[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
301
[15] B i o s y n t h e t i c M e t h o d for I n t r o d u c i n g U n n a t u r a l A m i n o A c i d s S i t e - S p e c i f i c a l l y into P r o t e i n s By JON ELLMAN, DAVID MENDEL, SPENCER ANTHONY-CAHILL, CHRISTOPHER J. N O R E N , and PETER G. SCHULTZ
Introduction Oligonucleotide-directed mutagenesis is a powerful technique which allows any amino acid in a protein to be substituted with one of the other 19 natural amino acids. Characterization of the resulting mutant proteins has greatly increased our insight into the nature of molecular recognition and catalysis in biological systems. The scope of this method would be expanded if, in addition to the natural amino acids, unnatural amino acids with novel steric, electronic, or chemical properties could also be incorporated into proteins. We have recently reported a general biosynthetic method to site specifically incorporate unnatural amino acids into proteins (Fig. 1)) -4 This method involves replacement of the codon for the amino acid of interest with the amber nonsense codon by conventional oligonucleotide-directed mutagenesis. The amber nonsense codon is not recognized by any of the common tRNAs involved in protein synthesis and thus can be viewed as a "blank" in the genetic code. A suppressor tRNA is then constructed which recognizes the amber nonsense codon. The suppressor tRNA is then chemically aminoacylated with the desired unnatural amino acid and is added to an in oitro transcription-translation system programmed with the mutagenized DNA. This results in the specific incorporation of the unnatural amino acid at the position corresponding to the amber mutation. This article describes preparation of the suppressor tRNA, general methodology for chemical aminoacylation of the suppressor tRNA, and an in vitro transcription-translation system optimized for the incorporation of unnatural amino acids into proteins. Efforts aimed at simplifying the i C. J. N o r e n , S. J. Anthony-Cahill, M. C. Griffith, a n d P. G. Schultz, Science 244, 182 (1989). 2 S. A. Robertson, C. J. N o r e n , S. J. Anthony-Cahill, M. C. Griffith, and P. G. Schultz, Nucleic Acids Res. 17, 9649 (1989). 3 S. A. Robertson, J. A. Ellman, and P. G. Schultz, J. Am. Chem. Soc. 113, 2722 (1991). 4 C. J. N o r e n , S. J. Anthony-Cahill, D. J. Suich, K. A. Noren, M. C. Griffith, and P. G. Schultz, Nucleic Acids Res. 18, 83 (1990).
METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991by AcademicPress, Inc. All fights of reproduction in any form reserved.
302
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
Codon for residue of interest AGC
[15]
Nonsense codon TAG
Oligonucleotide-directed mutagenesis
(aa)NA dC C pdCpA-aa,
I
(m RNA ) ---".--.-.~'---~ I
G
~
_.~
~, In vitro
transcription
3'12
SuppressortRNA (-CA)
A
In vitro
translation
Mutant enzymewith unnatural amino acid site-specifically incorporated.
FIG. 1. Strategy for biosynthetic incorporation of unnatural amino acids (aa) into proteins. A, Adenosine; C, cytidine; G, guanosine; T, thymidine; U, uridine.
