- Email: [email protected]
Real-time monitoring of cell-free protein synthesis on a surface plasmon resonance chip
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 366 (2007) 170–174 www.elsevier.com/locate/yabio
Real-time monitoring of cell-free protein synthesis on a surface plasmon resonance chip Kyung-Ho Lee
a,1
, Hyou-Arm Joung b,1, Jin-Ho Ahn c, Kyeong-Ohn Kim a, In-Seok Oh c, Yong-Beom Shin b, Min-Gon Kim b,*, Dong-Myung Kim a,*
a Department of Fine Chemical Engineering and Chemistry, Chungnam National University, Daejeon 305-764, Korea BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon, Korea Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, Seoul 151-742, Korea b
c
Received 22 December 2006 Available online 3 May 2007
Abstract Taking advantage of the ‘‘open’’ nature of cell-free protein synthesis, this study investigated the direct analysis of protein expression using a surface plasmon resonance sensor. During the on-chip incubation of the reaction mixture for cell-free protein synthesis, the expressed protein molecules were immobilized onto the surface of the chip, giving rise to a sensorgram signal, which enabled on-line monitoring of protein expression. In addition, we found that the expression of the aggregation-prone proteins could be effectively monitored. The ability to monitor these proteins was most likely through the instant isolation of the expressed protein molecules onto the solid surface of the chip. Ó 2007 Elsevier Inc. All rights reserved. Keywords: SPR; Cell-free protein synthesis; Real-time monitoring
The techniques of cell-free protein synthesis are now readily accepted as a powerful tool for parallel expression of multiple proteins. In particular, recent progress in the direct expression of PCR-amplified genes [1,2] makes the idea of genome-scale protein expression more feasible. However, large-scale expression of proteins should be accompanied with a method for rapid and parallel analysis of the expressed proteins also. Unfortunately, due to the intensive requirements for time and labor, conventional electrophoretic and blotting techniques cannot be readily scaled-up for simultaneous handling of large numbers of proteins. In this respect, a method that enables the direct and instant analysis of protein expression is necessary to reach the high potential of cell-free protein synthesis as a platform to translate the genome-scale sequence information into their corresponding protein molecules. *
Corresponding authors. Fax: +82 42 879 8594 (M.-G. Kim), +82 42 823 7692 (D.-M. Kim). E-mail addresses: [email protected] (M.-G. Kim), dmkim@ cnu.ac.kr (D.-M. Kim). 1 These authors equally contributed to this work. 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.04.044
Several methods based on the measurement of fluorescence have been developed for in situ, real-time monitoring of protein expression. For example, fluorescence correlation spectroscopy has been used to monitor expression of the green fluorescent protein and its fusion proteins [3,4]. More recently, a real-time method based on binding-dependent fluorescence of metal ions (Lumio Technology, Invitrogen, Carlsbad, CA) has been commercialized for analysis of cell-free protein synthesis. However, applications of such protocols are limited by the requirements for large fusion partners or specific tags that selectively bind to expensive fluorescent probes. In addition, only soluble and/or properly folded protein accumulation can be monitored. In this study, we attempted a real-time measurement of protein expression by conducting cell-free synthesis reactions in a surface plasmon resonance (SPR)2 instrument. 2
Abbreviations used: SPR, surface plasmon resonance; PCR, polymerase chain reaction; NTA, nitrilotriacetic acid; SAM, self-assembled monolayer; MUA, 11-mercaptoundecanoicacid; TCA, trichloroacetic acid; NHS, N-hydroxysuccinimide; EGFP, enhanced green fluorescent protein.
