Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position

Daisuke Kajihara1,2, Takahiro Hohsaka2,3,4 and Masahiko Sisido1,4

1Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, 2School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 and 3PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan

4 To whom correspondence should be addressed. E-mail: hohsaka{at}jaist.ac.jp; sisido{at}cc.okayama-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.

Keywords: cell-free translation/four-base codon/green fluorescent protein/non-natural amino acid/random mutation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Position-specific incorporation of non-natural amino acids into proteins is useful for creating artificial proteins with specialty functions. Techniques for synthesizing non-natural mutant proteins have been developed and the mutants are finding a wide range of applications in chemistry and biology (Noren et al., 1989Go; Arslan et al., 1997Go; Kanamori et al., 1999Go; Beene et al., 2003Go; Zhang et al., 2004Go). We have developed a technique for efficient incorporation of multiple non-natural amino acids by using four-base codon/anticodon pairs (Hohsaka et al., 1996Go, 1999Go, 2001Go) and applied the technique to various proteins by introducing non-natural amino acids of various specialty side groups (Murakami et al., 1998Go; Taki et al., 2001Go; Muranaka et al., 2002Go; Hohsaka et al., 2004Go).

In this study, non-natural mutants of green fluorescent protein (GFP, Figure 1a) were synthesized by position-specific incorporation of non-natural amino acids (Figure 1b). GFP and its variants have been widely used as useful biological tools (Cubitt et al., 1995Go). GFP contains an intrinsic Ser65–Tyr66–Gly67 sequence, which forms a chromophore after intermolecular cyclization and subsequent oxidation. Various GFP mutants that show different fluorescent properties have been reported (Heim et al., 1994Go, 1995Go; Heim and Tsien, 1996Go; Wachter et al., 1997Go). In particular, substitutions of Tyr66 by histidine and tryptophan resulted in blue-shifted fluorescent proteins BFP and CFP (Heim et al., 1994Go; Wachter et al., 1997Go). These findings raise the possibility of obtaining alternative fluorescent proteins by incorporating aromatic non-natural amino acids into the Tyr66 position of GFP. This possibility was suggested by Wachter et al. (1998)Go and some GFP mutants containing tyrosine and tryptophan analogues have been synthesized in Escherichia coli cells (Bae et al., 2003Go; Wang et al., 2003Go).



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Fig. 1. (a) Three-dimensional structure of GFP (PDB: 1EMA). Chromophores formed with Ser65–Tyr66–Gly67 and Tyr145 are represented in stick model form. Non-natural amino acids were incorporated in the Tyr66 position. (b) Structure of aromatic non-natural amino acids used in this study.

 
Here, we report the incorporation of different types of aromatic non-natural amino acids into the Tyr66 position in an E.coli cell-free translation system and the acceptability of the non-natural mutation to GFP. In addition, we developed a method for natural mutations around a non-natural amino acid to improve its adaptation. In previous studies, incorporation of non-natural amino acids sometimes caused a decrease or disappearance of the original activity of target proteins. Non-natural residues may disrupt the microenvironment of proteins or destroy the whole protein structures. One solution to this problem is to incorporate non-natural amino acids into selected positions where the original activity is maintained. In our previous studies, non-natural amino acids were incorporated into various positions of streptavidin and horseradish peroxidase and then the proteins that show both desired functions were selected (Murakami et al., 1998Go; Taki et al., 2001Go; Muranaka et al., 2002Go; Hohsaka et al., 2004Go). On the other hand, when a non-natural amino acid should be introduced into a specific position (e.g. Tyr66 of GFP), optimization of the amino acid sequence around the non-natural residue will be effective.