methodology and improving the efficiency of protein synthesis are also discussed. Construction of Suppressor tRNA The suppressor t R N A used to deliver the unnatural amino acid to the growing peptide chain on the ribosome must meet two criteria: it must efficiently insert the amino acid in response to the U A G message, and it must be neither acylated nor deacylated by any of the E s c h e r i c h i a c o l i aminoacyl-tRNA synthetases present in the in v i t r o transcription-translation system. The first condition is necessary for producing q u a n t i t i e s o f protein that can be purified and further studied, and the second condition
[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
303
is required to ensure that only the desired unnatural amino acid and not one or more of the 20 natural amino acids in the in vitro reaction are inserted into the protein. An amber suppressor tRNA derived from yeast tRNA Phe was expected to meet these requirements): Anticodon loop replacement. Two methods have been used for the generation of this suppressor tRNA, runoff transcription and a chemical/ enzymatic anticodon loop replacement procedure. The former has the advantage of simplicity and high yield, while the latter preserves modified bases of the tRNA, the importance of which remains unclear. Anticodon loop replacement was initially used to construct the suppressor tRNA, tRNA~z~A. The protocol reported by Bruce and Uhlenbeck 7,8 was optimized for the production of t R N A ~ A on a 15- to 20-mg scale. Starting with commercially available yeast tRNA Phe, the amber suppressor Phe was prepared according to the synthetic strategy shown in tRNAcuA Fig. 2. Treatment of intact yeast tRNA Phe (1) with HCI at pH 2.9 for 3 hr at 37°, followed by aniline hydrochloride at pH 4.5 for 5 hr at 25°, results in nearly complete cleavage of the tRNA (and concomitant removal of Y-37), yielding intermediate 2 [half-molecules of 36 nucleotides (the 5' half-molecule) and 39 nucleotides (the 3' half-molecule)]. Partial RNase A digestion removes the anticodon GAA (residues 34-36) and the acceptor stem nucleotides C-75 and A-76 (intermediate 3). The four excised anticodon loop nucleotides 34-37 are replaced by ligation of the synthetic tetramer 5'CpUpApA-3' onto the 3' half-molecule to yield intermediate 4.The anticodon loop can be resynthesized with T4 RNA ligase if the phosphorylation states of the 3' and 5' termini of intermediate 4 are first reversed. This can be accomplished by treating the tRNA species 4 with T4 polynucleotide kinase, which, at pH 6.9, possesses 3'-phosphatase activity in addition to 5'-kinase activity. 8 The resulting tRNA (intermediate 5) is not isolated. T4 RNA ligase is added directly to the kinase reaction to yield a "one pot" conversion of 4 to 6. This tRNA species still lacks the two 3'-terminal acceptor stem nucleotides pCpA [tRNA~A(-CA)]. The t R N A ~ A ( - C A ) obtained after the final ligation can be purified, as needed, by preparative gel electrophoresis and recovered from the gel by elution. In our laboratory, 600 /zg of crude 6 yielded 90-300/xg of purified t R N A ~ A ( - C A ) . The yield of gel-purified product varied from batch to batch, such that the overall yield for the synthesis of 5 y . Kwok and J. T. Wong, Can. J. Biochem. 58, 213 (1980). 6 M. Yarus, Science 218, 646 0982). 7 A. G. Bruce, J. F. Atkins, N. Wills, O. Uhlenbeck, and R. F. Gesteland, Proc. Natl. Acad. Sci. U.S.A. 79, 7127 (1982). 8 A. G. Bruce and O. C. Uhlenbeck, Biochemistry 21, 855 (1982).
304
[15]
PROTEINS AND PEPTIDES; PRINCIPLES AND METHODS
cAoH C
AOH
3t
C
A
A
.5'
I1 pH2.9
RNoseA
2 ) Aniline
-4> Cm Ap Up 5~ 3~
c.,p GmAA
G,aAApX
!
Cp A C
2
3 COH A
Cp
A
RNALiqose
T4 Kinase-Phosphatase CmA A
Cm A UoH A pCU
Up A
U COH
4
CA oH
RNA LJgasei r
pG C
~
~
AOH C C A
ATP CTP
Nucleotidyltransferose P
C~ A U A CuA
Cm A U A CuA
6
7
FIG. 2. S u p p r e s s o r construction by anticodon loop r e p l a c e m e n t o f y e a s t t R N A Phe. Modified from Bruce and U h l e n b e c k ?
Phe - CA) starting from yeast tRNA Phe was 12-20%. The procedure tRNA~tjA( can be carried out on a 15-mg scale (starting yeast tRNA Phe) and typically requires 1 to 2 weeks. A 1 mg/ml solution of suppressor tRNA in doubly distilled water (dd H20) can be kept at - 80° for months without noticeable loss of potency as measured by in vitro assay for/3-1actamase activity. The crude 6 is always stored as a lyophilized solid at - 8 0 °. Runoff transcription. 4 More recently, we have prepared Phe tRNAcuA(-CA) in vitro by T7 RNA polymerase-directed runoff transcription9']° of plasmid pYPhe2. 4 Although this method produces 9 0 . C. U h l e n b e c k and R. I. G u m p o r t , The Enzymes 15, 31 (1982). 10 R. Silber, V. G. Malathi, and J. Hurwitz, Proc. Natl. Acad. Sci. U.S.A. 69, 3009 (1972).
