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• W3197366172 abstract "Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein. Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein. Biochemical and structural studies require large amounts of pure proteins and protein complexes. When these cannot be purified from natural sources, scientists typically opt for protein expression in bacteria, and N- or C-terminal affinity tags are commonly used for protein purification. These tags are often left on proteins during subsequent studies, and when they are enzymatically removed, extra amino acids often remain (1Waugh D.S. An overview of enzymatic reagents for the removal of affinity tags.Protein Expr. Purif. 2011; 80: 283-293Crossref PubMed Scopus (220) Google Scholar). While these approaches have proven powerful and will likely continue to be used in the future, there are many circumstances in which they fail to produce accurate results. For example, most human proteins undergo multiple types of posttranslational modifications (PTMs), including acetylation, phosphorylation, and methylation (2Khoury G.A. Baliban R.C. Floudas C.A. Proteome-wide post-translational modification statistics: Frequency analysis and curation of the swiss-prot database.Sci. Rep. 2011; 1: 90Crossref Scopus (570) Google Scholar). Such PTMs play crucial roles in the regulation of protein function, including protein stability, protein–protein interactions, and enzymatic activity (3Bah A. Forman-Kay J.D. Modulation of intrinsically disordered protein function by post-translational modifications.J. Biol. Chem. 2016; 291: 6696-6705Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 4Hunter T. Tyrosine phosphorylation: Thirty years and counting.Curr. Opin. Cell Biol. 2009; 21: 140-146Crossref PubMed Scopus (476) Google Scholar, 5Johnson L.N. Lewis R.J. Structural basis for control by phosphorylation.Chem. Rev. 2001; 101: 2209-2242Crossref PubMed Scopus (506) Google Scholar, 6Walsh C.T. Garneau-Tsodikova S. Gatto Jr., G.J. Protein posttranslational modifications: The chemistry of proteome diversifications.Angew. Chem. Int. Ed. Engl. 2005; 44: 7342-7372Crossref PubMed Scopus (994) Google Scholar). Most PTMs present in human proteins cannot be processed by expression systems such as bacteria, yeast, or even insect cells (7Amann T. Schmieder V. Faustrup Kildegaard H. Borth N. Andersen M.R. Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms.Biotechnol. Bioeng. 2019; 116: 2778-2796Crossref PubMed Scopus (23) Google Scholar, 8Brown C.W. Sridhara V. Boutz D.R. Person M.D. Marcotte E.M. Barrick J.E. Wilke C.O. Large-scale analysis of post-translational modifications in E. coli under glucose-limiting conditions.BMC Genomics. 2017; 18: 301Crossref PubMed Scopus (27) Google Scholar, 9Higel F. Seidl A. Sorgel F. Friess W. N-glycosylation heterogeneity and the influence on structure, function and pharmacokinetics of monoclonal antibodies and Fc fusion proteins.Eur. J. Pharm. Biopharm. 2016; 100: 94-100Crossref PubMed Scopus (197) Google Scholar, 10McKenzie E.A. Abbott W.M. Expression of recombinant proteins in insect and mammalian cells.Methods. 2018; 147: 40-49Crossref PubMed Scopus (26) Google Scholar, 11Tang H. Wang S. Wang J. Song M. Xu M. Zhang M. Shen Y. Hou J. Bao X. N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae.Sci. Rep. 2016; 6: 25654Crossref PubMed Scopus (39) Google Scholar, 12Weis B.L. Guth N. Fischer S. Wissing S. Fradin S. Holzmann K.H. Handrick R. Otte K. Stable miRNA overexpression in human CAP cells: Engineering alternative production systems for advanced manufacturing of biologics using miR-136 and miR-3074.Biotechnol. Bioeng. 2018; 115: 2027-2038Crossref PubMed Scopus (6) Google Scholar). These expression systems may also lack the adequate machinery to fold certain mammalian proteins (13Dunn A.Y. Melville M.W. Frydman J. Review: Cellular substrates of the eukaryotic chaperonin TRiC/CCT.J. Struct. Biol. 2001; 135: 176-184Crossref PubMed Scopus (116) Google Scholar, 14Liu Z. Tyo K.E. Martinez J.L. Petranovic D. Nielsen J. Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae.Biotechnol. Bioeng. 