In this study, Ser65 and Tyr145, located around Tyr66, were randomly mutated to improve the adaptability of the non-natural amino acid and the fluorescence properties of the GFP mutant. These residues have been found to be sensitive to the fluorescence properties of the wild-type and blue-shifted GFPs (Tsien, 1998Go; Yang et al., 1998Go; Cubitt et al., 1999Go). To carry out the random mutations, a method for cell-free expression of non-natural proteins with natural random mutations was developed. This method will be useful not only for non-natural GFP mutants, but also for a variety of non-natural proteins in which the inherent functions are suppressed by the introduction of non-natural amino acid.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Preparation of tRNACCCG aminoacylated with non-natural amino acids

Transfer RNAs containing a CCCG anticodon and aminoacylated with 19 kinds of non-natural amino acids were synthesized by a chemical aminoacylation technique as described previously (Hohsaka et al., 1999Go). A truncated tRNA that contained a CCCG anti-codon and lacked a CA dinucleotide unit at the 3'-terminus was prepared by using T7 RNA polymerase. The resulting tRNACCCG(-CA) was ligated with aminoacyl-pdCpAs by using T4 RNA ligase.

Preparation of mutant genes encoding GFP mutants containing a four-base codon

The coding region of GFP was obtained from pGFPuv (Clontech) and inserted into an expression vector containing T7 promoter and T7-tag sequence at the N-terminus (Hohsaka et al., 1999Go). Histidine-tag sequence was added to the C-terminus for purification and a single CGG codon at Arg80 position was replaced by a CGT codon. Then, a CGGG four-base codon was introduced into the Tyr66 position.

Cell-free synthesis and fluorescence measurement of GFP mutants containing a non-natural amino acid at the Tyr66 position

GFP mRNA containing CGGG four-base codon at the Tyr66 position was prepared using T7 RNA polymerase and added to an E.coli cell-free translation system together with tRNACCCG charged with non-natural amino acids. A 10 µl reaction mixture contained 55 mM HEPES–KOH (pH 7.5), 210 mM potassium glutamate, 6.9 mM ammonium acetate, 12 mM magnesium acetate, 1.7 mM DTT, 1.2 mM ATP, 0.28 mM GTP, 26 mM phosphoenolpyruvate, 1 mM spermidine, 1.9% poly(ethylene glycol) 8000, 35 µg/ml folinic acid, 0.1 mM each of amino acids except arginine, 0.01 mM arginine, 16 µg of the mRNA, 0.1 nmol of the aminoacyl-tRNACCCG and 2 µl of E.coli S30 extract (Promega, S30 extract for linear template). After incubation at 37°C for 60 min, expression of full-length GFP mutants was confirmed by western blot analysis using anti-T7 tag antibody (Novagen) and alkaline phosphatase-labelled anti-mouse IgG. The translation reaction mixture (5 µl) was diluted with 20 mM Tris–HCl, 150 mM NaCl, pH 7.5 (320 µl) and fluorescence spectra were measured at 25°C on a Spex-Jobin-Yvon Fluoromax-2 instrument. A translation reaction mixture without mRNA and tRNACCCG was also prepared and used as a baseline.

Construction of library of mutant GFP genes by random mutation

The Ser65 and Tyr145 positions of mutant GFP gene containing CGGG codon at Tyr66 position were replaced by random codon NNK (K = G or T) by using random mutation primers GAAAAGCATTGAACACCCCCGMNNGAAAGTAGTGACAAGTGTTGG (M = A or C) for Ser65 and GAAAAGCATTGAACACCCCCGMNNGAAAGTAGTGACAAGTGTTGG for Tyr145. The mutated genes were recloned into the same vector, with which E.coli DH10B was transformed and cultivated overnight on a SOB plate.

Expression and fluorescence measurement of random mutants containing p-aminophenylalanine

Forty-two colonies were picked up, suspended in PCR mixtures and then PCR was carried out to amplify the coding region of GFP mutant. The PCR mixtures (2 µl) were directly added to a cell-free transcription/translation reaction mixture (5 µl) that contained 55 mM HEPES–KOH (pH 7.5), 210 mM potassium glutamate, 6.9 mM ammonium acetate, 12 mM magnesium acetate, 1.7 mM DTT, 1.2 mM ATP, 0.28 mM GTP, 26 mM phosphoenolpyruvate, 1 mM spermidine, 1.9% poly(ethylene glycol) 8000, 35 µg/ml folinic acid, 0.1 mM each of amino acids except arginine, 0.6 µM arginine, 1.25 mM of NTPs and 50 units of T7 RNA polymerase. After incubation at 37°C for 60 min, p-aminophenylalanyl-tRNACCCG (0.05 nmol) and E.coli S30 extract (1 µl) were added to the reaction mixture and incubated at 37°C for 60 min. The reaction mixture (5 µl) was diluted with 20 mM Tris–HCl, 150 mM NaCl, pH 7.5 (320 µl) and fluorescence spectra were measured at 25°C.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Incorporation of various non-natural amino acids into GFP at the Tyr66 position using an E.coli cell-free translation system

