Directed Improvement of i-Photina Bioluminescence Properties, an Efficient Calcium-Regulated Photoprotein

Document Type: Article


1 Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Biochemistry, Faculty of Biological Sciences, Tarbiat modares University, Tehran, Iran

3 Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan, Iran

4 Biotechnology Research Center, Maleke Ashtar University, Tehran, Iran

5 Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran


  Photoproteins are excellent reporter systems because they don’t have virtually background signal. Aequorin is the most well-known photoprotein. Three improved engineered photoproteins photina, i-photina and c-photina, were also recently developed and optimized for generation of Ca2+ mobilization assays precisely. The total light emission is greater than aequorin and their reaction kinetics is also lower. Thus they have improved the applications of flash luminescence assays in High-Throughput Screening (HTS). These photoproteins have recently been commercialized by several companies. So we selected i-photina having the highest luminescence signal and good stability in comparison with two others. Subsequently, to produce i-Photina variants with improved analytical properties such as alternative emission colors, two mutants (F91Y and W95F mutants) were prepared by using site directed mutagenesis. Results showed as both substitutions shifted i-Photina bioluminescence to shorter wavelengths, photoprotein luminescence activity of F91Y and W95F mutants was increased and decreased, respectively. Moreover, while Ca2+ sensitivity and decay half-life time were increased in both mutants in comparison with i-Photina, F91Y mutant presented more stability and higher bioluminescence activity. So, F91Y mutant is an improved version of photoproteins that in many ways is superior to the other Ca2+ indicators such as aequorin and i-Photina for HTS and simultaneous assays.  

Graphical Abstract

Directed Improvement of i-Photina Bioluminescence Properties, an Efficient Calcium-Regulated Photoprotein