2012; 109: 1259-1268Crossref PubMed Scopus (91) Google Scholar, 15Resnicow D.I. Deacon J.C. Warrick H.M. Spudich J.A. Leinwand L.A. Functional diversity among a family of human skeletal muscle myosin motors.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 1053-1058Crossref PubMed Scopus (70) Google Scholar, 16Thomas J.A. Tate C.G. Quality control in eukaryotic membrane protein overproduction.J. Mol. Biol. 2014; 426: 4139-4154Crossref PubMed Scopus (42) Google Scholar). Many proteins also require native N- and C-termini and cannot tolerate the presence of extra amino acids from purification tags for proper function (17Brault V. Reedy M.C. Sauder U. Kammerer R.A. Aebi U. Schoenenberger C. Substitution of flight muscle-specific actin by human (beta)-cytoplasmic actin in the indirect flight muscle of Drosophila.J. Cell Sci. 1999; 112: 3627-3639Crossref PubMed Google Scholar, 18Sliwinska M. Skorzewski R. Moraczewska J. Role of actin C-terminus in regulation of striated muscle thin filament.Biophys. J. 2008; 94: 1341-1347Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 19Chant A. Kraemer-Pecore C.M. Watkin R. Kneale G.G. Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site.Protein Expr. Purif. 2005; 39: 152-159Crossref PubMed Scopus (102) Google Scholar, 20Suh-Lailam B.B. Hevel J.M. Efficient cleavage of problematic tobacco etch virus (TEV)-protein arginine methyltransferase constructs.Anal. Biochem. 2009; 387: 130-132Crossref PubMed Scopus (9) Google Scholar, 21Wu J. Filutowicz M. Hexahistidine (His6)-tag dependent protein dimerization: A cautionary tale.Acta Biochim. Pol. 1999; 46: 591-599Crossref PubMed Scopus (115) Google Scholar). We were confronted with these problems while studying tropomyosin (Tpm), a protein of central importance in the actin cytoskeleton (22Gunning P.W. Hardeman E.C. Lappalainen P. Mulvihill D.P. Tropomyosin - master regulator of actin filament function in the cytoskeleton.J. Cell Sci. 2015; 128: 2965-2974Crossref PubMed Scopus (166) Google Scholar).Tpm is a coiled coil dimer that polymerizes head-to-tail to form two contiguous chains, one on each side of the actin filament (F-actin). Through alternative splicing of four different genes (TPM1–4), up to 40 distinct Tpm isoforms can be expressed (23Geeves M.A. Hitchcock-DeGregori S.E. Gunning P.W. A systematic nomenclature for mammalian tropomyosin isoforms.J. Muscle Res. Cell Motil. 2015; 36: 147-153Crossref PubMed Scopus (120) Google Scholar). Most isoforms are ∼280-amino acids long (called long isoforms), comprising seven pseudo-repeats of ∼40 amino acids, with each pseudo-repeat spanning the length of one actin subunit along the long-pitch helix of the actin filament. Long Tpms decorate the contractile actin filaments of muscle cells. In nonmuscle cells, the majority of actin filaments are also decorated with Tpm (24Meiring J.C.M. Bryce N.S. Wang Y. Taft M.H. Manstein D.J. Liu Lau S. Stear J. Hardeman E.C. Gunning P.W. Co-polymers of actin and tropomyosin account for a major fraction of the human actin cytoskeleton.Curr. Biol. 2018; 28: 2331-2337.e2335Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Some of the genes encoding nonmuscle Tpm isoforms lack exon 2 and as a consequence such Tpm isoforms are one pseudo-repeat shorter (called short isoforms). Because the six human actin isoforms are very similar, sharing 93 to 99% sequence identity, it has been proposed that the decoration of actin filaments with distinct Tpm isoforms provides a mechanism for filaments to assume different identities and become functionally segregated in cells (25Gunning P.W. Ghoshdastider U. Whitaker S. Popp D. Robinson R.C. The evolution of compositionally and functionally distinct actin filaments.J. Cell Sci. 2015; 128: 2009-2019Crossref PubMed Scopus (189) Google Scholar). Tpm can move azimuthally on actin, helping to regulate the interactions of most actin-binding proteins, including myosin (26Lehman W. Hatch V. Korman V. Rosol M. Thomas L. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments.J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar, 27McKillop D.F. Geeves M.A. Regulation of the interaction between actin and myosin subfragment 1: Evidence for three states of the thin filament.Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (652) Google Scholar).Although there is high sequence similarity among Tpm isoforms, gene knockout and isoform overexpression studies have shown that they often play nonredundant roles (22Gunning P.W. Hardeman E.C. Lappalainen P. Mulvihill D.P. Tropomyosin - master regulator of actin filament function in the cytoskeleton.J. Cell Sci. 2015; 128: 2965-2974Crossref PubMed Scopus (166) Google Scholar). For example, Tpm4.2 knockout causes low platelet count (28Cheng C. Nowak R.B. Amadeo M.B. Biswas S.K. Lo W.-K. Fowler V.M. Tropomyosin 3.5 protects the F-actin networks required for tissue biomechanical properties.J. Cell Sci. 2018; 131jcs222042Crossref PubMed Scopus (14) Google Scholar), whereas Tpm3.5 knockout causes softer, less mechanically resilient lenses in the eye (29Pleines I. Woods J. Chappaz S. Kew V. Foad N. Ballester-Beltrán J. Aurbach K. Lincetto C. Lane R.M. Schevzov G. Alexander W.S. Hilton D.J. Astle W.J. Downes K. Nurden P. et al.Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia.J. Clin. Invest. 2017; 127: 814-829Crossref PubMed Scopus (44) Google Scholar), indicating that other Tpm isoforms cannot compensate for the lack of Tpm4.2 or Tpm3.5. Similarly, different Tpm isoforms have been implicated in processes such as muscle contraction (30Kee A.J. Gunning P.W. Hardeman E.C. A cytoskeletal tropomyosin can compromise the structural integrity of skeletal muscle.Cell Motil. Cytoskeleton. 2009; 66: 710-720Crossref PubMed Scopus (8) Google Scholar, 31Vlahovich N. Kee A.J. Van der Poel C. Kettle E. Hernandez-Deviez D. Lucas C. Lynch G.S. Parton R.G. Gunning P.W. Hardeman E.C. Cytoskeletal tropomyosin Tm5NM1 is required for normal excitation-contraction coupling in skeletal muscle.Mol. Biol. Cell. 2009; 20: 400-409Crossref PubMed Scopus (42) Google Scholar), cytokinesis (32Eppinga R.D. Li Y. Lin J.L. Lin J.J. Tropomyosin and caldesmon regulate cytokinesis speed and membrane stability during cell division.Arch. Biochem. Biophys. 2006; 456: 161-174Crossref PubMed Scopus (25) Google Scholar, 33Thoms J.A. Loch H.M. Bamburg J.R. Gunning P.W. Weinberger R.P. A tropomyosin 1 induced defect in cytokinesis can be rescued by elevated expression of cofilin.Cell Motil. Cytoskeleton. 2008; 65: 979-990Crossref PubMed Scopus (12) Google Scholar), embryogenesis (34Hook J. Lemckert F. Qin H. Schevzov G. Gunning P. Gamma tropomyosin gene products are required for embryonic development.Mol. Cell. Biol. 2004; 24: 2318-2323Crossref PubMed Scopus (51) Google Scholar, 35Hook J. Lemckert F. Schevzov G. Fath T. Gunning P. Functional identity of the gamma tropomyosin gene: Implications for embryonic development, reproduction and cell viability.Bioarchitecture. 2011; 1: 49-59Crossref PubMed Scopus (25) Google Scholar), and endocytosis (36Dalby-Payne J.R. O'Loughlin E.V. Gunning P. Polarization of specific tropomyosin isoforms in gastrointestinal epithelial cells and their impact on CFTR at the apical surface.Mol. Biol. Cell. 2003; 14: 4365-4375Crossref PubMed Scopus (35) Google Scholar). The temporal expression of Tpm isoforms is also tightly controlled. Thus, 16 isoforms are up- or downregulated at different stages of brain development in rodents (37Dufour C. Weinberger R.P. Gunning P. Tropomyosin isoform diversity and neuronal morphogenesis.Immunol. Cell Biol. 1998; 76: 424-429Crossref PubMed Scopus (19) Google Scholar, 38Had L. Faivre-Sarrailh C. Legrand C. Rabie A. The expression of tropomyosin genes in pure cultures of rat neurons, astrocytes and oligodendrocytes is highly cell-type specific and strongly regulated during development.Brain Res. Mol. Brain Res. 1993; 18: 77-86Crossref PubMed Scopus (28) Google Scholar). Tpm3.1 and Tpm3.2 are found in the axon in developing neurons, whereas in mature neurons they are expressed only in cell bodies and replaced in axons by Tpm1.12 and Tpm4.2 (39Hannan A.J. Gunning P. Jeffrey P.L. Weinberger R.P. Structural compartments within neurons: Developmentally regulated organization of microfilament isoform mRNA and protein.Mol. Cell. Neurosci. 1998; 11: 289-304Crossref PubMed Scopus (55) Google Scholar).PTMs, and in particular N-terminal acetylation (Nt-acetylation) (40Heald R.W. Hitchcock-DeGregori S.E. The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin.J. Biol. Chem. 1988; 263: 5254-5259Abstract Full Text PDF PubMed Google Scholar, 41Palm T. Greenfield N.J. Hitchcock-DeGregori S.E. Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes.Biophys. J. 2003; 84: 3181-3189Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 42Maytum R. Geeves M.A. Konrad M. Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification.Biochemistry. 2000; 39: 11913-11920Crossref PubMed Scopus (44) Google Scholar) and phosphorylation (43Lehman W. Medlock G. Li X.E. Suphamungmee W. Tu A.Y. Schmidtmann A. Ujfalusi Z. Fischer S. Moore J.R. Geeves M.A. Regnier M. Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain.Arch. Biochem. Biophys. 2015; 571: 10-15Crossref PubMed Scopus (21) Google Scholar, 44Palani S. Köster D.V. Hatano T. Kamnev A. Kanamaru T. Brooker H.R. Hernandez-Fernaud J.R. Jones A.M.E. Millar J.B.A. Mulvihill D.P. Balasubramanian M.K. Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement.J. Cell Biol. 2019; 218: 3548-3559Crossref PubMed Scopus (9) Google Scholar, 45Rajan S. Jagatheesan G. Petrashevskaya N. Biesiadecki B.J. Warren C.M. Riddle T. Liggett S. Wolska B.M. Solaro R.J. Wieczorek D.F. Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy.J. Biol. Chem. 2019; 294: 2913-2923Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 46Rao V.S. Marongelli E.N. Guilford W.H. Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit.Cell Motil. Cytoskeleton. 2009; 66: 10-23Crossref PubMed Scopus (37) Google Scholar), have been shown to critically regulate Tpm function. Proper head-to-tail assembly of Tpm coiled coils along actin filaments requires native N- and C-termini, i.e., Nt-acetylated and free of extra amino acids from tags (40Heald R.W. Hitchcock-DeGregori S.E. The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin.J. Biol. Chem. 1988; 263: 5254-5259Abstract Full Text PDF PubMed Google Scholar, 47Brooker H.R. Geeves M.A. Mulvihill D.P. Analysis of biophysical and functional consequences of tropomyosin-fluorescent protein fusions.FEBS Lett. 2016; 590: 3111-3121Crossref PubMed Scopus (7) Google Scholar), whereas the phosphorylation state of Tpm can also modulate the stiffness and affinity of the head-to-tail interaction as well as the F-actin-binding affinity (43Lehman W. Medlock G. Li X.E. Suphamungmee W. Tu A.Y. Schmidtmann A. Ujfalusi Z. Fischer S. Moore J.R. Geeves M.A. Regnier M. Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain.Arch. Biochem. Biophys. 2015; 571: 10-15Crossref PubMed Scopus (21) Google Scholar, 44Palani S. Köster D.V. Hatano T. Kamnev A. Kanamaru T. Brooker H.R. Hernandez-Fernaud J.R. Jones A.M.E. Millar J.B.A. Mulvihill D.P. Balasubramanian M.K. Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement.J. Cell Biol. 2019; 218: 3548-3559Crossref PubMed Scopus (9) Google Scholar, 46Rao V.S. Marongelli E.N. Guilford W.H. Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit.Cell Motil. Cytoskeleton. 2009; 66: 10-23Crossref PubMed Scopus (37) Google Scholar). Despite the importance of these factors, previous biochemical studies have focused on one or two Tpm isoforms purified from muscle or Escherichia coli-expressed full-length and peptide fragments of Tpm isoforms that lack PTMs and native N- and C-termini. While extra amino acids have been added at the N-terminus to substitute for Nt-acetylation (40Heald R.W. Hitchcock-DeGregori S.E. The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin.J. Biol. Chem. 1988; 263: 5254-5259Abstract Full Text PDF PubMed Google Scholar, 41Palm T. Greenfield N.J. Hitchcock-DeGregori S.E. Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes.Biophys. J. 2003; 84: 3181-3189Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 48Frye J. Klenchin V.A. Rayment I. Structure of the tropomyosin overlap complex from chicken smooth muscle: Insight into the diversity of N-terminal recognition.Biochemistry. 