A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions.

Split intein-mediated protein trans-splicing has found extensive applications in chemical biology, protein chemistry, and biotechnology. However, an enduring limitation of all well-established split inteins has been the requirement to carry out the reaction in a reducing environment due to the presence of 1 or 2 catalytic cysteines that need to be in a reduced state for splicing to occur. The concomitant exposure of the fused proteins to reducing agents severely limits the scope of protein trans-splicing by excluding proteins sensitive to reducing conditions, such as those containing critical disulfide bonds. Here we report the discovery, characterization, and engineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at ambient temperatures, without a denaturation step, and in the absence of reducing agents. We demonstrate its utility for the site-specific chemical modification of nanobodies and an antibody Fc fragment by N- and C-terminal trans-splicing with short peptide tags (CysTag) that consist of only a few amino acids and have been prelabeled on a single cysteine using classical cysteine bioconjugation. We also synthesized the short N-terminal fragment of the atypically split CL intein by solid-phase peptide synthesis. Furthermore, using the CL intein in combination with a nanobody-epitope pair as a high-affinity mediator, we showed chemical labeling of the extracellular domain of a cell surface receptor on living mammalian cells with a short CysTag containing a synthetic fluorophore. The CL intein thus greatly expands the scope of applications for protein trans-splicing.

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein.

Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of non-redundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

In Cellulo Protein Semi-Synthesis from Endogenous and Exogenous Fragments Using the Ultra-Fast Split Gp41-1 Intein.

Protein semi-synthesis inside live cells from exogenous and endogenous parts offers unique possibilities for studying proteins in their native context. Split-intein-mediated protein trans-splicing is predestined for such endeavors and has seen some successes, but a much larger variety of established split inteins and associated protocols is urgently needed. We characterized the association and splicing parameters of the Gp41-1 split intein, which favorably revealed a nanomolar affinity between the intein fragments combined with the exceptionally fast splicing rate. Following bead-loading of a chemically modified intein fragment precursor into live mammalian cells, we fluorescently labeled target proteins on their N- and C-termini with short peptide tags, thus ensuring minimal perturbation of their structure and function. In combination with a nuclear-entrapment strategy to minimize cytosolic fluorescence background, we applied our technique for super-resolution imaging and single-particle tracking of the outer mitochondrial protein Tom20 in HeLa cells.

Impact of Lens Opacity on Optical Coherence Tomography Angiography Metrics.

PURPOSE: To evaluate the impact of lens opacity on the reliability of optical coherence tomography angiography metrics and to find a vessel caliber threshold that is reproducible in cataract patients. METHODS: A prospective cohort study of 31 patients, examining one eye per patient, by applying 3 × 3 mm macular optical coherence tomography angiography before (18.94 ± 12.22 days) and 3 months (111 ± 23.45 days) after uncomplicated cataract surgery. We extracted superficial (SVC) and deep vascular plexuses (DVC) for further analysis and evaluated changes in image contrast, vessel metrics (perfusion density, flow deficit and vessel-diameter index) and foveal avascular area (FAZ). RESULTS: After surgery, the blood flow signal in smaller capillaries was enhanced as image contrast improved. Signal strength correlated to average lens density defined by objective measurement in Scheimpflug images (Pearson's r: -.40, p: .027) and to flow deficit (r= -.70, p < .001). Perfusion density correlated to the signal strength index (r = .70, p < .001). Vessel metrics and FAZ area, except for FAZ area in DVC, were significantly different after cataract surgery, but the mean change was approximately 3-6%. A stepwise approach in extracting vessels according to their pixel caliber showed a threshold of > 6 pixels caliber (∼20-30 µm) was comparable before and after lens removal. CONCLUSION: In patients with cataract, OCTA vessel metrics should be interpreted with caution. In addition to signal strength, contrast and pixel properties can serve as supplementary quality metrics to improve the interpretation of OCTA metrics. Vessels with ∼20-30 µm in caliber seem to be reproducible.

Mitochondria-targeting fluorescent molecules for high efficiency cancer growth inhibition and imaging.

Fluorescent mitochondria-accumulating delocalized lipophilic cations (DLCs) for cancer therapy have drawn significant attention in the field of cancer theranostics. One of the most promising fluorescent DLCs, F16, can selectively trigger the apoptosis and necrosis of cancer cells, making it an attractive targeted theranostic drug candidate. However, it suffers from low clinical translation potential, largely due to its inefficient anti-cancer activity (IC50 in the µM range) and poorly understood structure-activity relationship (SAR). In this report, eleven indole-ring substituted F16 derivatives (F16s) were synthesized. Among these derivatives, 5BMF was identified as a highly effective theranostic agent, with in vitro studies showing a low IC50 of ∼50 nM (to H2228 cells) and high cancer to normal cell selectivity index of 225. In vivo studies revealed that tumors treated with 5BMF were significantly suppressed (almost no growth over the treatment period) compared to the PBS treated control group, and also no obvious toxicity to mice was found. In addition, the tumor imaging capability of 5BMF was demonstrated by in vivo fluorescence imaging. Finally, we report for the first time a proposed SAR for F16 DLCs. Our work lays down a solid foundation for translating 5BMF into a novel and highly promising DLC for cancer theranostics.