The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes.DOI: http://dx.doi.org/10.7554/eLife.15312.001
Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli. These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.
ABSTRACT. The study aimed to investigate the effect of intrathecal injections of Tanshinone IIA on thermal hyperalgesia in a mouse model of bone cancer-pain. Spinal IL-1β, IL-6, TNF-α expression levels were analyzed. C3H/HeNCrlVr male mice were assigned to groups that either received dose-dependent injections of Tanshinone IIA, or the DMSO + Sham, Tanshinone IIA + Sham, DMSO + Tumor, and Control groups. Paw withdrawal thermal latency (PWTL) was measured with a radiant heat stimulus and mRNA expression levels were determined using real-time PCR. Fourteen days post-injection, PWTL in the DMSO + Tumor group was lower than that in the controls (P < 0.05). Twentyone days post-injection, compared with the Control group, there was no significant difference in PWTL and IL-1β, IL-6, and TNF-α expression levels between the Tanshinone IIA + Sham and DMSO + Sham groups (P > 0.05). PWTL in the DMSO + Tumor group was significantly lower than the Control group (P < 0.05), while the expression levels of IL-1β, IL-6, and TNF-α were significantly higher than controls. Compared with the DMSO + Tumor group, PWTLs were higher in the Tanshinone B.X. Ren et al. 2134©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (1): 2133-2138 (2015) IIA -20-μg and 40-μg groups, while expression levels of IL-1β, IL-6, and TNF-α were significantly lower (P < 0.05). These measures were not significantly different between the Tanshinone IIA 10 μg and the DMSO + Tumor groups (P > 0.05). In conclusion, Tanshinone IIA may inhibit the release of inflammatory cytokines, such as, IL-1 β, IL-6 α, TNF-α.
Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of originally intracellularly unstable nanobodies, without compromising target-binding function. Additionally, we identified distinct sequence features impacting nanobody intracellular stability, and demonstrated mutationally stabilized nanobody expression in vivo, in the murine retina and in E. coli. This work will allow for standardized nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.
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