Temporary Solubilizing Tags Method for the Chemical Synthesis of Hydrophobic Proteins

Author(s): Dong-Dong Zhao, Xiao-Wen Fan, He Hao, Hong-Li Zhang, Ye Guo*.

Journal Name: Current Organic Chemistry

Volume 23 , Issue 1 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Hydrophobic proteins, as one of the cellular protein classifications, play an essential function in maintaining the normal life cycle of living cells. Researches on the structure and function of hydrophobic proteins promote the exploration of the causes of major diseases, and development of new therapeutic agents for disease treatment. However, the poor water solubility of hydrophobic proteins creates problems for their preparation, separation, characterization and functional studies. The temporary solubilizing tags are considered a practical strategy to effectively solve the poor water solubility problem of hydrophobic proteins. This strategy can significantly improve the water solubility of hydrophobic peptides/proteins, making them like water-soluble peptides/proteins easy to be purified, characterized. More importantly, the temporary solubilizing tags can be removed after protein synthesis, so thus the structure and function of the hydrophobic proteins are not affected. At present, temporary solubilizing tags have been successfully used to prepare many important hydrophobic proteins such as membrane proteins, lipoproteins and chaperones. In this review, we summarize the recent researches and applications of temporary solubilizing tags.

Keywords: Hydrophobic proteins, membrane proteins, chemical protein synthesis, NCL, temporary solubilizing tags, lipoproteins.

Ho, B.; Baryshnikova, A.; Brown, G.W. Unification of protein abundance datasets yields a quantitative. Saccharomyces cerevisiae Proteome. Cell Syst., 2018, 6, 192-205.
(a)Gouaux, E.; MacKinnon, R. Principles of selective ion transport in channels and pumps. Science, 2005, 310, 1461-1465.
(b)Watson, H. Biological membranes. Essays Biochem., 2015, 59, 43-69.
(c)Venkatakrishnan, A.J.; Deupi, X.; Lebon, G.; Tate, C.G.; Schertler, G.F.; Babu, M.M. Molecular signatures of G-protein-coupled receptors. Nature, 2013, 494, 185-194.
(a)Bill, R.M.; Henderson, P.J.; Iwata, S.; Kunji, E.R.; Michel, H.; Neutze, R.; Newstead, S.; Poolman, B.; Tate, C.G.; Vogel, H. Overcoming barriers to membrane protein structure determination. Nat. Biotechnol., 2011, 29, 335-340.
(b)Bao, P.; Cartron, M.L.; Sheikh, K.H.; Johnson, B.R.G.; Hunter, C.N.; Evans, S.D. Controlling transmembrane protein concentration and orientation in supported lipid bilayers. Chem. Commun., 2017, 53, 4250-4253.
Takayama, H.; Chelikani, P.; Reeves, P.J.; Zhang, S.; Khorana, H.G. High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81. PLoS One, 2008, 3, e2314.
Wingfield, P.T. Overview of the purification of recombinant proteins. Curr. Protoc. Protein Sci., 2015, 80, 1-35.
(a)Evans, T.C.; Benner, Jr , J.; Xu, M.Q. Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci., 1998, 7, 2256-2264.
(b)Junge, F.; Schneider, B.; Reckel, S.; Schwarz, D.; Dötsch, V.; Bernhard, F. Large scale production of functional membrane proteins. Cell. Mol. Life Sci., 2008, 65, 1729-1755.
Schwarz, D.; Junge, F.; Durst, F.; Frölich, N.; Schneider, B.; Reckel, S.; Sobnanifar, S.; Dötsch, V.; Bernhard, F. Preparative scale expression of membrane proteins in Escherichia Coli-based continuous exchange cell-free systems. Nat. Protoc., 2007, 2, 2945-2957.
Muir, T.W.; Kent, S.B. The chemical synthesis of proteins. Curr. Opin. Biotechnol., 1993, 4, 420-4273.
(a)Zheng, J-S.; Tang, S.; Qi, Y-K.; Wang, Z-P.; Liu, L. Chemical synthesis of proteins using peptide hydrazides as thioester surrogates. Nature Protocols.., 2013, 8, 2483-2495.