Direct analysis of protein expression using a SPR chip / K.-H. Lee et al. / Anal. Biochem. 366 (2007) 170–174
The reaction mixture for cell-free protein synthesis was loaded onto a gold surface that had been modified to carry the Ni-NTA moieties. Since the plasmid construct added a 6x histidine tag at the C terminus of the expressed protein, cell-free synthesized protein molecules were instantly bound to the modified gold surface. A linear increase of the resonance signal was observed as the protein synthesis reaction proceeded. To the best of our knowledge, this is the first study to employ the SPR technology for on-line monitoring of protein synthesis. The presented method allows in situ analysis of protein expression in a simple and direct manner without any requirements for purification steps or fusion partners. Moreover, by use of a multiplexed SPR chip, the described method could be readily applied for rapid and parallel analysis of the expression of multiple proteins, laying the groundwork for global analysis of genome expression in vitro. Materials and methods
171
Preparation of Ni–NTA-coated SPR chips A self-assembled monolayer (SAM) of 11-mercaptoundecanoicacid (MUA) was formed on a bare gold surface (K-MAC, Korea) that had been cleaned in Piranha solution (75% H2SO4/25% H2O2, v/v). The Piranha-cleaned substrate was soaked in MUA solution (10 mM) for 12 h which resulted in formation of the SAM. The terminal carboxylic acid group of MUA was activated by placing the SAM-coated substrate in the aqueous solution of 0.1 M EDC/0.025 M NHS for 15 min. NHS-activated substrate was then transferred into the solution of 0.1 mg/ml amino dextran (Invitrogen; MW 500,000) in 10 mM phosphate buffer (pH 7.0) and incubated for 1 h. The substrate was then washed with distilled water and placed in 1 M ethanol amine (pH 8.5) for 15 min to block the NHS-activated carboxyl groups. For the introduction of a biotin group onto the substrate-bound dextran, the substrate was incubated with 0.1 mg/ml of NHS–biotin (Pierce, Rockford, IL) for 30 min in phosphate–buffered saline solution. After being
Materials
In the experiments for the real-time analysis of cell-free protein synthesis, open reading frames of the target proteins were cloned into the plasmid pIVEX2.3d (Roche Diagnostics, Indianapolis, IN) and used as the templates for protein synthesis. The standard reaction mixture for cell-free protein synthesis consisted of the following components in a total volume of 50 ll; 57 mM Hepes–KOH (pH 8.2), 1.2 mM ATP, 0.85 mM each GTP, UTP, and CTP, 1.7 mM dithiothreitol, 80 mM ammonium acetate, 0.17 mg/ml E. coli total tRNA mixture (from strain MRE 600), 34 lg/ml L-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid), 1.5 mM each 20 amino acids, 0.3 U/ml creatine kinase, 67 mM creatine phosphate, 0.3 lg of plasmid, and 13 ll of S30 extract. Protein synthesis reactions were conducted at 37 °C for 2 h. The synthesized EGFP was quantified by measuring the TCA-precipitable radioactivities as described in our previous report [5]. Molecular weight of the expressed protein was confirmed on a 13% Tricine–SDS–polyacrylamide gel [6]. In the experiments for EGFP, the fluorescence from the synthesized EGFP was quantified using the GenePix 4200A (Molecular Devices, CA) fluorescence spectrophotometer.
3000 2500
Angle shift (mD)
Cell-free protein synthesis reactions
A
2000 1500
2400 No DNA
EGFP
1000 500
1900 0
0 0
500
1000 1000
1500
2000
Time (sec)
B 100
Synthesized EGFP (µg/ml)
ATP, GTP, UTP, CTP, creatine phosphate, creatine kinase, and Escherichia coli total tRNA mix were purchased from Roche Applied Science (Indianapolis, IN). All other reagents were purchased from Sigma (St. Louis, MO). The S30 extracts were prepared from the strain BL21-Star (DE3) (Invitrogen, Carlsbad, CA) according to the procedures described earlier [2,5].
50
0 0
500
1000
1500
2000
Time (sec)
Fig. 1. Real-time monitoring of cell-free expressed EGFP molecules. (A) Solid line, on-chip cell-free expression of the plasmid pIVEX2.3d-EGFP; dotted line, control reaction without DNA. The inset shows fluorescence images of the incubated reaction mixture with and without DNA. (B) Cellfree expression of the plasmid pIVEX2.3d-EGFP in a conventional batch cell-free synthesis reaction.