[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
305
Phe - CA) lacking modified bases of the natural product, the unmodtRNA~tJA( ified transcript stringently meets the criteria for efficient and accurate suppression. Runoff transcription may be considered the method of choice because it can produce tens of milligrams of suppressor tRNA within a few days and is less costly and less labor-intensive than the anticodon loop replacement procedure. However, as a note of caution, it has recently been demonstrated that tRNA base modifications help prevent misacylation by noncognate tRNA synthetases in the case of yeast tRNAAsp, l~ Though the E. coli-derived transcription-translation system we employ performs equally with native and unmodified suppressor tRNAs, it is possible that other expression systems may require modified suppressor to avoid editing or misacylation by endogenous aminoacyl tRNA synthetases.
General Methods
Enzymes are stored at - 20° in buffered glycerol solutions unless otherwise noted. RNase A is obtained as a powder from Sigma (St. Louis, MO) and stored at - 20° as a stock solution of 10 mg/ml in doubly distilled water. T4 RNA ligase is either obtained from Takara Shuzo Inc. or prepared as described by Heaphy et al. 12 T4 polynucleotide kinase is either obtained from New England Biolabs (Beverly, MA) or prepared as described by Richardson 13 with the modifications of Wu and Kaiser, 14 H~inggi et al., 15 and Cameron and Uhlenbeck.16 Yeast phenylalanine-specific tRNA (yeast tRNA Phe) is obtained from Boehringer Mannheim (Indianapolis, IN). All enzyme reaction buffers are made up with sterile diethylpyrocarbonate (DEPC) treated doubly distilled water. The pH values of the buffers are adjusted at room temperature, and the buffers are filtered through 0.22-/zm sterile filters prior to use. Polypropylene SS-34 centrifuge tubes (Sorvall, Norwalk, CT) are soaked in 0.1% (v/v) diethyl pyrocarbonate in ethanol for several hours, then autoclaved prior to use. Phenol (Bethesda Research Laboratories, Gaithersburg, MD) used in phenol extractions is prepared as described. ~7Etha-
11 V. Perret, A. Garcia, H. Grosjean, J.-P. Ebel, C. Florentz, and R. Geige, Nature (London) 344, 787 (1990). 12 S. Heaphy, M. Singh, M. J. Gait, Biochemistry 26, 1688 (1987). 13 C. C. Richardson, Proc. Natl. Acad. Sci. U.S.A. 54, 158 (1965). 14 R. Wu and A. D. Kaiser, Proc. Natl. Acad. Sci. U.S.A. 57, 170 (1967). 15 U. J. Hanggi, R. E. Streeck, H. P. Voigt, and H. G. Zachau, Biochim. Biophys. Acta 217, 278 (1970). 16 V. Cameron and O. C. Uhlenbeck, Biochemistry 16, 5120 (1977). 17 E. F. Sambrook, E. F. Fritsch, and T. Maniatis, in "Molecular Cloning: A Laboratory Manual, 2nd E d . " Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
306
PROTEINS AND PEPTIDES" PRINCIPLES AND METHODS
[15]
nol precipitations are carried out by the addition of 0. I volumes of sterile aqueous 2.5 M sodium acetate, pH 4.5, to an aqueous solution of tRNA, followed by addition of 2.5-3 volumes of ice-cold ethanol. Transfer RNA suspensions precipitated in Eppendorf tubes are chilled on dry ice for 10 min prior to microcentrifugation (10 min); tRNA suspensions precipitated in Sorvall SS-34 tubes are chilled at - 2 0 ° for 2-10 hr then spun at 15,000 rpm at 4 ° for 10-30 min in an SS-34 rotor. Transfer RNA is quantitated by measuring the optical density of an aqueous solution at 260 nm (OD260), where 1 OD260 unit corresponds to a concentration of 40 txg tRNA/ml. Denaturing polyacrylamide gel electrophoresis (DPAGE) of tRNA is carried out on analytical (0.75 mm) and preparative (1.5 mm) gels. Analytical DPAGE gels contain 