AJMB  Vol.5 No.2 , April 2015
Inteins—A Focus on the Biotechnological Applications of Splicing-Promoting Proteins
Abstract: The main aim of this mini-review is to illustrate strategies and industrial applications based on inteins (INTErnal proteINS), which belong to a class of autocatalytic enzymes that are able to perform a catalytic reaction on a single substrate. However, since practical applications of inteins are strongly guided by a detailed understanding of their biological mechanisms and functions, the first part of this review will thus briefly discuss the physiological roles of inteins, describing what is currently known about their mechanisms of action. In the second part, specific biotechnological applications of inteins will be outlined (i.e. their use for (i) the purification of recombinant proteins, (ii) the cyclization of proteins and (iii) the production of seleno-proteins), paying attention to both potential strengths and weaknesses of this technology.
Cite this paper: Miraula, M. , Enculescu, C. , Schenk, G. and Mitić, N. (2015) Inteins—A Focus on the Biotechnological Applications of Splicing-Promoting Proteins. American Journal of Molecular Biology, 5, 42-56. doi: 10.4236/ajmb.2015.52005.

[1]   Gogarten, J.P., Senejani, A.G., Zhaxybayeva, O., Olendzenski, L. and Hilario, E. (2002) Inteins: Structure, Function, and Evolution. Annual Review of Microbiology, 56, 263-287.

[2]   Shih, C.K., Wagner, R., Feinstein, S., Kanik-Ennulat, C. and Neff, N. (1988) A Dominant Trifluoperazine Resistance Gene from Saccharomyces cerevisiae Has Homology with F0F1 ATP Synthase and Confers Calcium-Sensitive Growth. Molecular and Cellular Biology, 8, 3094-3103.

[3]   Perler, F.B. (1999) InBase, the New England Biolabs Intein Database. Nucleic Acids Research, 27, 346-347.

[4]   Perler, F.B., Olsen, G.J. and Adam, E. (1997) Compilation and Analysis of Intein Sequences. Nucleic Acids Research, 25, 1087-1093.

[5]   Pietrokovski, S. (1998) Modular Organization of Inteins and C-Terminal Autocatalytic Domains. Protein Science, 7, 64-71.

[6]   Dalgaard, J.Z., Moser, M.J., Hughey, R. and Mian, I.S. (1997) Statistical Modeling, Phylogenetic Analysis and Structure Prediction of a Protein Splicing Domain Common to Inteins and Hedgehog Proteins. Journal of Computational Biology, 4, 193-214.

[7]   Cooper, A.A. and Stevens, T.H. (1995) Protein Splicing: Self-Splicing of Genetically Mobile Elements at the Protein Level. Trends in Biochemical Science, 20, 351-356.

[8]   Perler, F.B., Davis, E.O., Dean, G.E., Gimble, F.S., Jack, W.E., Neff, N., Noren, C.J., Thorner, J. and Belfort, M. (1994) Protein Splicing Elements: Inteins and Exteins—A Definition of Terms and Recommended Nomenclature. Nucleic Acids Research, 22, 1125-1127.

[9]   Noren, C.J., Wang, J. and Perler, F.B. (2000) Dissecting the Chemistry of Protein Splicing and Its Applications. Angewandte Chemie International Edition, 39, 450-466.<450::AID-ANIE450>3.0.CO;2-F

[10]   Elleuche, S. and Poggeler, S. (2010) Inteins, Valuable Genetic Elements in Molecular Biology and Biotechnology. Applied Microbiology and Biotechnology, 87, 479-489.

[11]   Barzel, A., Naor, A., Privman, E., Kupiec, M. and Gophna, U. (2011) Homing Endonucleases Residing within Inteins: Evolutionary Puzzles Awaiting Genetic Solutions. Biochemical Society Transaction, 39, 169-173.

[12]   Liu, X.Q. (2000) Protein-Splicing Intein: Genetic Mobility, Origin, and Evolution. Annual Review of Genetics, 34, 61-76.

