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Single-site phosphorylation within the His-tag sequence attached to a recombinant protein
Analytical Biochemistry 570 (2019) 62–64
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Single-site phosphorylation within the His-tag sequence attached to a recombinant protein
T
Himanshu Singha,*, Deepshikha Vermaa, Benjamin Bardiauxb a b
Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400005, India Institut Pasteur, Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, CNRS UMR 3528, C3BI USR 3756, Paris, France
ARTICLE INFO
ABSTRACT
Keywords: pET28a Phosphorylation His-tag Recombinant protein NMR MALDI-TOF
We report the observation of single-site phosphorylation in a His-tag sequence N-terminally attached to a recombinant protein (UVI31+) in vitro. This modification was detected at position 23 at a serine residue of the Histag sequence encoded by the vector pET28a. Furthermore, the phosphorylated tag sequence was found to be dephosphorylated by the action of alkaline phosphatases. The functional activity and dynamics of the protein carrying the His-tag sequence were unchanged after phosphorylation. The possibility of phosphorylation within the N-terminal His-tag demonstrates that care has to be taken upon analysis of post-translational modifications via mass spectrometry for recombinant protein expression strategies.
Protein phosphorylation is an important event of signal transduction pathways that regulate many aspects of cell function [1]. Recombinant proteins are widely used in drug discovery and fundamental research where recombinant constructs are often expressed in mammalian, insect, yeast or bacterial cells, and purified for subsequent biophysical characterization, activity assays, structural and dynamic studies. Proteins may be phosphorylated to different extent when overexpressed in eukaryotic cells, and some are found to be phosphorylated when kinases are expressed in E. coli [2,3]. In order to understand the consequences of different patterns of phosphorylation on enzymatic functions, dynamics and interactions, it is important to characterize the extent and sites of phosphorylation [4]. Identification of the phosphorylation sites also permits to design constructs preventing undesired phosphorylations [5]. NMR and mass spectrometry (MS) are the methods of choice for probing protein phosphorylation, while other methods used to detect the same are Eastern blotting and 32P-radionucleotide incorporation [6–10,14]. In eukaryotes, phosphorylation of Ser and Thr are more common than that of Tyr and His. Mass spectrometry analysis reported a significant mass difference of 80 Da between the expected protein mass and kinase treated protein [2,3]. Histidine-tag (His-tag) is the most frequently used tag to label and purify recombinant proteins using affinity chromatography. The vector pET28a derived His-tag sequence, a construct often used from molecular and structural biology up to high-throughput methods, contains six serine residues in a 23 residues long tag sequence (“1MRGSHHHHHHGSHVISSIASRGS23”). When analyzing protein phosphorylation
*
events via mass spectrometric approaches, it appears that phosphorylation can take place not only on the protein of interest itself but also on vector-derived His-tag sequences. These modifications can thus lead to misinterpretation of the data with regard to physiological protein phosphorylation. In this study, MALDI-TOF-MS of N-terminal His-tagged full-length UVI31 + protein overexpressed under reported conditions [11,13] in E. coli cells gave a mass of 13,459 Da, a result in agreement with the theoretical mass of the protein (Fig. 1). The protein was incubated with protein kinase A for phosphorylation reaction and resulted a product containing singly phosphorylated form of modified mass 13,539 Da, with 80 Da (HPO3 = 80 Da) mass interval between the species, indicating single-site phosphorylation. When alkaline phosphatase was added to the phosphorylated protein mixture, the phosphorylated Histagged UVI31 + protein was converted back to its unphosphorylated form (Fig. S1). Analysis of His-tagged 15N and 13C/15N labeled UVI31 + purified from E. coli by NMR resulted in live monitoring of the phosphorylation events in a site-specific manner and chemical shift assignments were confirmed using standard backbone assignment experiments [12]. The phosphorylation site was determined as Ser23. As other sites of the Histag residues participate in electrostatic interactions after such modifications, significant chemical shift perturbations were observed also at Gly11, Ser12, His13, Met24, Ala25, Glu26 and His27 (Fig. 2). The phosphorylated site Ser23 shows the maximum shift perturbation of 0.68 ppm (Fig. S2).