(b)Li, Y.M.; Li, Y.T.; Pan, M.; Kong, X.Q.; Huang, Y.C.; Hong, Z.Y.; Liu, L. Irreversible site-specific hydrazinolysis of proteins by use of sortase. Angew. Chem. Int. Ed., 2014, 53, 2198-2202.
(c)Fang, G.M.; Li, Y.M.; Shen, F.; Huang, Y.C.; Li, J.B.; Lin, Y.; Cui, H.K.; Liu, L. Protein chemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int. Ed., 2011, 50, 7645-7649. d) Fang, G.M.; Wang, J.X.; Liu, L. Convergent chemical synthesis of proteins by ligation of peptide hydrazides. Angew. Chem. Int. Ed., 2012, 51, 10347-10350.
Dawson, P.E.; Muir, T.W.; Clark-Lewis, I.; Kent, S.B. Synthesis of proteins by native chemical ligation. Science, 1994, 266, 776-778.
(a)Baumruck, A.C.; Tietze, D.; Steinackera, L.K.; Tietze, A.A. Chemical synthesis of membrane proteins: a model study on the influenza virus B proton channel. Chem. Sci., 2018, 9, 2365-2375.
(b)Tang, S.; Zuo, C.; Huang, D.L.; Cai, X.Y.; Zhang, L.H.; Tian, C.L.; Zheng, J.S.; Liu, L. Chemical synthesis of membrane proteins by the removable backbone modification method. Nat. Protoc., 2017, 12, 2554-2569.
(a)Gao, S.; Pan, M.; Zheng, Y.; Huang, Y.C.; Zheng, Q.Y.; Sun, D.M.; Lu, L.N.; Tan, X.D.; Tan, X.L.; Lan, H.; Wang, J.X.; Wang, T.; Wang, J.W.; Liu, L. Monomer/oligomer quasi-racemic protein crystallography. J. Am. Chem. Soc., 2016, 138, 14497-14502.
(b)Pan, M.; Gao, S.; Zheng, Y.; Tan, X.D.; Lan, H.; Tan, X.; Sun, D.M.; Wang, J.W.; Liu, L. Quasi-racemic x-ray structures of K27-linked ubiquitin chains prepared by total chemical synthesis. J. Am. Chem. Soc., 2016, 138, 7429-7435.
(a)Nagorny, P.; Sane, N.; Fasching, B.; Aussedat, B.; Danishefsky, S.J. Probing the frontiers of glycoprotein synthesis: The fully elaborated β-Subunit of the human follicle-stimulating hormone. Angew. Chem. Int. Ed., 2012, 51, 975-979.
(b)Hien, N.M.; Izumi, M.; Sato, H.; Okamoto, R.; Kajihara, Y. Chemical synthesis of glycoproteins with the specific installation of gradient-enriched 15N-labeled amino acids for getting insights into glycoprotein behavior. Chemistry., 2017, 23, 6579-6585.
Thapa, P.; Zhang, R.Y.; Menon, V.; Bingham, J.P. Native chemical ligation: A boon to peptide chemistry. Molecules, 2014, 19, 14461-14483.
Shen, F.; Huang, Y.C.; Tang, S. Chen, Y.X.; Liu, L. Chemical Synthesis of Integral Membrane Proteins: Methods and Applications. Isr. J. Chem., 2011, 51, 940-952.
Li, J.B.; Tang, S.; Zheng, J.S.; Tian, C.L.; Liu, L. Removable backbone modification method for the chemical synthesis of membrane proteins. Acc. Chem. Res., 2017, 50, 1143-1153.
Dittmann, M.; Sadek, M.; Seidel, R.; Engelhard, M. Native chemical ligation in dimethylformamide can be performed chemoselectively without racemization. J. Pept. Sci., 2012, 18, 312-316.
Dittmann, M.; Sauermann, J.; Seidel, R.; Zimmermann, W.; Engelhard, M. Native chemical ligation of hydrophobic peptides in organic solvents. J. Pept. Sci., 2010, 16, 558-562.
Kochendoerfer, G.G.; Salom, D.; Lear, J.D.; Wilk-Orescan, R.; Kent, S.B.; DeGrado, W.F. Total chemical synthesis of the integral membrane protein influenza A virus M2: Role of its C-terminal domain in tetramer assembly. Biochemistry, 1999, 38, 11905-11913.
