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.

The receptor subunit Tom20 is dynamically associated with the TOM complex in mitochondria of human cells

“…Roughly, ~30% of Tom20-FP1 were spliced to FP2-Tom7, ~50% to FP2-hFis. For the crosslinking of Tom20-mCherry-Int N and Int C -mCitrine-hFis, we suggest that the driving force for reaction was the high affinity between the split-Inteins (KD=9.2 ± 1.3 nM) (Bhagawati et al, 2020), since no interaction between the proteins is expected (STRING-consortium, 2020). However, we cannot conclusively say which was the driving force for the formation of the Tom20-Tom7 crosslinking product, since kon/koff rates for the interaction between Tom20 and the GIP or Tom7 are unknown.…”

“…Since the process requires folding of the of the intein domain, it is also a quality control step for correct import of the fusion proteins into the OMM. The Gp41-1 split-intein is the fastest split intein with protein trans-splicing occurring within a few seconds and a nanomolar affinity between the intein fragments (Bhagawati et al, 2020;Carvajal-Vallejos et al, 2012). We genetically generated Tom20-FP1-Int N and Int C -FP2-Tom7 with FP1 and FP2 as fluorescent proteins.…”

“…We found that the median mobility of Tom20 was higher than that of Tom7. In order to obtain the diffusion constant of Tom20 associated with the GIP, Tom20 was then covalently ligated with Tom7 on the post-translational level by using the recently introduced ultra-fast self-splicing split-Intein Gp41-1 system (Bhagawati et al, 2020). The traceless split-Intein system allows ligation in situ and avoids the challenge of correct import and assembly of a genetically encoded Tom20-HT-Tom7 fusion protein.…”

Multiplexed Delivery of Synthetic (Un)Conjugatable Ubiquitin and SUMO2 Enables Simultaneous Monitoring of Their Localization and Function in Live Cells

“…To best of our knowledge, the most simple, mild and cost effective physical method utilizes glass beads to enable direct cytosolic delivery in a process termed bead loading (BL) [14] . Using this method, various groups have reported successful delivery of synthetic peptides to achieve live cell protein engineering, [15,16] catalytic installation of PTMs to histones [17] imaging of histone PTMs [18] and mRNA translation by ribosomes [19] . Importantly, treated cells exhibited normal proliferation rate, emphasizing the low toxicity generated by loading cells using this approach [20] …”

Front Cover: Multiplexed Delivery of Synthetic (Un)Conjugatable Ubiquitin and SUMO2 Enables Simultaneous Monitoring of Their Localization and Function in Live Cells (ChemBioChem 11/2022)

“…[13] To best of our knowledge, the most simple, mild and cost effective physical method utilizes glass beads to enable direct cytosolic delivery in a process termed bead loading (BL). [14] Using this method, various groups have reported successful delivery of synthetic peptides to achieve live cell protein engineering, [15,16] catalytic installation of PTMs to histones [17] imaging of histone PTMs [18] and mRNA translation by ribosomes. [19] Importantly, treated cells exhibited normal proliferation rate, emphasizing the low toxicity generated by loading cells using this approach.…”