Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing.

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, NaV1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

Optical control of the antigen translocation by synthetic photo-conditional viral inhibitors.

The immune system makes use of major histocompatibility complex class I (MHC I) molecules to present peptides to other immune cells, which can evoke an immune response. Within this process of antigen presentation, the MHC I peptide loading complex, consisting of a transporter associated with antigen processing TAP, MHC I, and chaperones, is key to the initiation of immune response by shuttling peptides from the cytosol into the ER lumen. However, it is still enigmatic how the flux of antigens is precisely coordinated in time and space, limiting our understanding of antigen presentation pathways. Here, we report on the development of a synthetic viral TAP inhibitor that can be cleaved by light. This photo-conditional inhibitor shows temporal blockade of TAP-mediated antigen translocation, which is unleashed upon illumination. The recovery of TAP activity was monitored at single-cell resolution both in human immune cell lines and primary cells. The development of a photo-conditional TAP inhibitor thus expands the repertoire of chemical intervention tools for immunological processes.

Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies.

Accurate labeling of endogenous proteins for advanced light microscopy in living cells remains challenging. Nanobodies have been widely used for antigen labeling, visualization of subcellular protein localization and interactions. To facilitate an expanded application, we present a scalable and high-throughput strategy to simultaneously target multiple endogenous proteins in living cells with micro- to nanometer resolution. For intracellular protein labeling, we advanced nanobodies by site-specific and stoichiometric attachment of bright organic fluorophores. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing enabled high-throughput delivery with less than 10% dead cells. This strategy allowed for the dual-color imaging of distinct endogenous cellular structures, and culminated in super-resolution imaging of native protein networks in genetically non-modified living cells. The simultaneous delivery of multiple engineered nanobodies does not only offer exciting prospects for multiplexed imaging of endogenous protein, but also holds potential for visualizing native cellular structures with unprecedented accuracy.

Dynamic blue light-switchable protein patterns on giant unilamellar vesicles.

The blue light-dependent interaction between the proteins iLID and Nano allows recruiting and patterning proteins on GUV membranes, which thereby capture key features of patterns observed in nature. This photoswitchable protein interaction provides non-invasive, reversible and dynamic control over protein patterns of different sizes with high specificity and spatiotemporal resolution.

Nanomolar affinity protein trans-splicing monitored in real-time by fluorophore-quencher pairs.

High background originating from non-reacted, 'always-on' fluorescent probes remains a key unsolved problem in life science since washing procedures are not easily applicable. Covalent labeling approaches with simultaneous activation of fluorescence are advantageous to increase sensitivity and to reduce background signal. Here, we combined high-affinity protein trans-splicing with fluorophore/quencher pairs for online detection of covalent N-terminal 'traceless' protein labeling under physiological conditions in cellular environments. Substantial fluorescence enhancement at nanomolar probe concentrations was achieved.

'Traceless' tracing of proteins - high-affinity trans-splicing directed by a minimal interaction pair.

Protein trans-splicing mediated by split inteins is a powerful technique for site-specific protein modification. Despite recent developments there is still an urgent need for ultra-small high-affinity intein tags for in vitro and in vivo approaches. To date, only very few in-cell applications of protein trans-splicing have been reported, all limited to C-terminal protein modifications. Here, we developed a strategy for covalent N-terminal intein-mediated protein labeling at (sub) nanomolar probe concentrations. Combined with a minimal synthetic lock-and-key element, the affinity between the intein fragments was increased more than 50-fold to 10 nM. Site-specific and efficient 'traceless' protein modification by high-affinity trans-splicing is demonstrated at nanomolar concentrations in living mammalian cells.

Acute hypertension after renal allograft rejection therapy with OKT3 monoclonal antibody.

Acute rejection of renal allografts was treated with the monoclonal antibody OKT3 given intravenously. A variety of adverse events were observed on days 1 and 2 following treatment with 5 mg/day OKT3 for 10 days including heart failure, pulmonary oedema and hypertension. Continuous monitoring of 19 patients treated with OKT3 for acute renal allograft rejection detected a transient increase, lasting 2 h, in systolic and diastolic blood pressures on day 1. A larger increase in systolic and diastolic pressures lasting 11-13 h was observed on day 2. Treatment with 5 mg OKT3 on day 3 did not significantly increase systolic or diastolic pressure. It is concluded that OKT3 can aggravate hypertension and hypertensive emergencies may be encountered during the initial phase of OKT3 treatment.