Main Subjects


[1]       L. Chen, L. Jin, N. Zhou, Exp. Opinion on Drug Discov. 7 (2012) 791.
[2]       L. Deng, S.V. Markova, E.S. Vysotski, Z.-J. Liu, J. Lee, J. Rose, B.-C. Wang, J. Biol. Chem. 279 (2004) 33647.
[3]       E. Vysotski, S. Markova, L. Frank, Molekuliarnaia Biologiia. 40 (2006) 404.
[4]       J.C. Lewis, S. Daunert, Fresenius' J. Anal. Chem. 366 (2000) 760.
[5]       J.K. Konstantou, P.C. Ioannou, T.K. Christopoulos, Eur. J. Hum. Genet. 17 (2008) 105.
[6]       M. Mirasoli, S.K. Deo, J.C. Lewis, A. Roda, S. Daunert, Anal. Biochem. 306 (2002) 204.
[7]       D. Scott, K.T. Hamorsky, C.M. Ensor, K.W. Anderson, S. Daunert, Bioconjugate Chem. 22 (2011) 475.
[8]       K.T. Hamorsky, C.M. Ensor, P. Pasini, S. Daunert, Anal. Biochem. 421 (2012) 172.
[9]       S. Inouye, J. Sato, Protein Expres. Purif. 83 (2012) 205.
[10]   S. Inouye, J. Sato, Anal. Biochem. 378 (2008) 105.
[11]   M.S. Petrovick, J.D. Harper, F.E. Nargi, E.D. Schwoebel, M.C. Hennessy, T.H. Rider, M.A. Hollis, Lincoln Laboratory J. 17 (2007) 63.
[12]   O. Shimomura, F.H. Johnson, Y. Saiga, J. Cell.  Comp. Physiol. 59 (1962) 223.
[13]   Z.J. Liu, E.S. Vysotski, C.J. Chen, J.P. Rose, J. Lee, B.C. Wang, Protein Sci. 9 (2000) 2085.
[14]   T.F. Fagan, Y. Ohmiya, J.R. Blinks, S. Inouye, F.I. Tsuji, FEBS lett. 333 (1993) 301.
[15]   S. Inouye, Y. Sahara, Protein Expres. Puri. 53 (2007) 384.
[16]   W.W. Ward, H.H. Seliger, Biochemistry 13 (1974) 1491.
[17]   S.V. Markova, L.P. Burakova, S. Golz, N.P. Malikova, L.A. Frank, E.S. Vysotski, FEBS J. 279 (2012) 856.
[18]   M.L. Powers, A.G. McDermott, N. Shaner, S.H. Haddock, Biochem. Bioph. Res. Co. 431 (2012) 360.
[19]   S. Inouye, J. Biochem. 143 (2008) 711.
[20]   S. Bovolenta, M. Foti, S. Lohmer, S. Corazza, J. Biomol. Screen. 12 (2007) 694.
[21]   N. Mastroianni, S. Cainarca, S. Corazza, EP Patent 1,858,921, 2009.
[22]   C. Nucci, S. Corazza, S. Lohmer, S. Cainarca, EP Patent 1,973,937, 2011; WO2006094805A1?cl=en
[23]   S. Cainarca, S. Fenu, C. Ferri, C. Nucci, P. Arioli, A. Menegon, L. Piemonti, S. Lohmer, L. Wrabetz, S. Corazza, PLoS One 5 (2010) e8882.
[24]   S. Veitinger, T. Veitinger, S. Cainarca, D. Fluegge, C.H. Engelhardt, S. Lohmer, H. Hatt, S. Corazza, J. Spehr, E.M. Neuhaus, J. Physiol. 589 (2011) 5033.
[25]   J. Comley, Drug Discov. 39 (2008) 433.
[26]   L. Rowe, E. Dikici, S. Daunert, Anal. Chem. 81 (2009) 8662.
[27]   L.A. Frank, V.V. Borisova, S.V. Markova, N.P. Malikova, G.A. Stepanyuk, E.S. Vysotski, Anal. Bioanal. Chem. 391 (2008) 2891.
[28]   N.P. Malikova, G.A. Stepanyuk, L.A. Frank, S.V. Markova, E.S. Vysotski, J. Lee, FEBS lett. 554 (2003) 184.
[29]   E. Dikici, X. Qu, L. Rowe, L. Millner, C. Logue, S. Deo, M. Ensor, S. Daunert, Protein Eng. Des. Sel. 22 (2009) 243.
[30]   Y. Ohmiya, M. Ohashi, F.I. Tsuji, FEBS lett. 301 (1992) 197.
[31]   G.A. Stepanyuk, S. Golz, S.V. Markova, L.A. Frank, J. Lee, E.S. Vysotski, FEBS lett. 579 (2005) 1008.
[32]    S.  Shrestha,  I.R.  Paeng,   S.K.   Deo,   S.    Daunert,Bioconjugate Chem. 13 (2002) 2695.
[33]   W. Wang, B.A. Malcolm, Biotechniques 26 (1999) 680.
[34]   J. Sambrook, D.W. Russell, Harbor Laboratory Press, New York, 3rd ed., Vol. 1, 2001, pp. 2344.
[35]   M.M. Bradford, Anal. Biochem.72 (1976) 248.
[36]   G. Semisotnov, N. Rodionova, O. Razgulyaev, V. Uversky, A. Gripas, R. Gilmanshin, Biopolymers 31 (1991) 119.
[37]   M. Eftink, C. Ghiron, Biochemistry 16 (1977) 5546.
[38]   M.S. Titushin, Y. Feng, G.A. Stepanyuk, Y. Li, S.V. Markova, S. Golz, B.-C. Wang, J. Lee, J. Wang, E.S. Vysotski, J. Biol. Chem. 285 (2010) 40891.
[39]   R. Rodriguez, G. Chinea, N. Lopez, T. Pons, G. Vriend, Bioinformatics 14 (1998) 523.
[40]   U.K. Laemmli, Nature 227 (1970) 680.
[41]   S. Inouye, Y. Sahara, Protein Expres. Purif. 66 (2009) 52.
[42]   L. Deng, E.S. Vysotski, Z.-J. Liu, S.V. Markova, N.P. Malikova, J. Lee, J. Rose, B.-C. Wang, FEBS lett. 506 (2001) 281.
[43]   E.S. Vysotski, Z.-J. Liu, S.V. Markova, J.R. Blinks, L. Deng,  L.A.  Frank,  M.  Herko,  N.P.   Malikova,  J.P. Rose, B.-C. Wang, Biochemistry 42 (2003) 6013.
[44]   P.V. Natashin, S.V. Markova, J. Lee, E.S. Vysotski, Z.J. Liu, FEBS J. 5 (2014) 1432.
[45]   A. Mahdavi, R.H. Sajedi, S. Hosseinkhani, M. Taghdir, R. Sariri, Photochem. Photobiol. Sci. 3 (2013) 467.
[46]   E.S. Vysotski, J. Lee, Accounts Chem. Res. 37 (2004) 405.
[47]   L. Deng, E.S. Vysotski, S.V. Markova, Z.J. Liu, J. Lee, J. Rose, B.C. Wang, Protein Sci. 14 (2005) 663.
[48]   L. Tricoire, K. Tsuzuki, O. Courjean, N. Gibelin, G. Bourout, J. Rossier, B. Lambolez, Proceedings of the National Academy of Sciences 103 (2006) 9500.
[49]   L.A. Frank, Sensors 10 (2010) 11287.
[50]   S.K. Deo, M. Mirasoli, S. Daunert, Anal. Bioanal. Chem. 381 (2005) 1387.
[51]   A. Chiesa, E. Rapizzi, V. Tosello, P. Pinton, M. De Virgilio, R. Rizzuto, Biochem. J. 355 (2001) 1.
[52]   J. Alvarez, M. Montero, Cell Calcium. 32 (2002) 251.
[53]   M. Bonora, C. Giorgi, A. Bononi, S. Marchi, S. Patergnani, A. Rimessi, R. Rizzuto, P. Pinton, Nature protocols 8 (2013) 2105.