10% (w/v) acrylamide, 19 : 1 (w/w) acrylamide : bisacrylamide, 7 M urea, 50 mM Tris-borate, pH 8.3, and 1 mM EDTA. DPAGE gels are preelectrophoresed at 600 V for 0.5-2 hr prior to loading the samples. Analytical gels are run at 15 or 20 W constant power, and 8% preparative gels are run at 25 or 30 W constant power. Analytical gels are visualized by staining with ethidium bromide followed by UV transillumination. Preparative gels are visualized by staining with 0.02% aqueous toluidine blue O (Aldrich, Milwaukee, WI) or by UV shadowing over an activated TLC plate. Nucleic acid is recovered from preparative gels by excising the desired band with a razor blade, mincing the gel slice, and soaking overnight at room temperature in 5 volumes of elution buffer [50 mM sodium acetate, pH 4.5, 1 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS)]. The supernatant is removed and saved, and an additional 5 volumes of elution buffer is added. After 6 hr of soaking, the supernatant is removed. The combined supernatants are filtered through a 0.45-tzm sterile filter, extracted once with phenol then once with chloroform, and then precipitated twice with ethanol (note that for the first ethanol precipitation it is not necessary to add sodium acetate prior to addition of the ethanol since the elution buffer already contains 50 mM sodium acetate). Alternatively, nucleic acid may be electroeluted from the excised gel slice and rapidly desalted on a sizing chromatography column. R u n o f f transcription o f t R N A ~ A ( - CA) 4 R u n o f f Transcription. Plasmid pYPhe2 is digested with F o k I and used as template for runoff transcription according to Noren et al. 4 Transcription reactions (300/.d) contain 30/zg FokI-cleaved pYPhe2, 40 mM Tris hydrochloride pH 8.1, 20 mM MgClz, 5 mM dithiothreitol (DTT); 50/xg/ ml bovine serum albumin, 1 mM spermidine, 4 mM each neutralized nucleoside triphosphate (NTP), 0.25 units inorganic pyrophosphatase, 0.08
[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
307
units/ml RNase inhibitor and 25-30 units//zl T7 RNA polymerase. Reactions are incubated for 150 min at 42 °, then supplemented with 200/xl of the same solution except containing 10 mM each NTP, 30 mM MgCI2,5-6 units/txl T7 polymerase and lacking template. Reactions are then further incubated overnight at 38°, quenched with 50 tzl of 0.5 M EDTA pH 8, extracted twice with (1 : 1 v/v) phenol : chloroform, once with chloroform and then precipitated with ethanol. Runoff tRNA prepared in this manner is purified on an 8% denaturing gel and yields - 1 I00/xg per 500 tzl reaction mixture. It is important for the T7 RNA polymerase to be sufficiently concentrated and active to produce accurate transcripts. We have noticed that older preparations of polymerase may "stutter" near the template 3' terminus resulting in a heterogeneous population of transcripts. T7 R N A polymerase purification. E. coli strain BL21, containing the plasmid pAR1219 is used for this preparation. Plasmid pAR1219 (amp l) contains the gene for T7 RNA polymerase under transcriptional control of the lac promoter. This strain is grown at 37° and maintained on plates containing ampicillin. Six 4-liter culture flasks are loaded with one liter each of autoclaved growth media containing 1 g NH4CI, 3 g KH2PO 4, 6 g Na2HPO 4, 0.5 g NaCI, 1% tryptone, 2 mM MgSO4, 0.1 mM CaCI2,0.2% glucose, and 100 mg ampicillin. The medium is equilibrated at 37° with vigorous shaking and inoculated with overnight starter culture (12 ml/liter) grown in the same medium. Cells are grown until A590 = 0.9 (2 hrs) after which is added 119 mg of isopropylthio-fl-D-galactoside (IPTG) dissolved in 5 ml of the same medium. Cells are then incubated for an additional 4 hrs, cooled, harvested (GS-3 rotor, 6000 rpm, 10 min, 4°), washed with 500 ml of buffer W (50 mM Tris hydrochloride, pH 8.0 and 25% sucrose) and frozen at - 80°. All further operations are carried out at 4 °. Cells (20.0 g) are thawed and suspended in 270 ml of Buffer L (1.18 g NH4Cl/liter, 20 mM Tris hydrochloride, 1 mM EDTA, 1 mM DTT, 2/xg/ ml phenylmethylsulfonyl fluoride (PMSF), 1/xg/ml leupeptin, and 5% (v/ v) glycerol), then lysed with hen egg white lysozyme solution. After a 30 min incubation, 5 ml of sodium deoxycholate solution (50 mg/ml) is added and the mixture is incubated for 30 min with shaking. The viscous lysate is spun at 40,000 rpm for 4 hrs in a Dupont T-865 rotor at 4 ° and supernatant is then carefully transferred to a GS-3 rotor bottle. Solid ammonium sulfate (80 g) is added slowly to the supernatant on ice (300 ml), and dissolved completely with gentle stirring for 30 min. After sitting an additional 20 min on ice, the white precipitate is collected by centrifugation in a GS-3 rotor at 9000 rpm for 1 hr. The precipitate is then dissolved in 20
308
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
[15]
ml of Buffer I (10 mM KH2PO4, pH 8.0, 0.I mM EDTA, 2 ~g/ml PMSF, 0.07% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, and 0.15 M NaCI) and dialyzed against 1 liter of Buffer I. The dialyzed ammonium sulfate fraction is spun down in an SS-34 rotor at 10,000 rpm for 20 min to remove particulates and the clear fraction is applied, at a flow rate of 0.5 ml/min, to a column (2.5 × 20 cm) of P11 pre-equilibrated in Buffer I. After collecting the loading fractions, the column is washed with 10 column volumes of Buffer I until the OD280 is less than 0.1. Bound proteins are then eluted with a 500 ml linear gradient from 0.25 to 1.0 M NaCI in Buffer I at a flow rate of 1 ml/min and fractions of 7 ml are collected. The absorbance at 280 nm is monitored, and the peak fractions again are analyzed by 7.5% SDS-PAGE. Peak fractions containing T7 RNA polymerase are pooled and concentrated with an Amicon concentrator (PM30 membrane; Lexington, MA) to a protein concentration of 1 mg/ml. The concentrated fraction is dialyzed against 2 × 1 liter changes of Buffer II (5.4 g NH4C1/liter, 0.05 mM EDTA, 10 mM Tris hydrochloride pH 8.0, 1 mM DTT, and 50% (v/v) glycerol. After the activity assay, 200/zl aliquots (specific activity: 256,000 units/mg, recovery yield: 21%) are prepared and stored at - 20°. Assay reactions (100/.rL) for T7 RNA polymerase contain: 40 mM Tris hydrochloride, pH 8.0, 20 mM MgCI2, 5 mM DTT, 0.4 mM each NTP, 50 /zg/ml BSA, 36 gtg/ml plasmid pSG1, and diluted enzyme. Reactions are supplemented with [3H]-UTP (13.7 Ci/mmol) to a final specific activity of 18 mCi/mmol UTP. Enzyme fractions are diluted 1 : 100 to 1 : 1000 with 10 mM Tris hydrochloride, pH 7.5, I0 mM 2-mercaptoethanol, 1 mg/ml BSA such that each assay reaction ultimately contains 10-100 ng enzyme. Reactions are incubated at 37° for 15 min, and acid-insoluble radioactivity is determined by diluting the reactions with I ml of cold 5% (w/v) trichloroacetic acid (TCA) and allowed to remain on ice for 30 min. Precipitated material is collected on Whatman GF-C glass fiber filters (premoistened with 5% TCA). Filters are washed successively with 5 ml each of 5% TCA, a 9 : 1 mixture of 5% TCA: saturated sodium pyrophosphate, aqueous 1 N HC1, and 95% ethanol. Filters are then dried and counted. One unit of enzyme activity is defined as the amount of enzyme required to convert 1 nmol [3H]-UTP to acid-insoluble form in 60 min.