172
Direct analysis of protein expression using a SPR chip / K.-H. Lee et al. / Anal. Biochem. 366 (2007) 170–174
washed with distilled water, the substrate was incubated in 0.1 mg/ml of avidin (NeutrAvidin; Pierce) solution for 30 min. Finally, for the introduction of Ni-nitrilotriacetic acid onto the surface, the substrate was sequentially soaked in a 10-lg/ml solution of biotin-X–NTA (Invitrogen) and 50 mM solution of NiCl2 for 15 min each. Real-time monitoring of cell-free protein synthesis on a SPR chip A Ni–NTA-coated SPR chip was fixed on a half-cylinder prism with refractive-index matching oil (nD = 1.517) and installed in the AutoLab Esprit instrument (Eco Chemie, Utrecht, Netherlands). A complete reaction mixture for cell-free protein synthesis reaction (50 ll) was loaded onto the chip and incubated at 37 °C without mixing. Time-dependent change in the angle shift was monitored throughout the incubation period.
Results and discussion Real-time measurement of protein accumulation during cellfree protein synthesis on a SPR chip One hundred microliters of the reaction mixture for cellfree expression of the plasmid pIVEX2.3d-EGFP was prepared as described under Materials and methods. Half of the complete reaction mixture was incubated in a water bath with intermittent samplings, while the other half was incubated on a SPR chip installed in the Esprit instrument. The synthesis reactions were conducted at 37 °C in both cases. As shown in the sensorgram (Fig. 1A), the angle shift continued to increase, indicating that the cell-free expressed EGFP molecules were successfully immobilized onto the
Cell-free protein synthesis on a SPR imaging chip The Piranha-cleaned gold surface was soaked in biotin– HPDP (Pierce) solution (0.05 mM in ethanol) for 12 h which resulted in formation of the SAM. After being washed with ethanol and distilled water, the substrate was incubated in 0.1 mg/ml of avidin (Pierce) solution for 30 min. Finally, for the introduction of Ni–NTA onto the surface, the substrate was sequentially soaked in a 10 lg/ml solution of biotin-X–NTA (Invitrogen) and 50 mM solution of NiCl2 for 15 min each. The Ni–NTA-coated SPR chip was washed with distilled water and dried by N2 gas. Complete reaction mixtures for cell-free protein synthesis reaction (0.2 ll) were spotted onto the chip and incubated at 37 °C for 40 min. The chip was then sequentially washed with phosphate buffer (0.1 M, pH 7.0) containing 0.15 M sodium chloride and distilled water. After being dried by N2 gas, the chip was loaded onto a SPR imaging instrument (SPRi; K-MAC, Korea).
A
Fig. 3. SPR imaging analysis comparing the binding affinity of the separately expressed protein and on-chip expressed AD transaminase molecules. (A) The reaction mixture for cell-free expression of the AD transaminase was loaded onto the SPR chip after 2 h of separate incubation. (B) The AD transaminase was expressed on the SPR chip for the in situ isolation of the expressed protein onto the chip surface. A reaction mixture without DNA was used for a control reaction.
B 2500
40 kDa 30 kDa 20 kDa 15 kDa
S
P
2000
Angle shift (mD)
Markers
2200
1500
1000
500
1900 500
2000
0 0
2000
4000
6000
8000
Time (sec)
Fig. 2. On-chip synthesis and analysis of an aggregation-prone protein. (A) The plasmid pIVEX2.3d-AD was incubated in the reaction mixture for cellfree protein synthesis for 2 h at 37 °C. After centrifugation (12,000 RCF) for 15 min, pellet (P) and supernatant (S) fractions were analyzed by Western blotting. (B) Solid line, on-chip cell-free protein synthesis of the AD transaminase; dotted line, SPR analysis of AD transaminase after a separate expression.
Direct analysis of protein expression using a SPR chip / K.-H. Lee et al. / Anal. Biochem. 366 (2007) 170–174
A
B
No DNA
TrpA
TrpB
I L-2
TNF-
p24
41(O)-1
EGFP
TrpA
TrpB
I L-2
S
S
S
P
P
p24
TNFP
S
P
S
P
41(O)-1
EGFP
S
S
P
173
P
Fig. 4. SPR imaging analysis of multiple proteins expressed on-chip. (A) SPR images after incubation of the reaction mixtures on a SPR chip. (B) Western blotting analysis. A reaction mixture without DNA was used for a control reaction.
