Khoo KK , Galleano I , Gasparri F , Wieneke R , Harms H , Poulsen MH , Chua HC , Wulf M , Tampé R , Pless SA .

	Fig. 1: Schematic diagram of the tPTS strategy to incorporate recombinant and synthetic proteins. The chosen split inteins used in this study, inteins A (CfaDnaE) and B (SspDnaBM86), are indicated by square and round symbols, respectively. Flanking +1, +2, +3 extein residues for each intein are indicated in italics at their respective positions in the top panel. The +1 extein residue (underlined) is a critical requirement for splicing to occur. X denotes that the type of residue at that position is not critical for splicing, although they might affect the kinetics or splicing efficiency.
	Fig. 2: Insertion of recombinantly expressed peptides into the DIII–IV linker of NaV1.5. a Schematic presentation of the strategy to reconstruct full-length NaV1.5 from recombinantly expressed N-/C-terminal fragments (N and C) and a recombinantly expressed peptide corresponding to amino acids 1472 to 1502 of the NaV1.5 DIII–DIV linker (X) in Xenopus laevis oocytes. Inteins A (CfaDnaE) and B (SspDnaBM86) are indicated by square and round symbols, respectively. Note that we cannot exclude the possibility that splicing takes place at a different subcellular location than depicted here. b Representative sodium currents in response to sodium channel activation protocol (see methods; only voltage steps from −50 to +10 mV in 10 mV steps are displayed), demonstrating expression of functional NaV1.5 only in the presence of all three components (N + C + X), along with WT and N1472C channels (the latter mutation was introduced to create an optimized splice site for intein A. c Immunoblots verifying the presence of fully spliced NaV1.5 only when all three components (N + C + X) were co-expressed (using antibody against NaV1.5 C-terminus). NaV1.5 band was not detected when one component was missing (left blot) or when non-splicing mutation (N + C + X mut.) was introduced to prevent splicing (right blot; see Supplementary Fig. 2). Black arrows indicate band positions of the respective constructs (Actual MW of constructs: WT, 227 kDa; C-construct, 79 kDa; C + X, 65 kDa; C-terminal cleavage product, C*, 58 kDa). Note that X and XREC refer to XNav1.5REC in this panel. d Steady-state inactivation and conductance–voltage (G–V) relationships for functional constructs tested. e Comparison of values for half-maximal (in)activation (V50) (values are displayed as mean + /– SD; WT, n = 12; N1472C, n = 14; N + C + XNav1.5REC, n = 16). Significant differences were determined by one-way ANOVA with a Tukey post-hoc test. *p < 0.03 (WT vs. N1472C, p = 0.014; WT vs. N + C + XNav1.5REC, p = 0.012). Source data are provided as a Source data file.
	Fig. 3: Insertion of synthetic peptides into NaV1.5. a Schematic of strategy to reconstruct full-length NaV1.5 from recombinantly expressed N-/C-terminal fragments (N and C) and a synthetic peptide (XNav1.5SYN) in Xenopus laevis oocytes. Inteins A (CfaDnaE) and B (SspDnaBM86) indicated by square and round symbols, respectively. Note: we cannot exclude that splicing takes place at a different subcellular location than depicted. b Peptide XNav1.5SYN sequence corresponding to amino acids replaced in the NaV1.5 DIII–DIV linker and chemical structures of native amino acids and PTM mimics (tAcK/phY) incorporated via chemical synthesis of peptide XNav1.5SYN. The N1472C (underlined) mutation was introduced to optimize splicing (Supplementary Fig. 1). c Immunoblot verifying the presence of fully spliced NaV1.5 only when peptide XNav1.5SYN was co-injected with N and C. Black arrows indicate band positions of the respective constructs (Actual MW of constructs: WT, 227 kDa; C, 79 kDa; C + X, 65 kDa; C-terminal cleavage product, C*, 58 kDa). Band at ~150 kDa is possibly a dimer or aggregate of C. d Representative sodium currents (see Methods; only voltage steps from −50 to +10 mV in 10 mV steps are displayed), demonstrating expression of functional NaV1.5 when Xenopus laevis oocytes expressing N and C constructs were injected with synthetic peptides containing non-modifiable side chains in positions 1479 and 1495 (K1479R and Y1495F, NM), tAcK1479 or phY1495 or both PTM mimics together. e Average current amplitudes recorded at −35 mV from oocytes expressing N and C constructs and injected with synthetic peptide variant NM or phY1495 depicted as a bar plot (mean + /– SD; WT, n = 8; NM, n = 9; phY, n = 8; N + C, n = 6). Currents normalized to mean currents measured from oocytes expressing the full-length WT construct. To ensure adequate control of voltage clamp, [Na+] in the extracellular recording solution was reduced (see Supplementary Fig. 3 for details). f Steady-state inactivation and conductance–voltage (G–V) relationships for PTM-modified/non-modified constructs (values displayed as mean + /– SD; N1472C, n = 15; NM, n = 10; tAcK1479, n = 21; phY1495, n = 19; tAcK1479 + phY1495, n = 14). Source data are provided as a Source data file.