Corresponding author. Department of Chemistry and Pharmacy, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, 81377, Munich, Germany. E-mail addresses: [email protected], [email protected] (H. Singh).
https://doi.org/10.1016/j.ab.2019.02.001 Received 25 December 2018; Received in revised form 3 February 2019; Accepted 4 February 2019 Available online 05 February 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
Analytical Biochemistry 570 (2019) 62–64
H. Singh, et al.
2D-[15N-1H]-nOe experiments were used to probe changes of dynamics on picosecond to nanosecond time scales of His-tagged regions of the protein with and without phosphorylation. This experiment indicated only reduced mobility in the His-tag regions after phosphorylation and did not affect the overall protein dynamics (Fig. S3). Molecular interactions of His-tagged charged residues with the phosphoryl group of serine 23 were mapped after performing MD simulations. The statistics on interactions between the oxygen atoms of the phosphoryl group of pSer23 and side-chain nitrogen (or N-terminal nitrogen for Gly11) atoms of charged residues in the N-terminal His-tag region of phosphorylated Ser23-UVI31 + in MD trajectories are shown in Fig. S4 and Table S1. We also confirmed that the endonuclease function of UVI31 + protein was unaffected after such modification of His-tag sequence (Fig. S5). Taken together, our results show that the recombinant protein derived from pET28a vector show phosphorylation at the His-tag sequence at serine 23 position and this site is dephosphorylated by the action of alkaline phosphatase. This result demonstrates that single-site phosphorylation at the tag sequence derived from the current commercial pET28a expression vectors can interfere with unambiguous phosphorylation detection in a protein of interest. Acknowledgments This manuscript is dedicated to Prof. K.V.R. Chary's 65th birth anniversary, IISER Berhampur, Odisha, India. HS would like to thank Prof. Dr. Rasmus Linser, Dr. Aracelys L Castilla and Dr. Anand K Das for critical reading of the manuscript. This work was supported by National Facility for High-Field NMR at Tata Institute of Fundamental Research, Mumbai and Raman Charpak Fellowship, CEFIPRA, New Delhi, India. Fig. 1. MALDI-TOF mass spectra of UVI31 + protein at different incubation times (0–9 h). 100 μM protein, 10 mM MgCl2, 1.5 mM ATP was incubated with 1 Unit of protein kinase A at pH 6.4 at 37 °C.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.02.001. References [1] T. Hunter, Signaling—2000 and beyond, Cell 100 (2000) 113–127. [2] P. Du, P. Loulakis, C. Luo, A. Mistry, S.P. Simons, P.K. LeMotte, F. Rajamohan, K. Rafidi, K.G. Coleman, K.F. Geoghegan, Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function, Protein Expr. Purif. 44 (2005) 121–129. [3] R. Lomas-Lopez, A.J. Cozzone, B. Duclos, A modified His-tag vector for the production of recombinant protein kinases, Anal. Biochem. 377 (2008) 272–273. [4] L.N. Johnson, The regulation of protein phosphorylation, Biochem. Soc. Trans. 37 (2009) 627–641. [5] Y. Oda, K. Huang, F. Cross, D. Cowburn, B. Chait, Accurate quantitation of protein expression and site-specific phosphorylation, Proc. Natl. Acad. Sci. Unit. States Am. 96 (1999) 6591–6596. [6] F.-X. Theillet, H.M. Rose, S. Liokatis, A. Binolfi, R. Thongwichian, M. Stuiver, P. Selenko, Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts, Nat. Protoc. 