surface of the SPR chip. After 30 min of incubation the angle shift in the plasmid-containing reaction reached approximately 572.6 mD, while that of the control reaction (without DNA) did not exceed 84.2 mD. The kinetics of protein accumulation correlated well with the result of the conventional reaction, in which the accumulation of the expressed protein was estimated by measuring the TCAinsoluble radioactivity of the reaction samples (Fig. 1B). Expression of correctly folded proteins was confirmed using the fluorescence of the completed reactions. The background angle shift in the control reaction is likely to be caused by the time-dependent deposition of endogenous proteins in the cell extract. We previously reported the presence of several contaminating proteins in the E. coli S30 extract that non specifically bind to the Ni–NTA-treated surface [7]. Nevertheless, the angle shift due to binding of the expressed proteins was high enough to monitor the progress of the cell-free protein synthesis reaction. In situ binding onto the chip surface enables the detection of aggregation-prone proteins We initially expected that the method of on-chip analysis of protein synthesis would be applicable only to the proteins that were solubly expressed in our cell-free synthesis system because the aggregated proteins would fail to bind onto the chip surface. To test this hypothesis, we expressed a transaminase cloned from Alcaligenes denitrificans (AD) to be used as a representative insoluble protein in our collection (Fig. 2A). Interestingly, a linear increase of the sensorgram signal in a pattern similar to that obtained in the expression of EGFP was observed (Fig. 2B). We reasoned that these
results may be due to the instant immobilization of the expressed proteins onto the chip surface, which would prevent them from aggregating to each other. Conversely, when the SPR chip was loaded with the reaction mixture in which the AD transaminase had been separately expressed, the increase of the SPR angle shift was minimal (Fig. 2B), which supports our hypothesis that the in situ expression/isolation process facilitated the detection of the insoluble protein molecules through binding of the nascent polypeptide onto the chip surface. The same phenomenon was observed when the protein synthesis reaction was analyzed using SPR imaging. SPR imaging utilizes collimated illumination of the entire surface, with the reflected beam imaged onto a two-dimensional array detector [8]. While sequential expression and SPR image analysis fail to allow the detection of the expressed proteins (Fig. 3A), the cell-free synthesis reaction conducted on the chip resulted in a clear image of protein deposition (Fig. 3B). SPR imaging techniques enable parallel analyses of the expression of multiple proteins. As shown in Fig. 4A, we were able to analyze the expression of seven different proteins on a single chip. Although the solubility of the proteins expressed in solution phase varied depending upon the proteins (Fig. 4B), all of the examined proteins gave clear imaging signals when they were expressed on the SPR chip. Conclusions In this study, we found that accumulation of cell-free synthesized proteins can be directly monitored by conducting the synthesis reaction on an appropriately modified surface of a SPR chip. Time-dependent increase of the SPR angle shift was observed in the reactions programmed with
174
Direct analysis of protein expression using a SPR chip / K.-H. Lee et al. / Anal. Biochem. 366 (2007) 170–174
different plasmids. Moreover, it was found that the described on-chip analysis protocols enabled monitoring of the expression of aggregation-prone proteins, most likely through the instant binding of the nascent polypeptides onto the chip surface. We believe that the combination of the flexibility of cellfree synthesis coupled with the sensitivity and throughput of SPR technology will provide an excellent platform for large-scale functional analysis of gene products. Acknowledgments Authors gratefully acknowledge the financial support from the Center for Advanced Bioseparation Technology, Inha University, Korea and the KRIBB Research Initiative Program. References [1] P. Angenendt, J. Kreutzberger, J. Glo¨kler, J.D. Hoheisel, Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR, J. Proteome Res. 5 (2006) 1658–1666.