GFP mutants Y66W and Y66H emit blue-shifted fluorescence compared with wild-type GFP (Heim et al., 1994Go; Wachter et al., 1997Go). The blue shift has been interpreted as the result of a breakdown change of {pi} conjugation of the chromophore caused by the replacement of a phenol group with an indole or imidazole group. However, the substitution of Tyr66 with natural aromatic amino acids has been limited to Trp, His and Phe. Here, we attempted to incorporate non-natural amino acids carrying various aromatic rings to produce novel fluorescent proteins. Previously we reported that various kinds of non-natural amino acids with large aromatic side groups can be incorporated into streptavidin in an E.coli cell-free translation system (Hohsaka et al., 1999Go). Incorporation of these non-natural amino acids into Tyr66 of GFP will give alternative {pi} conjugate systems which emit different fluorescent colours. Non-natural amino acids used in this study are shown in Figure 1b. Introduction of the expanded {pi} conjugate systems will produce a red-shifted chromophore, although these large residues may retard or inhibit the chromophore formation reaction. Incorporation of tyrosine analogues, on the other hand, will have a smaller influence on the chromophore formation but provide relatively small changes in fluorescence.

Incorporation of non-natural amino acids into Tyr66 of GFP was carried out by using a four-base codon, CGGG (Hohsaka et al., 1999Go). When a CGGG codon is decoded by a tRNACCCG aminoacylated with non-natural amino acids, a full-length GFP will be synthesized. On the other hand, when the first three bases, CGG, are decoded as arginine, the reading frame shifts in a +1 direction, which causes an irregular termination of peptide elongation at a stop codon downstream. Therefore, full-length GFP will be obtained only when non-natural amino acids are incorporated into the Tyr66 position.

In this study, GFPuv, one of the GFP variants, was used as ‘wild-type’ GFP and CGGG codon was introduced into the Tyr66 position of the GFP gene. Transfer RNAs aminoacylated with non-natural amino acids were prepared as described previously (Hohsaka et al., 1999Go) and were added to an E.coli cell-free translation system with GFP mRNA containing CGGG codon at the Tyr66 position. The expression of full-length GFP mutants was confirmed by western blot analysis using anti-T7 tag antibody (Figure 2) and the yields of the GFP mutants were determined by comparing the band intensity of GFP mutants on the western blot with those of serial dilutions of wild-type GFP. In the presence of non-natural aminoacyl-tRNACCCG, the full-length GFP mutants observed around 27 kDa were obtained. In the absence of tRNACCCG, on the other hand, a negligible band was detected in the absence of aminoacyl-tRNACCCG. The yield of the readthrough product was estimated to be 5% relative to the wild-type GFP. This result indicated that the four-base codon was successfully decoded by the aminoacyl-tRNACCCG and a GFP mutant containing the non-natural amino acid at the Tyr66 position was produced. The yields varied depending on the structure of non-natural amino acids, in the range 5–80%. These values were in good agreement with those obtained in the case of the Tyr83 position of streptavidin (Hohsaka et al., 1999Go), suggesting that the efficiency of four-base decoding depends mainly on the structures of amino acids. However, some amino acids were incorporated into the Tyr66 position of GFP more efficiently than the Tyr83 position of streptavidin, suggesting that the efficiency may also depend on the context of mRNA sequences.