[13]   Chong, S.R. and Xu, M.Q. (1997) Protein Splicing of the Saccharomyces cerevisiae VMA Intein without the Endonuclease Motifs. Journal of Biological Chemistry, 272, 15587-15590.

[14]   Derbyshire, V., Wood, D.W., Wu, W., Dansereau, J.T., Dalgaard, J.Z. and Belfort, M. (1997) Genetic Definition of a Protein-Splicing Domain: Functional Mini-Inteins Support Structure Predictions and a Model for Intein Evolution. Proceedings of the National Academy of Sciences of the United States of America, 94, 11466-11471.

[15]   Shingledecker, K., Jiang, S.Q. and Paulus, H. (1998) Molecular Dissection of the Mycobacterium tuberculosis RecA Intein: Design of a Minimal Intein and of a Trans-Splicing System Involving Two Intein Fragments. Gene, 207, 187-195.

[16]   Perler, F.B. (2000) InBase, the Intein Database. Nucleic Acids Research, 28, 344-345.

[17]   Pietrokovski, S. (1994) Conserved Sequence Features of Inteins (Protein Introns) and Their Use in Identifying New Inteins and Related Proteins. Protein Science, 3, 2340-2350.

[18]   Tori, K., Dassa, B., Johnson, M.A., Southworth, M.W., Brace, L.E., Ishino, Y., Pietrokovski, S. and Perler, F.B. (2010) Splicing of the Mycobacteriophage Bethlehem DnaB Intein. Identification of a New Mechanistic Class of Inteins That Contain an Obligate Block F Nucleophile. Journal of Biological Chemistry, 285, 2515-2526.

[19]   Southworth, M.W., Benner, J. and Perler, F.B. (2000) An Alternative Protein Splicing Mechanism for Inteins Lacking an N-Terminal Nucleophile. EMBO Journal, 19, 5019-5026.

[20]   Johnson, M.A., Southworth, M.W., Herrmann, T., Brace, L., Perler, F.B. and Wuthrich, K. (2007) NMR Structure of a KlbA Intein Precursor from Methanococcus jannaschii. Protein Science, 16, 1316-1328.

[21]   Xu, M.Q. and Perler, F.B. (1996) The Mechanism of Protein Splicing and Its Modulation by Mutation. EMBO Journal, 15, 5146-5153.

[22]   Chong, S.R., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H. and Xu, M.Q. (1997) Single-Column Purification of Free Recombinant Proteins Using a Self-Cleavable Affinity Tag Derived from a Protein Splicing Element. Gene, 192, 271-281.

[23]   Southworth, M.W., Amaya, K., Evans, T.C., Xu, M.Q. and Perler, F.B. (1999) Purification of Proteins Fused to Either the Amino or Carboxy Terminus of the Mycobacterium xenopi Gyrase A Intein. Biotechniques, 27, 110-114.

[24]   Wu, H., Xu, M.Q. and Liu, X.Q. (1998) Protein Trans-Splicing and Functional Mini-Inteins of a Cyanobacterial dnaB Intein. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1387, 422-432.

[25]   Kwon, Y., Coleman, M.A. and Camarero, J.A. (2006) Selective Immobilization of Proteins onto Solid Supports through Split-Intein-Mediated Protein Trans-Splicing. Angewandte Chemie International Edition, 45, 1726-1729.

[26]   Shi, J. and Muir, T.W. (2005) Development of a Tandem Protein Trans-Splicing System Based on Native and Engineered Split Inteins. Journal of American Chemical Society, 127, 6198-6206.

[27]   Kurpiers, T. and Mootz, H.D. (2008) Site-Specific Chemical Modifi-cation of Proteins with a Prelabelled Cysteine Tag Using the Artificially Split Mxe GyrA Intein. ChemBioChem, 9, 2317-2325.

[28]   Kanno, A., Ozawa, T. and Umezawa, Y. (2011) Detection of Protein-Protein Interactions in Bacteria by GFP-Fragment Reconstitution. Methods in Molecular Biology, 705, 251-258.