2010; 49: 4908-4920Crossref PubMed Scopus (55) Google Scholar, 49Monteiro P.B. Lataro R.C. Ferro J.A. Reinach Fde C. Functional alpha-tropomyosin produced in Escherichia coli. A dipeptide extension can substitute the amino-terminal acetyl group.J. Biol. Chem. 1994; 269: 10461-10466Abstract Full Text PDF PubMed Google Scholar), it is unclear how precisely this approach mimics acetylation. Tpm coexpression with the yeast acetyltransferase NatB in E. coli has been also used to acetylate the N-terminus (50Johnson M. Coulton A.T. Geeves M.A. Mulvihill D.P. Targeted amino-terminal acetylation of recombinant proteins in E. coli.PLoS One. 2010; 5e15801Crossref PubMed Scopus (79) Google Scholar). However, while yeast NatB can acetylate the N-terminus in E. coli, the extent of this modification varies for different Tpm isoforms, being as low as 30% for certain mammalian Tpm isoforms (50Johnson M. Coulton A.T. Geeves M.A. Mulvihill D.P. Targeted amino-terminal acetylation of recombinant proteins in E. coli.PLoS One. 2010; 5e15801Crossref PubMed Scopus (79) Google Scholar). Moreover, E. coli cannot add other types of PTMs (8Brown C.W. Sridhara V. Boutz D.R. Person M.D. Marcotte E.M. Barrick J.E. Wilke C.O. Large-scale analysis of post-translational modifications in E. coli under glucose-limiting conditions.BMC Genomics. 2017; 18: 301Crossref PubMed Scopus (27) Google Scholar). Furthermore, as we show here, for many Tpm isoforms the initiator methionine is removed by mammalian N-terminal aminopeptidases and Nt-acetylation occurs on the second residue. E. coli lacks the necessary regulatory and modification machineries to perform such multistep Nt-processing reactions (51Wingfield P.T. N-terminal methionine processing.Curr. Protoc. Protein Sci. 2017; 88: 6.14.1-6.14.3Crossref Scopus (48) Google Scholar). We finally show here that Nt-acetylation mimics are not always capable of recapitulating the F-actin-binding properties of native Tpm isoforms.Here, we describe a method that combines expression in human Expi293F cells, which grow in suspension to high density and contain the native PTM machinery, with an intein-based affinity tag that can be precisely removed through self-cleavage after purification. We applied this method to the expression of six nonmuscle Tpm isoforms and one muscle isoform (Tpm1.1). We demonstrate the general applicability of this method to other proteins by also showing expression of α-synuclein, a protein implicated in several neurodegenerative disorders and whose function is critically regulated by numerous PTMs (52Schmid A.W. Fauvet B. Moniatte M. Lashuel H.A. Alpha-synuclein post-translational modifications as potential biomarkers for Parkinson disease and other synucleinopathies.Mol. Cell. Proteomics. 2013; 12: 3543-3558Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 53Zhang J. Li X. Li J.D. The roles of post-translational modifications on alpha-synuclein in the pathogenesis of Parkinson's diseases.Front. Neurosci. 2019; 13: 381Crossref PubMed Scopus (81) Google Scholar). We performed a functional analysis of the expressed Tpms in comparison with their E. coli-expressed counterparts, revealing significant differences in their ability to bind F-actin. Further, a comprehensive proteomics analysis of the human-cell-expressed Tpms showed the type and location of various PTMs, including Nt-acetylation and phosphorylation sites. We finally show that nonmuscle Tpm isoforms can form heterodimers in vitro and in cells, as previously shown for muscle isoforms purified from tissues.ResultsHuman cell expression and intein-mediated purification method and its application to Tpm and α-synucleinThe expression method developed here combines mammalian cell expression with a self-cleavable affinity tag. The cells used are Expi293F, commercialized by Thermo Fisher Scientific, which are based on human embryonic kidney (HEK) cells and have been adapted to grow in suspension to high density (54Backliwal G. Hildinger M. Hasija V. Wurm F.M. High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI.Biotechnol. Bioeng. 2008; 99: 721-727Crossref PubMed Scopus (116) Google Scholar, 55Liu C. Dalby B. Chen W. Kilzer J.M. Chiou H.C. Transient transfection factors for high-level recombinant protein production in suspension cultured mammalian cells.Mol. Biotechnol. 2008; 39: 141-153Crossref PubMed Scopus (67) Google Scholar). These cells can be transiently transfected, display high protein expression levels, and have the endogenous protein folding and PTM machineries necessary for native expression and modification of human proteins. The self-cleavable affinity tag consists of a modified intein element followed by a chitin-binding domain (CBD) and is based on the IMPACT (intein-mediated purification with an affinity chitin-binding tag) system marketed by New England Biolabs (56Shah N.H. Muir T.W. Inteins: Nature's gift to protein chemists.Chem. Sci. 2014; 5: 446-461Crossref PubMed Scopus (227) Google Scholar, 57Southworth M.W. Amaya K. Evans T.C. Xu M.Q. Perler F.B. Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein.Biotechniques. 1999; 27 (116, 118-120): 110-114Crossref PubMed Scopus (191) Google Scholar). Both, N- and C-terminal intein-CBD tags are commercially available for bacterial protein expression, but we opted here to use a C-terminal intein-CBD tag for expression in mammalian cells to allow for N-terminal processing and acetylation, which occurs on ∼90% of human proteins (58Aksnes H. Drazic A. Marie M. Arnesen T. First things first: Vital protein marks by N-terminal acetyltransferases.Trends Biochem. Sci. 2016; 41: 746-760Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). After purification on a high-affinity chitin resin, self-cleavage of the intein takes place precisely after the last endogenous amino acid of the expressed target protein, leaving no extra amino acids from the tag. While both mammalian cell expression (59Gray D. Overview of protein expression by mammalian cells.Curr. Protoc. Protein Sci. 2001; (Chapter 5(1):Unit5.9)Google Scholar, 60Khan K.H. Gene expression in mammalian cells and its applications.Adv. Pharm. Bull. 2013; 3: 257-263PubMed Google Scholar, 61Tokmakov A.A. Kurotani A. Takagi T. Toyama M. Shirouzu M. Fukami Y. Yokoyama S. Multiple post-translational modifications affect heterologous protein synthesis.J. Biol. Chem. 2012; 287: 27106-27116Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and intein-CBD purification tags (62Wood D.W. Camarero J.A. Intein applications: From protein purification and labeling to metabolic control methods.J. Biol. Chem. 2014; 289: 14512-14519Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) have been previously used, to our knowledge this is the first time that the two are combined in a single method. Self-cleavage of the intein is thiol-catalyzed, meaning that a portion of the intein of bacterial origin is prematurely activated in the reducing environment of the cytoplasm of eukaryotic cells, likely explaining why they have not been used for mammalian cell expression. Yet, we show here that a significant portion of the expressed protein remains fused to the intein-CBD tag, and milligram yields of purified protein can be obtained.We built a new vector (named pJCX4), which incorporates the Mxe GyrA intein and CBD into a mammalian-cell expression vector (Fig. 1A). The Mxe GyrA intein is a 198-amino acid self-splicing protein from Mycobacterium xenopi (63Telenti A. Southworth M. Alcaide F. Daugelat S. Jacobs Jr., W.R. Perler F.B. The Mycobacterium xenopi GyrA protein splicing element: Characterization of a minimal intein.J. Bacteriol. 1997; 179: 6378-6382Crossref PubMed Google Scholar). In its native form, the intein catalyzes its own excision from the precursor protein, forming a new peptide bond between the N- and C-terminal fragments that flank the intein (64Hirata R. Ohsumk Y. Nakano A. Kawasaki H. Suzuki K. Anraku Y. Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae.J. Biol. Chem. 1990; 265: 6726-6733Abstract Full Text PDF PubMed Google Scholar, 65Kane P.M. Yamashiro C.T. Wolczyk D.F. Neff N. Goebl M. Stevens T.H. Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase.Science. 1990; 250: 651-657Crossref PubMed Scopus (384) Google Scholar). To harness this process for expression and purification of a target protein, the N- and C-terminal fragments of the intein precursor protein are substituted respectively" @default.
• W3197366172 created "2021-09-13" @default.
• W3197366172 creator A5063558144 @default.
• W3197366172 creator A5078781578 @default.
• W3197366172 creator A5084441912 @default.
• W3197366172 date "2021-10-01" @default.
• W3197366172 modified "2023-10-18" @default.
• W3197366172 title "Novel human cell expression method reveals the role and prevalence of posttranslational modification in nonmuscle tropomyosins" @default.
• W3197366172 cites W1481760785 @default.
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