[2] J.H. Ahn, H.S. Chu, T.W. Kim, I.S. Oh, C.Y. Choi, G.H. Hahn, C.G. Park, D.M. Kim, Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions, Biochem. Biophys. Res. Commun. 338 (2005) 1346–1352. [3] Y. Nomura, H. Tanaka, L. Poellinger, F. Higashino, M. Kinjo, Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy, Cytometry 44 (2001) 1–6. [4] M. Chalfie, Y. Tu, G. Euskirchen, W.W. Ward, D.C. Prasher, Green Fluorescent protein as a marker for gene expression, Science 263 (1994) 802–805. [5] D.M. Kim, T. Kigawa, C.Y. Choi, S. Yokoyama, A highly efficient cell-free protein synthesis system from Escherichia coli, Eur. J. Biochem. 239 (1996) 881–886. [6] H. Schagger, G. von Jagow, Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa, Anal. Biochem. 166 (1987) 368–379. [7] T.W. Kim, I.S. Oh, J.H. Ahn, C.Y. Choi, D.M. Kim, Cell-free synthesis and in situ isolation of recombinant proteins, Protein Expr. Purif. 45 (2006) 249–254. [8] S.O. Jung, H.S. Ro, B.H. Kho, Y.B. Shin, M.G. Kim, B.H. Chung, Surface plasmon resonance imaging-based protein arrays for highthroughput screening of protein-protein interaction inhibitors, Proteomics 5 (2005) 4427–4431.
Real-time monitoring of cell-free protein synthesis on a surface plasmon resonance chip Kyung-Ho Lee
a,1
, Hyou-Arm Joung b,1, Jin-Ho Ahn c, Kyeong-Ohn Kim a, In-Seok Oh c, Yong-Beom Shin b, Min-Gon Kim b,*, Dong-Myung Kim a,*
a Department of Fine Chemical Engineering and Chemistry, Chungnam National University, Daejeon 305-764, Korea BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon, Korea Interdisciplinary Program for Biochemical Engineering and Biotechnology, College of Engineering, Seoul National University, Seoul 151-742, Korea b
c
Received 22 December 2006 Available online 3 May 2007
Abstract Taking advantage of the ‘‘open’’ nature of cell-free protein synthesis, this study investigated the direct analysis of protein expression using a surface plasmon resonance sensor. During the on-chip incubation of the reaction mixture for cell-free protein synthesis, the expressed protein molecules were immobilized onto the surface of the chip, giving rise to a sensorgram signal, which enabled on-line monitoring of protein expression. In addition, we found that the expression of the aggregation-prone proteins could be effectively monitored. The ability to monitor these proteins was most likely through the instant isolation of the expressed protein molecules onto the solid surface of the chip. Ó 2007 Elsevier Inc. All rights reserved. Keywords: SPR; Cell-free protein synthesis; Real-time monitoring
The techniques of cell-free protein synthesis are now readily accepted as a powerful tool for parallel expression of multiple proteins. In particular, recent progress in the direct expression of PCR-amplified genes [1,2] makes the idea of genome-scale protein expression more feasible. However, large-scale expression of proteins should be accompanied with a method for rapid and parallel analysis of the expressed proteins also. Unfortunately, due to the intensive requirements for time and labor, conventional electrophoretic and blotting techniques cannot be readily scaled-up for simultaneous handling of large numbers of proteins. In this respect, a method that enables the direct and instant analysis of protein expression is necessary to reach the high potential of cell-free protein synthesis as a platform to translate the genome-scale sequence information into their corresponding protein molecules. *
Corresponding authors. Fax: +82 42 879 8594 (M.-G. Kim), +82 42 823 7692 (D.-M. Kim). E-mail addresses: [email protected] (M.-G. Kim), dmkim@ cnu.ac.kr (D.-M. Kim). 1 These authors equally contributed to this work. 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.04.044
Several methods based on the measurement of fluorescence have been developed for in situ, real-time monitoring of protein expression. For example, fluorescence correlation spectroscopy has been used to monitor expression of the green fluorescent protein and its fusion proteins [3,4]. More recently, a real-time method based on binding-dependent fluorescence of metal ions (Lumio Technology, Invitrogen, Carlsbad, CA) has been commercialized for analysis of cell-free protein synthesis. However, applications of such protocols are limited by the requirements for large fusion partners or specific tags that selectively bind to expensive fluorescent probes. In addition, only soluble and/or properly folded protein accumulation can be monitored. In this study, we attempted a real-time measurement of protein expression by conducting cell-free synthesis reactions in a surface plasmon resonance (SPR)2 instrument. 2
Abbreviations used: SPR, surface plasmon resonance; PCR, polymerase chain reaction; NTA, nitrilotriacetic acid; SAM, self-assembled monolayer; MUA, 11-mercaptoundecanoicacid; TCA, trichloroacetic acid; NHS, N-hydroxysuccinimide; EGFP, enhanced green fluorescent protein.