[29]   Jung, D., Min, K., Jung, J., Jang, W. and Kwon, Y. (2013) Chemical Biology-Based Approaches on Fluorescent Labeling of Proteins in Live Cells. Molecular Biosystem, 9, 862-872.

[30]   Fong, B.A., Wu, W.Y. and Wood, D.W. (2010) The Potential Role of Self-Cleaving Purification Tags in Commercial-Scale Processes. Trends in Biotechnology, 28, 272-279.

[31]   Otomo, T., Ito, N., Kyogoku, Y. and Yamazaki, T. (1999) NMR Observation of Selected Segments in a Larger Protein: Central-Segment Isotope Labeling through Intein-Mediated Ligation. Biochemistry, 38, 16040-16044.

[32]   Otomo, T., Teruya, K., Uegaki, K., Yamazaki, T. and Kyogoku, Y. (1999) Improved Segmental Isotope Labeling of Proteins and Application to a Larger Protein. Journal of Biomolecular NMR, 14, 105-114.

[33]   Evans, T.C., Martin, D., Kolly, R., Panne, D., Sun, L., Ghosh, I., Chen, L.X., Benner, J., Liu, X.Q. and Xu, M.Q. (2000) Protein Trans-Splicing and Cyclization by a Naturally Split Intein from the dnaE Gene of Synechocystis Species PCC6803. Journal of Biological Chemistry, 275, 9091-9094.

[34]   Scott, C.P., Abel-Santos, E., Wall, M., Wahnon, D.C. and Benkovic, S.J. (1999) Production of Cyclic Peptides and Proteins in Vivo. Proceedings of the National Academy of Sciences of the United States of America, 96, 13638-13643.

[35]   Scott, C.P., Abel-Santos, E., Jones, A.D. and Benkovic, S.J. (2001) Structural Requirements for the Biosynthesis of Backbone Cyclic Peptide Libraries. Chemistry & Biology, 8, 801-815.

[36]   Evans, T.C., Benner, J. and Xu, M.Q. (1998) Semisynthesis of Cytotoxic Proteins Using a Modified Protein Splicing Element. Protein Science, 7, 2256-2264.

[37]   Saraswat, M., Musante, L., Ravida, A., Shortt, B., Byrne, B. and Holthofer, H. (2013) Preparative Purification of Recombinant Proteins: Current Status and Future Trends. BioMed Research International, 2013, Article ID: 312709.

[38]   Straathof, A.J.J. (2011) 2.57. The Proportion of Downstream Costs in Fermentative Production Processes. Comprehensive Biotechnology (Second Edition), 811-814.

[39]   Wood, D.W. (2014) New Trends and Affinity Tag Designs for Recombinant Protein Purification. Current Opinion in Structural Biology, 26C, 54-61.

[40]   Young, C.L., Britton, Z.T. and Robinson, A.S. (2012) Recombinant Protein Expression and Purification: A Comprehensive Review of Affinity Tags and Microbial Applications. Biotechnology Journal, 7, 620-634.

[41]   Bornhorst, J.A. and Falke, J.J. (2000) Purification of Proteins Using Polyhistidine Affinity Tags. Applications of Chimeric Genes and Hybrid Proteins, 326, 245-254.

[42]   Schmidt, T.G.M. and Skerra, A. (2007) The Strep-Tag System for One-Step Purification and High-Affinity Detection or Capturing of Proteins. Nature Protocols, 2, 1528-1535.

[43]   Chong, S.R., Montello, G.E., Zhang, A.H., Cantor, E.J., Liao, W., Xu, M.Q. and Benner, J. (1998) Utilizing the C-Terminal Cleavage Activity of a Protein Splicing Element to Purify Recombinant Proteins in a Single Chromatographic Step. Nucleic Acids Research, 26, 5109-5115.

[44]   Lepage, P., Heckel, C., Humbert, S., Stahl, S. and Rautmann, G. (1993) Recombinant Technology as an Alternative to Chemical Peptide-Synthesis—Expression and Characterization of HIV-1 Rev Recombinant Peptides. Analytical Biochemistry, 213, 40-48.