8 (2013) 1416. [7] Y.-Q. Zhao, J. Yan, J.-B. Chao, A.-H. Liang, B.-S. Yang, The biochemical effect of Ser166 phosphorylation on Euplotes octocarinatus centrin, JBIC J. Biol. Inorgan. Chem. 18 (2013) 123–136. [8] M. Mann, S.-E. Ong, M. Grønborg, H. Steen, O.N. Jensen, A. Pandey, Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome, Trends Biotechnol. 20 (2002) 261–268. [9] I. Amata, M. Maffei, A. Igea, M. Gay, M. Vilaseca, A.R. Nebreda, M. Pons, Multiphosphorylation of the intrinsically disordered unique domain of c-src studied by in-cell and real-time NMR spectroscopy, Chembiochem 14 (2013) 1820–1827. [10] S. Liokatis, A. Dose, D. Schwarzer, P. Selenko, Simultaneous detection of protein phosphorylation and acetylation by high-resolution NMR spectroscopy, J. Am. Chem. Soc. 132 (2010) 14704–14705. [11] M. Shukla, R. Minda, H. Singh, S. Tirumani, K.V. Chary, B.J. Rao, UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii, PLoS One 7 (2012) e51913. [12] H. Singh, D. Verma, B. Rao, K.V. Chary, 1 H, 13 C and 15 N NMR assignments of Mg
Fig. 2. An overlay of a 2D [15N-1H] HSQC spectrum of the uniformly-15N-labelled His-tag containing UVI31+ (shown in blue) with phosphorylated His-tag UVI31+ (shown in red). The experiments were recorded at pH 6.4 and 310 K. The spectra were recorded on a Bruker Avance 800 MHz NMR spectrometer with 256 and 2048 complex points along t1 and t2 dimensions, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Analytical Biochemistry 570 (2019) 62–64
H. Singh, et al. 2+ bound form of UV inducible transcript protein (UVI31+) from Chlamydomonas reinhardtii, Biomolecular NMR Assignments, vol. 9, 2015, pp. 93–97. [13] A.K. Rout, H. Singh, S. Patel, V. Raghvan, S. Gautam, R. Minda, B.J. Rao, K.V.R. Chary, Structural characterization of a novel KH-domain containing plant
chloroplast endonuclease, Sci. Rep. 8 (2018). [14] Y.Q. Zhao, J. Yan, J.B. Chao, A.H. Liang, B.S. Yang, The biochemical effect of Ser166 phosphorylation on Euplotes octocarinatus centrin, J. Biol. Inorg. Chem. 18 (1) (2013) 123–136.
64
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Single-site phosphorylation within the His-tag sequence attached to a recombinant protein
T
Himanshu Singha,*, Deepshikha Vermaa, Benjamin Bardiauxb a b
Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400005, India Institut Pasteur, Structural Bioinformatics Unit, Department of Structural Biology and Chemistry, CNRS UMR 3528, C3BI USR 3756, Paris, France
ARTICLE INFO
ABSTRACT
Keywords: pET28a Phosphorylation His-tag Recombinant protein NMR MALDI-TOF
We report the observation of single-site phosphorylation in a His-tag sequence N-terminally attached to a recombinant protein (UVI31+) in vitro. This modification was detected at position 23 at a serine residue of the Histag sequence encoded by the vector pET28a. Furthermore, the phosphorylated tag sequence was found to be dephosphorylated by the action of alkaline phosphatases. The functional activity and dynamics of the protein carrying the His-tag sequence were unchanged after phosphorylation. The possibility of phosphorylation within the N-terminal His-tag demonstrates that care has to be taken upon analysis of post-translational modifications via mass spectrometry for recombinant protein expression strategies.