Direct analysis of protein expression using a SPR chip / K.-H. Lee et al. / Anal. Biochem. 366 (2007) 170–174
The reaction mixture for cell-free protein synthesis was loaded onto a gold surface that had been modified to carry the Ni-NTA moieties. Since the plasmid construct added a 6x histidine tag at the C terminus of the expressed protein, cell-free synthesized protein molecules were instantly bound to the modified gold surface. A linear increase of the resonance signal was observed as the protein synthesis reaction proceeded. To the best of our knowledge, this is the first study to employ the SPR technology for on-line monitoring of protein synthesis. The presented method allows in situ analysis of protein expression in a simple and direct manner without any requirements for purification steps or fusion partners. Moreover, by use of a multiplexed SPR chip, the described method could be readily applied for rapid and parallel analysis of the expression of multiple proteins, laying the groundwork for global analysis of genome expression in vitro. Materials and methods
171
Preparation of Ni–NTA-coated SPR chips A self-assembled monolayer (SAM) of 11-mercaptoundecanoicacid (MUA) was formed on a bare gold surface (K-MAC, Korea) that had been cleaned in Piranha solution (75% H2SO4/25% H2O2, v/v). The Piranha-cleaned substrate was soaked in MUA solution (10 mM) for 12 h which resulted in formation of the SAM. The terminal carboxylic acid group of MUA was activated by placing the SAM-coated substrate in the aqueous solution of 0.1 M EDC/0.025 M NHS for 15 min. NHS-activated substrate was then transferred into the solution of 0.1 mg/ml amino dextran (Invitrogen; MW 500,000) in 10 mM phosphate buffer (pH 7.0) and incubated for 1 h. The substrate was then washed with distilled water and placed in 1 M ethanol amine (pH 8.5) for 15 min to block the NHS-activated carboxyl groups. For the introduction of a biotin group onto the substrate-bound dextran, the substrate was incubated with 0.1 mg/ml of NHS–biotin (Pierce, Rockford, IL) for 30 min in phosphate–buffered saline solution. After being
Materials
In the experiments for the real-time analysis of cell-free protein synthesis, open reading frames of the target proteins were cloned into the plasmid pIVEX2.3d (Roche Diagnostics, Indianapolis, IN) and used as the templates for protein synthesis. The standard reaction mixture for cell-free protein synthesis consisted of the following components in a total volume of 50 ll; 57 mM Hepes–KOH (pH 8.2), 1.2 mM ATP, 0.85 mM each GTP, UTP, and CTP, 1.7 mM dithiothreitol, 80 mM ammonium acetate, 0.17 mg/ml E. coli total tRNA mixture (from strain MRE 600), 34 lg/ml L-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid), 1.5 mM each 20 amino acids, 0.3 U/ml creatine kinase, 67 mM creatine phosphate, 0.3 lg of plasmid, and 13 ll of S30 extract. Protein synthesis reactions were conducted at 37 °C for 2 h. The synthesized EGFP was quantified by measuring the TCA-precipitable radioactivities as described in our previous report [5]. Molecular weight of the expressed protein was confirmed on a 13% Tricine–SDS–polyacrylamide gel [6]. In the experiments for EGFP, the fluorescence from the synthesized EGFP was quantified using the GenePix 4200A (Molecular Devices, CA) fluorescence spectrophotometer.
3000 2500
Angle shift (mD)
Cell-free protein synthesis reactions
A
2000 1500
2400 No DNA
EGFP
1000 500
1900 0
0 0
500
1000 1000
1500
2000
Time (sec)
B 100
Synthesized EGFP (µg/ml)
ATP, GTP, UTP, CTP, creatine phosphate, creatine kinase, and Escherichia coli total tRNA mix were purchased from Roche Applied Science (Indianapolis, IN). All other reagents were purchased from Sigma (St. Louis, MO). The S30 extracts were prepared from the strain BL21-Star (DE3) (Invitrogen, Carlsbad, CA) according to the procedures described earlier [2,5].