[45]   Nygren, P.A., Stahl, S. and Uhlen, M. (1994) Engineering Proteins to Facilitate Bioprocessing. Trends in Biotechnology, 12, 184-188.

[46]   Mathys, S., Evans, T.C., Chute, I.C., Wu, H., Chong, S.R., Benner, J., Liu, X.Q. and Xu, M.Q. (1999) Characterization of a Self-Splicing Mini-Intein and Its Conversion into Autocatalytic N- and C-Terminal Cleavage Elements: Facile Production of Protein Building Blocks for Protein Ligation. Gene, 231, 1-13.

[47]   Wood, D.W., Wu, W., Belfort, G., Derbyshire, V. and Belfort, M. (1999) A Genetic System Yields Self-Cleaving Inteins for Bioseparations. Nature Biotechnology, 17, 889-892.

[48]   Zhao, Z., Lu, W., Dun, B., Jin, D., Ping, S., Zhang, W., Chen, M., Xu, M.Q. and Lin, M. (2008) Purification of Green Fluorescent Protein Using a Two-Intein System. Applied Microbiology and Biotechnology, 77, 1175-1180.

[49]   Guan, D., Ramirez, M. and Chen, Z. (2013) Split Intein Mediated Ultra-Rapid Purification of Tagless Protein (SIRP). Biotechnology and Bioengineering, 110, 2471-2481.

[50]   Ramirez, M., Valdes, N., Guan, D. and Chen, Z. (2013) Engineering Split Intein DnaE from Nostoc punctiforme for Rapid Protein Purification. Protein Engineering, Design and Selection, 26, 215-223.

[51]   Jiang, A., Jin, W., Zhao, F., Tang, Y., Sun, Z. and Liu, J.N. (2014) Split Ssp DnaB Mini-Intein Mediated Production of Recombinant Human Glucagon-Like Peptide-1/7-36. Biotechnology and Applied Biochemestry.

[52]   Setrerrahmane, S., Zhang, Y., Dai, G., Lv, J. and Tan, S. (2014) Efficient Production of Native Lunasin with Correct N-Terminal Processing by Using the pH-Induced Self-Cleavable Ssp DnaB Mini-Intein System in Escherichia coli. Applied Biochemestry and Biotechnology, 174, 612-622.

[53]   Mannucci, E., Ognibene, A., Cremasco, F., Bardini, G., Mencucci, A., Pierazzuoli, E., Ciani, S., Fanelli, A., Messeri, G. and Rotella, C.M. (2000) Glucagon-Like Peptide (GLP)-1 and Leptin Concentrations in Obese Patients with Type 2 Diabetes Mellitus. Diabetic Medicine, 17, 713-719.

[54]   Dia, V.P. and Gonzalez de Mejia, E. (2011) Lunasin Induces Apoptosis and Modifies the Expression of Genes Associated with Extracellular Matrix and Cell Adhesion in Human Metastatic Colon Cancer Cells. Molecular Nutrition & Food Research, 55, 623-634.

[55]   Dia, V.P. and Mejia, E.G. (2010) Lunasin Promotes Apoptosis in Human Colon Cancer Cells by Mitochondrial Pathway Activation and Induction of Nuclear Clusterin Expression. Cancer Letters, 295, 44-53.

[56]   Wu, W.Y., Mee, C., Califano, F., Banki, R. and Wood, D.W. (2006) Recombinant Protein Purification by Self-Cleaving Aggregation Tag. Nature Protocols, 1, 2257-2262.

[57]   Banki, M.R., Gerngross, T.U. and Wood, D.W. (2005) Novel and Economical Purification of Recombinant Proteins: Intein-Mediated Protein Purification Using in Vivo Polyhydroxybutyrate (PHB) Matrix Association. Protein Science, 14, 1387-1395.

