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

Abstract

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.

Photocaged Nanobodies Delivered into Cells for Light Activation of Biological Processes

“…Our experiences with bead‐loading for protein transduction showed that this technique is inexpensive, rapid and very simple to implement for well‐adherent cells. However, some cells will be lost during the procedure due to detachment and the protein delivery is difficult to control in terms of percentage of cells transduced (20 to 50 %) and intracellular protein concentration [12c] . Of note, these observations on the particular cellular delivery method do not affect our conclusions on the intracellular use of photobodies.…”

“…[8a] For the intracellular delivery of purified photobodies, we pursued the bead-loading technique, because it is easily and quickly performed without the need for any specialized equipment. [12] To this end, small glassbeads and the photobody or nanobody protein (25 μM) were added to HeLa cells, protein uptake was enforced by sharply hitting the culture dish on the bench (8 ×) and cell recovery was allowed for 2 min before washing.…”

Strategies for Site‐Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

“…Recently, Mootz and co-workers reported on the Gp41-1 split intein and in cellulo semisynthesis of intracellular proteins in live mammalian cells using relatively short peptide fragments (N-intein: 88 aa, C-intein: 37 aa). [97] This split intein displays rapid PTS (t 1/2~5 s at 37°C) with high yields. [174,175] However, split intein approaches are still limited to rather big POI modifications for efficient labeling, when targeting the N terminus of proteins like GPCRs and RTKs.…”

“…[96] Covalent modification: pre-incubation with 0.5 mM TCEP prior to addition of the labeling probe (0.5 μM) at room temperature. [95] Split intein [97] Gp41-1 system: N-intein, 88 aa; C-intein, 37 aa…”

“…The DnaB C (12–154) fragment (C‐intein) was fused to mRFP and labeled with 0.5 μM of the respective N‐intein, equipped with biotin, requiring 20 mM reduced glutathione and a reaction time of 8 h. Subsequent POI visualization was performed by anti‐biotin staining using fluorescent streptavidin. Recently, Mootz and co‐workers reported on the Gp41‐1 split intein and in cellulo semisynthesis of intracellular proteins in live mammalian cells using relatively short peptide fragments (N‐intein: 88 aa, C‐intein: 37 aa) [97] . This split intein displays rapid PTS (t 1/2 ∼5 s at 37 °C) with high yields [174,175] .…”