50
0 0
500
1000
1500
2000
Time (sec)
Fig. 1. Real-time monitoring of cell-free expressed EGFP molecules. (A) Solid line, on-chip cell-free expression of the plasmid pIVEX2.3d-EGFP; dotted line, control reaction without DNA. The inset shows fluorescence images of the incubated reaction mixture with and without DNA. (B) Cellfree expression of the plasmid pIVEX2.3d-EGFP in a conventional batch cell-free synthesis reaction.
172
Direct analysis of protein expression using a SPR chip / K.-H. Lee et al. / Anal. Biochem. 366 (2007) 170–174
washed with distilled water, the substrate was incubated in 0.1 mg/ml of avidin (NeutrAvidin; Pierce) solution for 30 min. Finally, for the introduction of Ni-nitrilotriacetic acid onto the surface, the substrate was sequentially soaked in a 10-lg/ml solution of biotin-X–NTA (Invitrogen) and 50 mM solution of NiCl2 for 15 min each. Real-time monitoring of cell-free protein synthesis on a SPR chip A Ni–NTA-coated SPR chip was fixed on a half-cylinder prism with refractive-index matching oil (nD = 1.517) and installed in the AutoLab Esprit instrument (Eco Chemie, Utrecht, Netherlands). A complete reaction mixture for cell-free protein synthesis reaction (50 ll) was loaded onto the chip and incubated at 37 °C without mixing. Time-dependent change in the angle shift was monitored throughout the incubation period.
Results and discussion Real-time measurement of protein accumulation during cellfree protein synthesis on a SPR chip One hundred microliters of the reaction mixture for cellfree expression of the plasmid pIVEX2.3d-EGFP was prepared as described under Materials and methods. Half of the complete reaction mixture was incubated in a water bath with intermittent samplings, while the other half was incubated on a SPR chip installed in the Esprit instrument. The synthesis reactions were conducted at 37 °C in both cases. As shown in the sensorgram (Fig. 1A), the angle shift continued to increase, indicating that the cell-free expressed EGFP molecules were successfully immobilized onto the
Cell-free protein synthesis on a SPR imaging chip The Piranha-cleaned gold surface was soaked in biotin– HPDP (Pierce) solution (0.05 mM in ethanol) for 12 h which resulted in formation of the SAM. After being washed with ethanol and distilled water, the substrate was incubated in 0.1 mg/ml of avidin (Pierce) solution for 30 min. Finally, for the introduction of Ni–NTA onto the surface, the substrate was sequentially soaked in a 10 lg/ml solution of biotin-X–NTA (Invitrogen) and 50 mM solution of NiCl2 for 15 min each. The Ni–NTA-coated SPR chip was washed with distilled water and dried by N2 gas. Complete reaction mixtures for cell-free protein synthesis reaction (0.2 ll) were spotted onto the chip and incubated at 37 °C for 40 min. The chip was then sequentially washed with phosphate buffer (0.1 M, pH 7.0) containing 0.15 M sodium chloride and distilled water. After being dried by N2 gas, the chip was loaded onto a SPR imaging instrument (SPRi; K-MAC, Korea).
A
Fig. 3. SPR imaging analysis comparing the binding affinity of the separately expressed protein and on-chip expressed AD transaminase molecules. (A) The reaction mixture for cell-free expression of the AD transaminase was loaded onto the SPR chip after 2 h of separate incubation. (B) The AD transaminase was expressed on the SPR chip for the in situ isolation of the expressed protein onto the chip surface. A reaction mixture without DNA was used for a control reaction.
B 2500
40 kDa 30 kDa 20 kDa 15 kDa
S
P
2000
Angle shift (mD)
Markers
2200
1500
1000
500
1900 500
2000
0 0
2000
4000
6000
8000
Time (sec)
Fig. 2. On-chip synthesis and analysis of an aggregation-prone protein. (A) The plasmid pIVEX2.3d-AD was incubated in the reaction mixture for cellfree protein synthesis for 2 h at 37 °C. After centrifugation (12,000 RCF) for 15 min, pellet (P) and supernatant (S) fractions were analyzed by Western blotting. (B) Solid line, on-chip cell-free protein synthesis of the AD transaminase; dotted line, SPR analysis of AD transaminase after a separate expression.