[58]   Wang, Z., Wu, H., Chen, J., Zhang, J., Yao, Y. and Chen, G.Q. (2008) A Novel Self-Cleaving Phasin Tag for Purification of Recombinant Proteins Based on Hydrophobic Polyhydroxyalkanoate Nanoparticles. Lab on a Chip, 8, 1957-1962.

[59]   Banki, M.R., Feng, L. and Wood, D.W. (2005) Simple Bioseparations Using Self-Cleaving Elastin-Like Polypeptide Tags. Nature Methods, 2, 659-661.

[60]   Meyer, D.E. and Chilkoti, A. (1999) Purification of Recombinant Proteins by Fusion with Thermally-Responsive Polypeptides. Nature Biotechnology, 17, 1112-1115.

[61]   Banki, M.R. and Wood, D.W. (2005) Inteins and Affinity Resin Substitutes for Protein Purification and Scale Up. Microbial Cell Factories, 4, 32.

[62]   Trabbic-Carlson, K., Liu, L., Kim, B. and Chilkoti, A. (2004) Expression and Purification of recombinant proteins from Escherichia coli: Comparison of an Elastin-Like Polypeptide Fusion with an Oligohistidine Fusion. Protein Science, 13, 3274-3284.

[63]   Barnard, G.C., McCool, J.D., Wood, D.W. and Gerngross, T.U. (2005) Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha. Applied and Environmental Microbiology, 71, 5735-5742.

[64]   Moldes, C., Garcia, P., Garcia, J.L. and Prieto, M.A. (2004) In Vivo Immobilization of Fusion Proteins on Bioplastics by the Novel Tag BioF. Applied and Environmental Microbiology, 70, 3205-3212.

[65]   Lin, M., Rose-John, S., Grotzinger, J., Conrad, U. and Scheller, J. (2006) Functional Expression of a Biologically Active Fragment of Soluble gp130 as an ELP-Fusion Protein in Transgenic Plants: Purification via Inverse Transition Cycling. Biochemical Journal, 398, 577-583.

[66]   Urry, D.W. (1992) Free Energy Transduction in Polypeptides and Proteins Based on Inverse Temperature Transitions. Progress in Biophysics and Molecular Biology, 57, 23-57.

[67]   Meyer, D.E. and Chilkoti, A. (2004) Quantification of the Effects of Chain Length and Concentration on the Thermal Behavior of Elastin-Like Polypeptides. Biomacromolecules, 5, 846-851.

[68]   Shen, Y., Ai, H.X., Song, R., Liang, Z.N., Li, J.F. and Zhang, S.Q. (2010) Expression and Purification of Moricin CM4 and Human Beta-Defensins 4 in Escherichia coli Using a New Technology. Microbiological Research, 165, 713-718.

[69]   Tian, L. and Sun, S.S. (2011) A Cost-Effective ELP-Intein Coupling System for Recombinant Protein Purification from Plant Production Platform. PLoS ONE, 6, e24183.

[70]   Trabbic-Carlson, K., Meyer, D.E., Liu, L., Piervincenzi, R., Nath, N., LaBean, T. and Chilkoti, A. (2004) Effect of Protein Fusion on the Transition Temperature of an Environmentally Responsive Elastin-Like Polypeptide: A Role for Surface Hydrophobicity? Protein Engineering, Design and Selection, 17, 57-66.

[71]   Lee, S.J., Park, J.P., Park, T.J., Lee, S.Y., Lee, S. and Park, J.K. (2005) Selective Immobilization of Fusion Proteins on Poly(Hydroxyalkanoate) Microbeads. Analitical Chemistry, 77, 5755-5759.

[72]   Yamada, M., Yamashita, K., Wakuda, A., Ichimura, K., Maehara, A., Maeda, M. and Taguchi, S. (2007) Autoregulator Protein PhaR for Biosynthesis of Polyhydroxybutyrate [P(3HB)] Possibly Has Two Separate Domains That Bind to the Target DNA and P(3HB): Functional Mapping of Amino Acid Residues Responsible for DNA Binding. Journal of Bacteriology, 189, 1118-1127.