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Fig. 2. Incorporation of various non-natural amino acids into the Tyr66 position of GFP by using CGGG four-base codon. Cell-free translation products were analysed by western blotting using anti-T7 tag antibody. The bands at 27 kDa correspond to full-length GFP: lane ‘wt’, wild-type mRNA; lane ‘–’, no tRNACCCG; lane 1, L-1-naphthylalanine; lane 2, L-2-naphthylalanine; lane 3, L-p-biphenylylalanine; lane 4, L-2-anthrylalanine; lane 5, L-2-pyrenylalanine; lane 6, L-p-nitrophenylalanine; lane 7, L-p-dimethylaminophenylalanine; lane 8, L-3-(9-ethylcarbazolyl)alanine; lane 9, L-azatryptophan; lane 10, L-kynurenine; lane 11, L-p-phenylazophenylalanine; lane 12, L-p-benzoylphenylalanine; lane 13, L-2-anthraquinonylalanine; lane 14, L-p-aminophenylalanine; lane 15, L-O-methyltyrosine. Lane M contained a prestained molecular weight marker.

 
Fluorescence of mutant GFPs containing different non-natural amino acids at the Tyr66 position

Fluorescence spectra of GFP mutants containing various non-natural amino acids were then measured. One portion of cell-free translation reaction mixture (1 µl) was diluted with 320 µl of Tris buffer, pH 7.5, and fluorescence spectra were measured with excitation wavelengths between 350 and 600 nm. No fluorescence was detected except for p-aminophenylalanine (aminoPhe) and O-methyltyrosine (OMeTyr) mutants, whereas a clear fluorescence spectrum was observed when wild-type GFP was expressed in the cell-free translation system. Prolonged incubation of the translation mixture or the diluted solution gave no fluorescence, either. Incorporation of large non-natural amino acids into the Tyr66 position may inhibit the chromophore formation or give non-fluorescent compounds. It should be noted that no fluorescence was detected in the absence of non-natural aminoacyl-tRNA, indicating that the readthrough product gave no fluorescence.

GFP mutants containing aminoPhe and OMeTyr at the Tyr66 position emitted fluorescence as shown in Figure 3. The raw spectra were subtracted by a baseline spectrum of the translation mixture without mRNA and tRNACCCG and normalized for relative concentration of GFP mutants determined by the western blotting. The fluorescence of non-natural GFP mutants was blue-shifted ({lambda}max = 497 nm for the aminoPhe mutant and {lambda}max = 462 nm for the OMeTyr mutant) from wild-type GFP ({lambda}max = 507 nm) (Table I). The substitution of hydroxylate anion by less electron-donating amino and methoxy groups will lead to less expanded {pi} conjugate chromophore systems. The finding that the OMeTyr chromophore gives a blue-shifted fluorescence like a protonated chromophore at 77 K (Chattoraj et al., 1996Go) supports the idea that the deprotonation of the GFP chromophore is essential to green fluorescence (Ormo et al., 1996Go; Brejc et al., 1997Go). These results are consistent with the results for in vivo expressed non-natural GFP mutants (Wang et al., 2003Go). However, the in vivo method using mutated aminoacyl-tRNA synthetases is available for only a limited type of non-natural amino acids. The cell-free method is more suitable than the in vivo method for synthesizing and screening non-natural GFP mutants incorporated with a wide variety of non-natural amino acids.



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Fig. 3. Fluorescence spectra of non-mutated GFP ({lambda}ex = 395 nm, {lambda}em = 506 nm) and mutants containing p-aminophenylalanine ({lambda}ex = 440 nm, {lambda}em = 497 nm) and O-methyltyrosine ({lambda}ex = 395 nm, {lambda}em = 462 nm) at the Tyr66 position in Tris–HCl buffer, pH7.5. The fluorescence intensities were normalized by the relative concentration of full-length GFP mutant determined by western blotting.

 

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Table I. Spectral properties of non-natural GFP mutants

 
Sequence optimization of non-natural GFP mutants

Although aminoPhe and OMeTyr mutants showed blue-shifted fluorescence, the fluorescence intensities of their {lambda}max (440 and 395 nm, respectively) with excitation at {lambda}max 497 and 462 nm were reduced (50% for aminoPhe mutant and 40% for OMeTyr mutant compared with wild-type GFP) (Table I). One of the reasons for the reduced intensity is that the amino acid residues around the Tyr66 position allow less efficient chromophore formation. Another possibility is that the polarity of the chromophore is altered by the substitution of Tyr66, which reduces the fluorescence quantum yield. Cubitt et al. reported that the fluorescence intensity of a GFP mutant with a single Y66W mutation can be improved by introducing second mutations around Trp66 (Cubitt et al., 1999Go). In this study, we tried to improve the fluorescence intensity of the aminoPhe mutant by introducing random mutations. The sequence optimization will be useful for improving functions of non-natural mutants of proteins that are synthesized only in cell-free translation systems.

