomplete chaperone machinery that can impair correct folding at high expression rate and the second is the consequence of the limited volume of the compartment which can lead to overcrowding. Alternative production of nanobodies and nanobodyfusion constructs has been successfully demonstrated in bacterial cytoplasm [85] and by using secretion routs [86,87] (see sections 5 and 6).…”

Section: Bacterial Periplasmmentioning

confidence: 99%

“…There is no necessity to purify the recombinant binders, no engineering is required to link them to the surface and they are already oriented outwards. The bacteria can be directly spotted on a surface and be used as immunocapture elements of biosensors or as both the capture and detection regents of an ELISA test [87,93]. As an alternative, their capacity of agglutinating in solution can be exploited to quantify the antigens present in liquid samples [94].…”

Section: A De Marcomentioning

confidence: 99%

“…Despite the impossibility to quantify exactly the nanobody concentration in an E. coli sample, the binder amount can be tuned by controlling the level of VHH expression [94]. Nanobody displaying bacteria can co-express cytoplasmic fluorescent proteins for obtaining colored living immunoreagents suitable for light microscopy and flow cytometry applications [87]. Nanobodies were also displayed on bacteria with the aim of facilitating the cell targeting in vivo.…”

Section: A De Marcomentioning

confidence: 99%

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Recombinant expression of nanobodies and nanobody-derived immunoreagents

Marco

2020

Protein Expression and Purification

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A B S T R A C TAntibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or posttranslational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.

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Section: Bacterial Periplasmmentioning

confidence: 99%

Section: A De Marcomentioning

confidence: 99%

Section: A De Marcomentioning

confidence: 99%

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Recombinant expression of nanobodies and nanobody-derived immunoreagents

Marco

2020

Protein Expression and Purification

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Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

et al. 2019

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Photocaged antibody fragments,t ermed photobodies,h ave been developed that are impaired in their antigenbinding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of as ingle tyrosine in an anobody (a single-domain antibody fragment). Tyrosine is af requently occurring residue in central positions of the paratope region. o-Nitrobenzyl-protected tyrosine variants were incorporated into four nanobodies,i ncluding examples directed against EGFR and HER2, and photodeprotection restores the native sequence.Ananti-GFP photobody exhibited an at least 10 000fold impaired binding affinity before photodeprotection compared with the parent nanobody.Abispecific nanobodyphotobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.

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Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

et al. 2019

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Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen‐binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single‐domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o‐Nitrobenzyl‐protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti‐GFP photobody exhibited an at least 10 000‐fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.

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Purification-independent immunoreagents obtained by displaying nanobodies on bacteria surface

Cited by 5 publications

References 43 publications

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

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