Shen, F.; Tang, S.; Liu, L. Hexafluoro-2-propanol as a potent co-solvent for chemical ligation of membrane proteins. Sci. China Chem., 2011, 54, 110-116.
Zuo, C.; Tang, S.; Zheng, J.S. Chemical synthesis and biophysical applications of membrane proteins. J. Pept. Sci., 2015, 21, 540-549.
Valiyaveetil, F.I.; MacKinnon, R.; Muir, T.W. Semisynthesis and folding of the potassium channel KcsA. J. Am. Chem. Soc., 2002, 124, 9113-9120.
Lahiri, S.; Brehs, M.; Olschewski, D.; Becker, C.F. Total chemical synthesis of an integral membrane enzyme: Diacylglycerol kinase from Escherichia coli. Angew. Chem. Int. Ed., 2011, 50, 3988-3992.
Olschewski, D.; Becker, C.F. Chemical synthesis and semisynthesis of membrane proteins. Mol. Biosyst., 2008, 4, 733-740.
Loo, R.R.; Dales, N.; Andrews, P.C. Surfactant effects on protein structure examined by electrospray ionization mass spectrometry. Protein Sci., 1994, 3, 1975-1983.
(a)Hilbich, C.; Kisters-Woike, B.; Reed, J.; Masters, C.L.; Beyreuther, K. Aggregation and secondary structure of synthetic amyloid βA4 peptides of Alzheimer’s disease. J. Mol. Biol., 1999, 218, 149-163.
(b)Némethy, G. Hydrophobic Interactions. Angew. Chem. Int. Ed., 1967, 6, 195-206.
Rauf, S.M.; Arvidsson, P.I.; Albericio, F.; Govender, T.; Maguire, G.E.; Kruger, H.G.; Honarparvar, B. The effect of N-methylation of amino acids (Ac-X-OMe) on solubility and conformation: a DFT study. Org. Biomol. Chem., 2015, 13, 9993-10006.
Simmonds, R.G. Use of the Hmb backbone-protecting group in the synthesis of difficult sequences. Int. J. Pept. Protein Res., 1996, 47, 36-41.
Johnson, E.C.; Kent, S.B. Studies on the insolubility of a transmembrane peptide from signal peptide peptidase. J. Am. Chem. Soc., 2006, 128, 7140-7141.
Levinson, A.M.; McGee, J.H.; Roberts, A.G.; Creech, G.S.; Wang, T.; Peterson, M.T.; Hendrickson, R.C.; Verdine, G.L.; Danishefsky, S.J. Total chemical synthesis and folding of all-L and all-D variants of oncogenic KRas(G12V). J. Am. Chem. Soc., 2017, 139, 7632-7639.
Liu, L.P.; Deber, C.M. Guidelines for membrane protein engineering derived from de novo designed model peptides. Biopolymers, 1998, 47, 41-62.
Paradís-Bas, M.; Tulla-Puche, J.; Albericio, F. Semipermanent Cterminal carboxylic acid protecting group: application to solubilizing peptides and fragment condensation. Org. Lett., 2015, 17, 294-297.
Bianchi, E.; Ingenito, R.; Simon, R.J.; Pessi, A. Engineering and chemical synthesis of a transmembrane protein: The HCV protease cofactor protein NS4A. J. Am. Chem. Soc., 1999, 121, 7698-7699.
Tan, Z.; Shang, S.; Danishefsky, S.J. Rational evelopment of a strategy for modifying the aggregatibility of proteins. Proc. Natl. Acad. Sci. USA, 2011, 108, 4297-4302.
Becker, C.W.; Oblatt-Montal, M.; Kochendoerfer, G.G.; Montal, M. Chemical synthesis and single channel properties of tetrameric and pentameric TASPs (template-assembled synthetic proteins) derived from the transmembrane domain of HIV Virus protein u (Vpu). J. Biol. Chem., 2004, 279, 17483-17489.
Paradis-Bas, M.; Tulla-Puche, J.; Albericio, F. The road to the synthesis of “difficult peptides”. Chem. Soc. Rev., 2016, 45, 631-654.