We chose Ser65 and Tyr145 as mutation sites, since they have been shown to be effective for improving the intensity of GFP and BFP variants (Tsien, 1998Go; Yang et al., 1998Go; Cubitt et al., 1999Go). GFP genes containing a CGGG codon at Tyr66 and an NNK codon (K = G or T) at Ser65 or Tyr145 were generated by two-step PCR. The resulting GFP genes were transformed into E.coli DH10B. DNA sequence analysis of random pool of the plasmids supported the introduction of NNK codon at the desired specific position. Forty-two clones were randomly chosen and subjected to colony-directed PCR, followed by a cell-free coupled transcription/translation reaction including aminoPhe-tRNACCCG. Western blot analysis indicated that full-length GFP mutants containing aminoPhe at Tyr66 were synthesized from almost all clones.

Fluorescence measurements of these mutants were carried out without purification. In the case of Ser65 mutants, no improved clone was obtained. On the other hand, in the Tyr145 mutants, strong fluorescence was observed in six out of the 42 clones. These mutants were found to have a Leu, Phe or Met mutation by DNA sequencing. As shown in Figure 4, the fluorescence intensities of the Y145L, Y145F and Y145 M mutants increased by 1.4-, 2.9- and 3.9-fold compared with Y145; the emission maxima were slightly blue-shifted ({lambda}max = 487 nm for Y145L, 484 nm for Y145 M and 494 nm for Y145F) (Table I). The side chain of Tyr145 is located around the Tyr66 moiety of the chromophore and makes an edge-face interaction in native GFP, which is maintained in improved green fluorescent variants (Tsien, 1998Go). On the other hand, the fluorescence intensity of a blue-shifted Y66H variant was enhanced by substitution of Tyr145 by phenylalanine (Yang et al., 1998Go). In a similar manner, the Y66 aminoPhe mutation was optimized by substitutions of Tyr145 by leucine, methionine and phenylalanine. These substitutions may induce a rearrangement of surrounding amino acid residues and result in efficient formation and/or an increased quantum yield of the aminoPhe chromophore.



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Fig. 4. (a) The expression of full-length GFP containing p-aminophenylalanine was examined by western blot analysis. The band at 27 kDa is a full-length GFP: lane ‘wt’, wild-type mRNA; lane 1, Tyr145; lane 2, Phe145; lane 3, Leu145; lane 4, Met145; lane 5, non-aminoacylated tRNA. Lane M contained a prestained molecular weight marker. (b) Fluorescence spectra of GFP mutants containing p-amino-phenylalanine at Tyr66 and Y145F, Y145L and Y145M mutations ({lambda}ex = 440 nm). The fluorescence intensities were normalized by the relative concentration of full-length GFP mutant determined by western blotting.

 
Conclusions

Various aromatic non-natural amino acids were incorporated into Tyr66 of GFP using a four-base codon method. Non-natural GFP mutants that contained relatively large aromatic amino acids were non-fluorescent, but mutants with aminoPhe and OMeTyr showed blue-shifted fluorescence. To improve the fluorescence intensity of the aminoPhe mutant, random mutation was introduced and improved clones were selected. As a result, three clones with improved fluorescence properties were successfully obtained. These results indicate that random mutation around non-natural amino acid is a useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here is applicable to other non-natural proteins and will be performed in a high-throughput manner by using an automated system.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This work was supported by a Grand-in-Aid for Scientific Research (S) from the Ministry of Education, Science, Sports and Culture, Japan (No. 15101008) and by the Industrial Technology Research Grant Program in '03 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.


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 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
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Received February 17, 2005; revised April 28, 2005; accepted May 3, 2005.

Edited by Don Hilvert





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