Protein phosphorylation is an important event of signal transduction pathways that regulate many aspects of cell function [1]. Recombinant proteins are widely used in drug discovery and fundamental research where recombinant constructs are often expressed in mammalian, insect, yeast or bacterial cells, and purified for subsequent biophysical characterization, activity assays, structural and dynamic studies. Proteins may be phosphorylated to different extent when overexpressed in eukaryotic cells, and some are found to be phosphorylated when kinases are expressed in E. coli [2,3]. In order to understand the consequences of different patterns of phosphorylation on enzymatic functions, dynamics and interactions, it is important to characterize the extent and sites of phosphorylation [4]. Identification of the phosphorylation sites also permits to design constructs preventing undesired phosphorylations [5]. NMR and mass spectrometry (MS) are the methods of choice for probing protein phosphorylation, while other methods used to detect the same are Eastern blotting and 32P-radionucleotide incorporation [6–10,14]. In eukaryotes, phosphorylation of Ser and Thr are more common than that of Tyr and His. Mass spectrometry analysis reported a significant mass difference of 80 Da between the expected protein mass and kinase treated protein [2,3]. Histidine-tag (His-tag) is the most frequently used tag to label and purify recombinant proteins using affinity chromatography. The vector pET28a derived His-tag sequence, a construct often used from molecular and structural biology up to high-throughput methods, contains six serine residues in a 23 residues long tag sequence (“1MRGSHHHHHHGSHVISSIASRGS23”). When analyzing protein phosphorylation
*
events via mass spectrometric approaches, it appears that phosphorylation can take place not only on the protein of interest itself but also on vector-derived His-tag sequences. These modifications can thus lead to misinterpretation of the data with regard to physiological protein phosphorylation. In this study, MALDI-TOF-MS of N-terminal His-tagged full-length UVI31 + protein overexpressed under reported conditions [11,13] in E. coli cells gave a mass of 13,459 Da, a result in agreement with the theoretical mass of the protein (Fig. 1). The protein was incubated with protein kinase A for phosphorylation reaction and resulted a product containing singly phosphorylated form of modified mass 13,539 Da, with 80 Da (HPO3 = 80 Da) mass interval between the species, indicating single-site phosphorylation. When alkaline phosphatase was added to the phosphorylated protein mixture, the phosphorylated Histagged UVI31 + protein was converted back to its unphosphorylated form (Fig. S1). Analysis of His-tagged 15N and 13C/15N labeled UVI31 + purified from E. coli by NMR resulted in live monitoring of the phosphorylation events in a site-specific manner and chemical shift assignments were confirmed using standard backbone assignment experiments [12]. The phosphorylation site was determined as Ser23. As other sites of the Histag residues participate in electrostatic interactions after such modifications, significant chemical shift perturbations were observed also at Gly11, Ser12, His13, Met24, Ala25, Glu26 and His27 (Fig. 2). The phosphorylated site Ser23 shows the maximum shift perturbation of 0.68 ppm (Fig. S2).
Corresponding author. Department of Chemistry and Pharmacy, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, 81377, Munich, Germany. E-mail addresses: [email protected], [email protected] (H. Singh).
https://doi.org/10.1016/j.ab.2019.02.001 Received 25 December 2018; Received in revised form 3 February 2019; Accepted 4 February 2019 Available online 05 February 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
Analytical Biochemistry 570 (2019) 62–64
H. Singh, et al.