(a)Sato, T.; Saito, Y.; Aimoto, S. Synthesis of the C-terminal region of opioid receptor like 1 in an SDS micelle by the native chemical ligation: effect of thiol additive and SDS concentration on ligation efficiency. J. Peptide . Sci., 2005, 11, 410-416.
(b)Johnsona, E.C.; Kent, S.B. Towards the total chemical synthesis of integral membrane proteins: A general method for the synthesis of hydrophobic peptide-αthioester building blocks. Tetrahedron Lett., 2007, 48, 1795-1799.
(a)Harris, P.W.; Brimble, M.A. Synthesis of an arginine tagged [Cys155–Arg180] fragment of NY-ESO-1: Elimination of an undesired by-product using ‘In house’ resins. Synthesis, 2009, 20, 3460-3466.
(b)Harris, P.W.; Brimble, M.A. Toward the total chemical synthesis of the cancer protein NYESO- 1. Biopolymers (Pept. Sci)., 2010, 94, 542-550.
(a)(Johnson, E.C.; Malito, E.; Shen, Y.; Rich, D.; Tang, W.J.; Kent, S.B. Modular total chemical synthesis of a human immunodeficiency virus type 1 protease. J. Am. Chem. Soc., 2007, 129, 11480-11490.
(b)Chemuru, S.; Kodali, R.; Wetzel, R. Improved chemical synthesis of hydrophobic Aβ peptides using addition of C terminal lysines later removed by carboxypeptidase B. Biopolymers, 2014, 102, 206-221.
(a)Johnson, E.C.; Kent, S.B. Insights into the mechanism and catalysis of the native chemical ligation reaction. J. Am. Chem. Soc., 2006, 128, 6640-6646.
(b)Tang, S.; Si, Y.Y.; Wang, Z.P.; Mei, K.R.; Chen, X.; Cheng, J.Y.; Zheng, J.S.; Liu, L. An efficient one-pot four-segment condensation method for protein chemical synthesis. Angew. Chem. Int. Ed., 2015, 54, 5713-5717.
(c)Wang, J.X.; Fang, G.M.; He, Y.; Qu, D.L.; Yu, M.; Hong, Z.Y.; Liu, L. Peptide o-aminoanilides as crypto-thioesters for protein chemical synthesis. Angew. Chem. Int. Ed., 2015, 54, 2194-2198.
(d)Wang, Z.; Xu, W.; Liu, L.; Zhu, T.F. A synthetic molecular system capable of mirror-image genetic replication and transcription. Nat. Chem., 2016, 8, 698-704.
Raz, R.; Burlina, F.; Ismail, M.; Downward, J.; Li, J.J.; Smerdon, S.J.; Quibell, M. White, P.D.; Offer, J. HF-free Boc synthesis of peptide thioesters for ligation and cyclization. Angew. Chem. Int. Ed., 2016, 55, 13174-13179.
Behrendt, R.; White, P.; Offer, J. Advances in Fmoc solid-phase peptide synthesis. J. Pept. Sci., 2016, 22, 4-27.
Choma, C.T.; Robillard, G.T.; Englebretsen, D.R. Synthesis of hydrophobic peptides: an Fmoc “solubilising tail” method. Tetrahedron Lett., 1998, 39, 2417-2420.
Miller, M.; Schneider, J.; Sathyanarayana, B.K.; Toth, M.V.; Marshall, G.R.; Clawson, L.; Selk, L.; Kent, S.B.; Wlodawer, A. Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution. Science, 1989, 246, 1149-1152.
Chatterjee, A.; Mridula, P.; Mishra, R.K.; Mittal, R.; Hosur, R.V. Folding regulates autoprocessing of HIV-1 protease precursor. J. Biol. Chem., 2005, 280, 11369-11378.
Linn, K.M.; Derebe, M.G.; Jiang, Y.; Valiyaveetil, F.I. Semisynthesis of NaK; a Na+ and K+ conducting ion channel. Biochemistry, 2010, 49, 4450-4456.
(a)Mix, K.A.; Lomax, J.E.; Raines, R.T. Cytosolic delivery of proteins by bioreversible esterification. J. Am. Chem. Soc., 2017, 139, 14396-14398.