[73]   Steinbuchel, A., Aerts, K., Babel, W., Follner, C., Liebergesell, M., Madkour, M.H., Mayer, F., Pieper-Furst, U., Pries, A., Valentin, H.E., et al. (1995) Considerations on the Structure and Biochemistry of Bacterial Polyhydroxyalkanoic Acid Inclusions. Canadian Journal of Microbiology, 41, 94-105.

[74]   Stuart, E.S., Tehrani, A., Valentin, H.E., Dennis, D., Lenz, R.W. and Fuller, R.C. (1998) Protein Organization on the PHA Inclusion Cytoplasmic Boundary. Journal of Biotechnology, 64, 137-144.

[75]   Sudesh, K., Abe, H. and Doi, Y. (2000) Synthesis, Structure and Properties of Polyhydroxyalkanoates: Biological Polyesters. Progress in Polymer Science, 25, 1503-1555.

[76]   Zhou, X., Song, Z., Liu, X, Jia, F. and Wang, Y. (2011) Production of Recombinant Porcine Interferon Alpha Using PHB-Intein-Mediated Protein Purification Strategy. Applied Biochemistry and Biotechnology, 163, 981-993.

[77]   Barbe, F., Saelens, X. Braeckmans, D., Lefevre, F. and Reeth, K.V. (2010) Role of IFN-Alpha during the Acute Stage of a Swine Influenza Virus Infection. Research in Veterinary Science, 88, 172-178.

[78]   Horisberger, M.A. (1992) Virus-Specific Effects of Recombinant Porcine Interferon-Gamma and the Induction of Mx Proteins in Pig Cells. Journal of Interferon and Cytokine Research, 12, 439-444.

[79]   Osterlund, P., Pirhonen, J., Ikonen, N., Ronkko, E., Strengell, M., Makela, S.M., Broman, M., Hamming, O.J., Hartmann, R., Ziegler, T. and Julkunen, I. (2010) Pandemic H1N1 2009 Influenza A Virus Induces Weak Cytokine Responses in Human Macrophages and Dendritic Cells and Is Highly Sensitive to the Antiviral Actions of Interferons. Journal of Virology, 84, 1414-1422.

[80]   Craik, D.J., Daly, N.L., Bond, T. and Waine, C. (1999) Plant Cyclotides: A Unique Family of Cyclic and Knotted Proteins That Defines the Cyclic Cystine Knot Structural Motif. Journal of Molecular Biology, 294, 1327-1336.

[81]   Lennard, K.R. and Tavassoli, A. (2014) Peptides Come Round: Using SICLOPPS Libraries for Early Stage Drug Discovery. Chemistry, 20, 10608-10614.

[82]   Thali, M. (1995) Cyclosporines: Immunosuppressive Drugs with Anti-HIV-1 Activity. Molecular Medicine Today, 1, 287-291.

[83]   Weber, G., Schorgendorfer, K., Schneiderscherzer, E. and Leitner, E. (1994) The Peptide Synthetase Catalyzing Cyclosporine Production in Tolypocladium niveum Is Encoded by a Giant 45.8-Kilobase Open Reading Frame. Current Genetics, 26, 120-125.

[84]   Cheriyan, M. and Perler, F.B. (2009) Protein Splicing: A Versatile Tool for Drug Discovery. Advanced Drug Delivery Review, 61, 899-907.

[85]   Mootz, H.D. (2009) Split Inteins as Versatile Tools for Protein Semisynthesis. ChemBioChem, 10, 2579-2589.

[86]   Camarero, J., Sort, J., Hoffmann, A., Garcia-Martin, J.M., Dieny, B., Miranda, R. and Nogues, J. (2005) Origin of the Asymmetric Magnetization Reversal Behavior in Exchange-Biased Systems: Competing Anisotropies. Physical Review Letters, 95, Article ID: 057204.