	Fig. 4: Insertion of a recombinantly expressed peptide into GFP expressed in mammalian cells. a Schematic presentation of the strategy applied to reconstitute GFP from recombinantly expressed N-/C-terminal fragments and recombinantly expressed peptide XGFPREC corresponding to amino acids 65–85 of GFP in HEK293 cells. Inteins A (CfaDnaE) and B (SspDnaBM86) are indicated by square and round symbols, respectively. b Bright-field (right panels) and fluorescence (left panels) images of HEK293 cells expressing the indicated constructs. Scale bars: 20 µm. GFP fluorescence was only detected when all three constructs (N + X + C) were co-transfected. GFP fluorescence was not detected when one of the three constructs was absent or when +1 extein residues of each split intein is mutated to alanine (C65A for intein A and S86A for intein B) to prevent splicing.
	Fig. 5: Insertion of synthetic peptides into GFP. a Schematic of strategy applied to reconstitute GFP from recombinantly expressed N-/C-terminal fragments and a synthetic peptide XGFPSYN in HEK293 cells. b Overlay of bright-field and fluorescence images taken of HEK293 cells transfected with only WT GFP, or N and C fragments and squeezed in the presence or absence of peptide XGFPSYN. Scale bars: 50 µm.
	Fig. 6: Insertion of recombinant and synthetic peptides into the P2X2R extracellular domain. a Schematic presentation of the strategy to reconstruct full-length P2X2Rs from recombinantly expressed N-/C-terminal fragments (N and C) and a recombinant (strategy 1, Pept XP2X2REC) or synthetic peptide (strategy 2, Pept XP2X2SYN) into the P2X2R extracellular domain in Xenopus laevis oocytes. Note that a faux transmembrane helix (faux TMD) was engineered into the C-terminal fragment to maintain its native membrane topology (see also Supplementary Fig. 9). Inteins A (CfaDnaE) and B (SspDnaBM86) indicated by square and round symbols, respectively. Peptide X was designed to include a C-terminal ER-targeting KDEL signal sequence, which is excised during the splicing process. However, we cannot exclude the possibility that splicing takes place at a different subcellular location than depicted here. b Representative current traces obtained by application of 30 µM ATP, verifying functional expression only when all three components were recombinantly co-expressed (strategy 1 in a). c Immunoblot of surface-purified proteins. Black arrows on the right indicate band positions of the respective constructs (actual MW of constructs: WT, 53 kDa; C-construct, 97 kDa). Red arrow indicates the band of the spliced full-length receptor. d ATP concentration-response curves (CRCs) for P2X2 WT (black) and K71Q (red) reconstituted from the three co-expressed constructs. Dashed lines represent the CRCs for the respective full-length proteins. e Structures of inserted side chains at position 71 (Lys, Orn, hLys) incorporated via synthetic peptide X (strategy 2 in a). f ATP CRCs indicate successful incorporation of synthetic WT peptide XP2X2SYN (black) with wild-type like ATP sensitivity, while synthetic peptides with Orn (pink) or hLys (blue) resulted in a decrease in ATP sensitivity. Dashed lines indicate ATP CRCs for spliced P2X2R WT (gray) or K71Q (purple) obtained with peptide XP2X2REC. Values in d and f are displayed as mean + /– SD; WT, n = 5; [WT]REC, n = 8; K71Q, n = 7; [K71Q]REC, n = 9; [WT]SYN, n = 7; [K71Orn]SYN, n = 9; [K71hLys]SYN, n = 5). Source data are provided as a Source data file.