(b)Vassiliou, G.; McPherson, R. Role of cholesteryl ester transfer protein in selective uptake of high density lipoprotein cholesteryl esters by adipocytes. J. Lipid Res., 2004, 45, 1683-1693.
Kabeya, Y.; Mizushima, N.; Ueno, T.; Yamamoto, A.; Kirisako, T.; Noda, T.; Kominami, E.; Ohsumi, Y.; Yoshimori, T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J., 2000, 19, 5720-5728.
Huang, Y.C.; Li, Y.M.; Chen, Y.; Pan, M.; Li, Y.T.; Yu, L.; Guo, Q.X.; Liu, L. Synthesis of autophagosomal marker protein LC3-II under detergent-free conditions. Angew. Chem. Int. Ed., 2013, 52, 4858-4862.
Maity, S.K.; Mann, G.; Jbara, M.; Laps, S.; Kamnesky, G.; Brik, A. Palladium-assisted removal of a solubilizing tag from a Cys side chain to facilitate peptide and protein synthesis. Org. Lett., 2016, 18, 3026-3029.
Brailsford, J.A.; Stockdill, J.L.; Axelrod, A.J.; Peterson, M.T.; Vadola, P.A.; Johnston, E.V.; Danishefsky, S.J. Total chemical synthesis of human thyroid-stimulating hormone (hTSH) b-subunit: Application of arginine-tagged acetamidomethyl (AcmR) protecting groups. Tetrahedron, 2018, 74, 1951-1956.
Tsuda, S.; Mochizuki, M.; Ishiba, H.; Yoshizawa-Kumagaye, K.; Nishio, H.; Oishi, S.; Yoshiya, T. Easy-to-attach/detach solubilizing tag-aided chemical synthesis of an aggregative capsid protein. Angew. Chem. Int. Ed., 2018, 57, 2105-2109.
Porterfield, J.Z.; Dhason, M.S.; Loeb, D.D.; Nassal, M.; Stray, S.J.; Zlotnick, A. Full-length hepatitis B virus core protein packages viral and heterologous RNA with similarly high levels of cooperativity. J. Virol., 2010, 84, 7174-7184.
Jacobsen, M.T.; Petersen, M.E.; Ye, X.; Galibert, M.; Lorimer, G.H.; Aucagne, V.; Kay, M.S. A helping hand to overcome solubility challenges in chemical protein synthesis. J. Am. Chem. Soc., 2016, 138, 11775-11782.
Ozinskas, A.J.; Rosenthal, G.A. Synthesis of L-canaline and gamma-functional 2-aminobutyric acid derivatives. J. Org. Chem., 1986, 51, 5047-5050.
Tsuda, S.; Nishio, H.; Yoshiya, T. Peptide self-cleavage at a canaline residue: Application to a solubilizing tag system for native chemical ligation. Chem. Commun., 2018, 54, 8861-8864.
Eilers, M.; Shekar, S.C. Internal packing of helical membrane proteins. J. Proc. Natl. Acad. Sci. USA, 2000, 97, 5796-5801.
Zheng, J.S.; Yu, M.; Qi, Y.K.; Tang, S.; Shen, F.; Wang, Z.P.; Xiao, L.; Zhang, L.; Tian, C.L.; Liu, L. Expedient total synthesis of small to medium-sized membrane proteins via Fmoc chemistry. J. Am. Chem. Soc., 2014, 136, 3695-3704.
Zheng, J.S.; He, Y.; Zuo, C.; Cai, X.Y.; Tang, S.; Wang, Z.P.; Zhang, L.H.; Tian, C.L.; Liu, L. Robust chemical synthesis of membrane proteins through a general method of removable backbone modification. J. Am. Chem. Soc., 2016, 138, 3553-3561.
Zuo, C.; Tang, S.; Si, Y.Y.; Wang, Z.A.; Tian, C.L.; Zheng, J.S. Efficient synthesis of longer Aβ peptides via removable backbone modification. Org. Biomol. Chem., 2016, 14, 5012-5018.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [2 - 13]
Pages: 12
DOI: 10.2174/1385272822666181211121758
Price: $58

Article Metrics

PDF: 35