[87]   Evans, T.C. and Xu, M.Q. (1999) Intein-Mediated Protein Ligation: Harnessing Natures’ Escape Artists. Biopolymers, 51, 333-342.<333::AID-BIP3>3.0.CO;2-#

[88]   Sancheti, H. and Camarero, J.A. (2009) “Splicing Up” Drug Discovery. Cell-Based Expression and Screening of Genetically-Encoded Libraries of Backbone-Cyclized Polypeptides. Advanced Drug Delivery Reviews, 61, 908-917.

[89]   Xu, M.Q. and Evans, T.C. (2001) Intein-Mediated Ligation and Cyclization of Expressed Proteins. Methods, 24, 257-277.

[90]   Tavassoli, A. and Benkovic, S.J. (2007) Split-Intein Mediated Circular Ligation Used in the Synthesis of Cyclic Peptide Libraries in E. coli. Nature Protocols, 2, 1126-1133.

[91]   Baldwin, J.J. (1996) Design, Synthesis and Use of Binary Encoded Synthetic Chemical Libraries. Molecular Diversity, 2, 81-88.

[92]   Huwe, C.M. (2006) Synthetic Library Design. Drug Discovery Today, 11, 763-767.

[93]   Irwin, J.J. (2006) How Good Is Your Screening Library? Current Opinion in Chemical Biology, 10, 352-356.

[94]   Deschuyteneer, G., Garcia, S., Michiels, B., Baudoux, B., Degand, H., Morsomme, P. and Soumillion, P. (2010) Intein-Mediated Cyclization of Randomized Peptides in the Periplasm of Escherichia coli and Their Extracellular Secretion. ACS Chemical Biology, 5, 691-700.

[95]   Driscoll, D.M. and Copeland, P.R. (2003) Mechanism and Regulation of Selenoprotein Synthesis. Annual Review of Nutrition, 23, 17-40.

[96]   Kryukov, G.V., Castellano, S., Novoselov, S.V., Lobanov, A.V., Zehtab, O., Guigo, R. and Gladyshev, V.N. (2003) Characterization of Mammalian Selenoproteomes. Science, 300, 1439-1443.

[97]   Stadtman, T.C. (1974) Selenium Biochemistry. Science, 183, 915-922.

[98]   Baranov, P.V., Gesteland, R.F. and Atkins, J.F. (2002) Recoding: Translational Bifurcations in Gene Expression. Gene, 286, 187-201.

[99]   Johansson, L., Gafvelin, G. and Arner, E.S. (2005) Selenocysteine in Proteins-Properties and Biotechnological Use. Biochimica and Biophysica Acta, 1726, 1-13.

[100]   Donovan, J. and Copeland, P.R. (2010) The Efficiency of Selenocysteine Incorporation Is Regulated by Translation Initiation Factors. Journal of Molecular Biology, 400, 659-664.

[101]   Bock, A., Forchhammer, K., Heider, J. and Baron, C. (1991) Selenoprotein Synthesis: An Expansion of the Genetic Code. Trends in Biochemical Science, 16, 463-467.

[102]   Labunskyy, V.M., Hatfield, D.L. and Gladyshev, V.N. (2014) Selenoproteins: Molecular Pathways and Physiological Roles. Physiology Review, 94, 739-777.

[103]   Berry, M.J., Banu, L., Harney, J.W. and Larsen, P.R. (1993) Functional Characterization of the Eukaryotic SECIS Elements which Direct Selenocysteine Insertion at UGA Codons. EMBO Journal, 12, 3315-3322.

[104]   Hondal, R.J. (2009) Using Chemical Approaches to Study Selenoproteins—Focus on Thioredoxin Reductases. Biochimica et Biophysica Acta (BBA)-General Subjects, 1790, 1501-1512.

[105]   Arner, E.S., Sarioglu, H., Lottspeich, F., Holmgren, A. and Bock, A. (1999) High-Level Expression in Escherichia coli of Selenocysteine-Containing Rat Thioredoxin Reductase Utilizing Gene Fusions with Engineered Bacterial-Type SECIS Elements and Co-Expression with the selA, selB and selC Genes. Journal of Molecular Biology, 292, 1003-1016.