Direct analysis of protein expression using a SPR chip / K.-H. Lee et al. / Anal. Biochem. 366 (2007) 170–174
A
B
No DNA
TrpA
TrpB
I L-2
TNF-
p24
41(O)-1
EGFP
TrpA
TrpB
I L-2
S
S
S
P
P
p24
TNFP
S
P
S
P
41(O)-1
EGFP
S
S
P
173
P
Fig. 4. SPR imaging analysis of multiple proteins expressed on-chip. (A) SPR images after incubation of the reaction mixtures on a SPR chip. (B) Western blotting analysis. A reaction mixture without DNA was used for a control reaction.
surface of the SPR chip. After 30 min of incubation the angle shift in the plasmid-containing reaction reached approximately 572.6 mD, while that of the control reaction (without DNA) did not exceed 84.2 mD. The kinetics of protein accumulation correlated well with the result of the conventional reaction, in which the accumulation of the expressed protein was estimated by measuring the TCAinsoluble radioactivity of the reaction samples (Fig. 1B). Expression of correctly folded proteins was confirmed using the fluorescence of the completed reactions. The background angle shift in the control reaction is likely to be caused by the time-dependent deposition of endogenous proteins in the cell extract. We previously reported the presence of several contaminating proteins in the E. coli S30 extract that non specifically bind to the Ni–NTA-treated surface [7]. Nevertheless, the angle shift due to binding of the expressed proteins was high enough to monitor the progress of the cell-free protein synthesis reaction. In situ binding onto the chip surface enables the detection of aggregation-prone proteins We initially expected that the method of on-chip analysis of protein synthesis would be applicable only to the proteins that were solubly expressed in our cell-free synthesis system because the aggregated proteins would fail to bind onto the chip surface. To test this hypothesis, we expressed a transaminase cloned from Alcaligenes denitrificans (AD) to be used as a representative insoluble protein in our collection (Fig. 2A). Interestingly, a linear increase of the sensorgram signal in a pattern similar to that obtained in the expression of EGFP was observed (Fig. 2B). We reasoned that these
results may be due to the instant immobilization of the expressed proteins onto the chip surface, which would prevent them from aggregating to each other. Conversely, when the SPR chip was loaded with the reaction mixture in which the AD transaminase had been separately expressed, the increase of the SPR angle shift was minimal (Fig. 2B), which supports our hypothesis that the in situ expression/isolation process facilitated the detection of the insoluble protein molecules through binding of the nascent polypeptide onto the chip surface. The same phenomenon was observed when the protein synthesis reaction was analyzed using SPR imaging. SPR imaging utilizes collimated illumination of the entire surface, with the reflected beam imaged onto a two-dimensional array detector [8]. While sequential expression and SPR image analysis fail to allow the detection of the expressed proteins (Fig. 3A), the cell-free synthesis reaction conducted on the chip resulted in a clear image of protein deposition (Fig. 3B). SPR imaging techniques enable parallel analyses of the expression of multiple proteins. As shown in Fig. 4A, we were able to analyze the expression of seven different proteins on a single chip. Although the solubility of the proteins expressed in solution phase varied depending upon the proteins (Fig. 4B), all of the examined proteins gave clear imaging signals when they were expressed on the SPR chip. Conclusions In this study, we found that accumulation of cell-free synthesized proteins can be directly monitored by conducting the synthesis reaction on an appropriately modified surface of a SPR chip. Time-dependent increase of the SPR angle shift was observed in the reactions programmed with
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different plasmids. Moreover, it was found that the described on-chip analysis protocols enabled monitoring of the expression of aggregation-prone proteins, most likely through the instant binding of the nascent polypeptides onto the chip surface. We believe that the combination of the flexibility of cellfree synthesis coupled with the sensitivity and throughput of SPR technology will provide an excellent platform for large-scale functional analysis of gene products. Acknowledgments Authors gratefully acknowledge the financial support from the Center for Advanced Bioseparation Technology, Inha University, Korea and the KRIBB Research Initiative Program. References [1] P. Angenendt, J. Kreutzberger, J. Glo¨kler, J.D. Hoheisel, Generation of high density protein microarrays by cell-free in situ expression of unpurified PCR, J. Proteome Res. 5 (2006) 1658–1666.
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