Ahern, The hitchhiker's guide to the voltage-gated sodium channel galaxy. 2016, Pubmed

Ahern, The hitchhiker's guide to the voltage-gated sodium channel galaxy. 2016, Pubmed
Ahern, Modulation of the cardiac sodium channel NaV1.5 by Fyn, a Src family tyrosine kinase. 2005, Pubmed
Appleby-Tagoe, Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution. 2011, Pubmed
Aranko, Nature's recipe for splitting inteins. 2014, Pubmed
Bai, Covalently-assembled single-chain protein nanostructures with ultra-high stability. 2019, Pubmed
Bang, A one-pot total synthesis of crambin. 2004, Pubmed
Beltran-Alvarez, Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel. 2015, Pubmed
Bhagawati, A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions. 2019, Pubmed
Braner, 'Traceless' tracing of proteins - high-affinity trans-splicing directed by a minimal interaction pair. 2016, Pubmed
David, Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins. 2015, Pubmed
Demonte, Postsynthetic Domain Assembly with NpuDnaE and SspDnaB Split Inteins. 2015, Pubmed
Ellmer, Single Posttranslational Modifications in the Central Repeat Domains of Tau4 Impact its Aggregation and Tubulin Binding. 2019, Pubmed
Elsässer, Genetic code expansion in stable cell lines enables encoded chromatin modification. 2016, Pubmed
Gasparri, Molecular determinants for agonist recognition and discrimination in P2X2 receptors. 2019, Pubmed , Xenbase
Girish, Affordable image analysis using NIH Image/ImageJ. 2004, Pubmed
Haj-Yahya, Protein Semisynthesis Provides Access to Tau Disease-Associated Post-translational Modifications (PTMs) and Paves the Way to Deciphering the Tau PTM Code in Health and Diseased States. 2018, Pubmed
Huang, Structure-based assessment of disease-related mutations in human voltage-gated sodium channels. 2017, Pubmed
Jillette, Split selectable markers. 2019, Pubmed
Kapplinger, Spectrum and prevalence of mutations from the first 2,500 consecutive unrelated patients referred for the FAMILION long QT syndrome genetic test. 2009, Pubmed
Khakh, P2X receptors as cell-surface ATP sensors in health and disease. 2006, Pubmed
Klippenstein, Optocontrol of glutamate receptor activity by single side-chain photoisomerization. 2017, Pubmed
Kollmannsperger, Live-cell protein labelling with nanometre precision by cell squeezing. 2016, Pubmed
Kratochvil, Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy. 2016, Pubmed
Kvist, The use of Xenopus oocytes in drug screening. 2011, Pubmed , Xenbase
Lueck, Atomic mutagenesis in ion channels with engineered stoichiometry. 2016, Pubmed , Xenbase
Lynagh, Acid-sensing ion channels emerged over 600 Mya and are conserved throughout the deuterostomes. 2018, Pubmed
Lynagh, A selectivity filter at the intracellular end of the acid-sensing ion channel pore. 2017, Pubmed , Xenbase
Marionneau, Regulation of the cardiac Na+ channel NaV1.5 by post-translational modifications. 2015, Pubmed
Muralidharan, Protein ligation: an enabling technology for the biophysical analysis of proteins. 2006, Pubmed
Poulsen, Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis. 2019, Pubmed
Rajabi, Dethioacylation by Sirtuins 1-3: Considerations for Drug Design Using Mechanism-Based Sirtuin Inhibition. 2020, Pubmed
Rannversson, Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter. 2016, Pubmed
Schindelin, Fiji: an open-source platform for biological-image analysis. 2012, Pubmed
Schütz, Click-tag and amine-tag: chemical tag approaches for efficient protein labeling in vitro and on live cells using the naturally split Npu DnaE intein. 2014, Pubmed
Shah, Extein residues play an intimate role in the rate-limiting step of protein trans-splicing. 2013, Pubmed
Sharei, A vector-free microfluidic platform for intracellular delivery. 2013, Pubmed
Shi, Development of a tandem protein trans-splicing system based on native and engineered split inteins. 2005, Pubmed
Shiraishi, Phosphorylation-induced conformation of β2-adrenoceptor related to arrestin recruitment revealed by NMR. 2018, Pubmed
Smith, Mechanism-based inhibition of Sir2 deacetylases by thioacetyl-lysine peptide. 2007, Pubmed
Stevens, A promiscuous split intein with expanded protein engineering applications. 2017, Pubmed
Stevens, Design of a Split Intein with Exceptional Protein Splicing Activity. 2016, Pubmed
Subramanyam, Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing. 2013, Pubmed
Vikram, Sirtuin 1 regulates cardiac electrical activity by deacetylating the cardiac sodium channel. 2017, Pubmed
Wright, Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins. 2019, Pubmed
Xiao, Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. 2013, Pubmed
Ye, Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis. 2008, Pubmed
Ye, FTIR analysis of GPCR activation using azido probes. 2009, Pubmed