2D-[15N-1H]-nOe experiments were used to probe changes of dynamics on picosecond to nanosecond time scales of His-tagged regions of the protein with and without phosphorylation. This experiment indicated only reduced mobility in the His-tag regions after phosphorylation and did not affect the overall protein dynamics (Fig. S3). Molecular interactions of His-tagged charged residues with the phosphoryl group of serine 23 were mapped after performing MD simulations. The statistics on interactions between the oxygen atoms of the phosphoryl group of pSer23 and side-chain nitrogen (or N-terminal nitrogen for Gly11) atoms of charged residues in the N-terminal His-tag region of phosphorylated Ser23-UVI31 + in MD trajectories are shown in Fig. S4 and Table S1. We also confirmed that the endonuclease function of UVI31 + protein was unaffected after such modification of His-tag sequence (Fig. S5). Taken together, our results show that the recombinant protein derived from pET28a vector show phosphorylation at the His-tag sequence at serine 23 position and this site is dephosphorylated by the action of alkaline phosphatase. This result demonstrates that single-site phosphorylation at the tag sequence derived from the current commercial pET28a expression vectors can interfere with unambiguous phosphorylation detection in a protein of interest. Acknowledgments This manuscript is dedicated to Prof. K.V.R. Chary's 65th birth anniversary, IISER Berhampur, Odisha, India. HS would like to thank Prof. Dr. Rasmus Linser, Dr. Aracelys L Castilla and Dr. Anand K Das for critical reading of the manuscript. This work was supported by National Facility for High-Field NMR at Tata Institute of Fundamental Research, Mumbai and Raman Charpak Fellowship, CEFIPRA, New Delhi, India. Fig. 1. MALDI-TOF mass spectra of UVI31 + protein at different incubation times (0–9 h). 100 μM protein, 10 mM MgCl2, 1.5 mM ATP was incubated with 1 Unit of protein kinase A at pH 6.4 at 37 °C.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ab.2019.02.001. References [1] T. Hunter, Signaling—2000 and beyond, Cell 100 (2000) 113–127. [2] P. Du, P. Loulakis, C. Luo, A. Mistry, S.P. Simons, P.K. LeMotte, F. Rajamohan, K. Rafidi, K.G. Coleman, K.F. Geoghegan, Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function, Protein Expr. Purif. 44 (2005) 121–129. [3] R. Lomas-Lopez, A.J. Cozzone, B. Duclos, A modified His-tag vector for the production of recombinant protein kinases, Anal. Biochem. 377 (2008) 272–273. [4] L.N. Johnson, The regulation of protein phosphorylation, Biochem. Soc. Trans. 37 (2009) 627–641. [5] Y. Oda, K. Huang, F. Cross, D. Cowburn, B. Chait, Accurate quantitation of protein expression and site-specific phosphorylation, Proc. Natl. Acad. Sci. Unit. States Am. 96 (1999) 6591–6596. [6] F.-X. Theillet, H.M. Rose, S. Liokatis, A. Binolfi, R. Thongwichian, M. Stuiver, P. Selenko, Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts, Nat. Protoc. 8 (2013) 1416. [7] Y.-Q. Zhao, J. Yan, J.-B. Chao, A.-H. Liang, B.-S. Yang, The biochemical effect of Ser166 phosphorylation on Euplotes octocarinatus centrin, JBIC J. Biol. Inorgan. Chem. 18 (2013) 123–136. [8] M. Mann, S.-E. Ong, M. Grønborg, H. Steen, O.N. Jensen, A. Pandey, Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome, Trends Biotechnol. 20 (2002) 261–268. [9] I. Amata, M. Maffei, A. Igea, M. Gay, M. Vilaseca, A.R. Nebreda, M. Pons, Multiphosphorylation of the intrinsically disordered unique domain of c-src studied by in-cell and real-time NMR spectroscopy, Chembiochem 14 (2013) 1820–1827. [10] S. Liokatis, A. Dose, D. Schwarzer, P. Selenko, Simultaneous detection of protein phosphorylation and acetylation by high-resolution NMR spectroscopy, J. Am. Chem. Soc. 132 (2010) 14704–14705. [11] M. Shukla, R. Minda, H. Singh, S. Tirumani, K.V. Chary, B.J. Rao, UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii, PLoS One 7 (2012) e51913. [12] H. Singh, D. Verma, B. Rao, K.V. Chary, 1 H, 13 C and 15 N NMR assignments of Mg
Fig. 2. An overlay of a 2D [15N-1H] HSQC spectrum of the uniformly-15N-labelled His-tag containing UVI31+ (shown in blue) with phosphorylated His-tag UVI31+ (shown in red). The experiments were recorded at pH 6.4 and 310 K. The spectra were recorded on a Bruker Avance 800 MHz NMR spectrometer with 256 and 2048 complex points along t1 and t2 dimensions, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
63
Analytical Biochemistry 570 (2019) 62–64
H. Singh, et al. 2+ bound form of UV inducible transcript protein (UVI31+) from Chlamydomonas reinhardtii, Biomolecular NMR Assignments, vol. 9, 2015, pp. 93–97. [13] A.K. Rout, H. Singh, S. Patel, V. Raghvan, S. Gautam, R. Minda, B.J. Rao, K.V.R. Chary, Structural characterization of a novel KH-domain containing plant
chloroplast endonuclease, Sci. Rep. 8 (2018). [14] Y.Q. Zhao, J. Yan, J.B. Chao, A.H. Liang, B.S. Yang, The biochemical effect of Ser166 phosphorylation on Euplotes octocarinatus centrin, J. Biol. Inorg. Chem. 18 (1) (2013) 123–136.
64