Chemical~Enzymatic Generation 0pf t R N A cPhe uA Y-37 Depurination and Backbone Cleavage. Yeast tRNA ehe (16 rag) is dissolved in 15.2 ml of sterile ddH20. Addition of 0.1 M HC1 (0.4 ml) brings the pH to 3.5; addition of 10 mM HC1 ( - 1 . 2 ml) brings the pH to
[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
309
2.9. It is important to titrate the pH of the solution slowly so as not to bring the pH below the desired value of 2.9. Readjustment of an overly acidic tRNA solution to pH 2.9 with dilute base (10 mM NaOH) results in nonspecific cleavage of the tRNA (as judged by analytical DPAGE). The acidified tRNA solution is incubated at 37° for 3 hr, then divided into two SS-34 tubes and precipitated with ethanol. Each pellet is resuspended in 4.5 ml doubly distilled water (added directly to the SS-34 tube), and 4.5 ml of 0.9 M aniline hydrochloride (pH 4.5) is added. After 5 hr at room temperature with occasional vortexing, the tRNA is recovered by ethanol precipitation. Cleaved tRNA is separated from intact tRNA by preparative DPAGE (0.15 x 16 x 42 cm; 5 mg crude tRNA is loaded onto each gel). The gel is stained with toluidine blue, and the bands are excised and eluted as described in the general methods section. The two tRNA half-molecules (intermediate 2) (271 OD260, 10.8 mg, 67%) are recovered by ethanol precipitation. Complete separation of uncleaved material (
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
301
[15] B i o s y n t h e t i c M e t h o d for I n t r o d u c i n g U n n a t u r a l A m i n o A c i d s S i t e - S p e c i f i c a l l y into P r o t e i n s By JON ELLMAN, DAVID MENDEL, SPENCER ANTHONY-CAHILL, CHRISTOPHER J. N O R E N , and PETER G. SCHULTZ
Introduction Oligonucleotide-directed mutagenesis is a powerful technique which allows any amino acid in a protein to be substituted with one of the other 19 natural amino acids. Characterization of the resulting mutant proteins has greatly increased our insight into the nature of molecular recognition and catalysis in biological systems. The scope of this method would be expanded if, in addition to the natural amino acids, unnatural amino acids with novel steric, electronic, or chemical properties could also be incorporated into proteins. We have recently reported a general biosynthetic method to site specifically incorporate unnatural amino acids into proteins (Fig. 1)) -4 This method involves replacement of the codon for the amino acid of interest with the amber nonsense codon by conventional oligonucleotide-directed mutagenesis. The amber nonsense codon is not recognized by any of the common tRNAs involved in protein synthesis and thus can be viewed as a "blank" in the genetic code. A suppressor tRNA is then constructed which recognizes the amber nonsense codon. The suppressor tRNA is then chemically aminoacylated with the desired unnatural amino acid and is added to an in oitro transcription-translation system programmed with the mutagenized DNA. This results in the specific incorporation of the unnatural amino acid at the position corresponding to the amber mutation. This article describes preparation of the suppressor tRNA, general methodology for chemical aminoacylation of the suppressor tRNA, and an in vitro transcription-translation system optimized for the incorporation of unnatural amino acids into proteins. Efforts aimed at simplifying the i C. J. N o r e n , S. J. Anthony-Cahill, M. C. Griffith, a n d P. G. Schultz, Science 244, 182 (1989). 2 S. A. Robertson, C. J. N o r e n , S. J. Anthony-Cahill, M. C. Griffith, and P. G. Schultz, Nucleic Acids Res. 17, 9649 (1989). 3 S. A. Robertson, J. A. Ellman, and P. G. Schultz, J. Am. Chem. Soc. 113, 2722 (1991). 4 C. J. N o r e n , S. J. Anthony-Cahill, D. J. Suich, K. A. Noren, M. C. Griffith, and P. G. Schultz, Nucleic Acids Res. 18, 83 (1990).
METHODS IN ENZYMOLOGY, VOL. 202
Copyright © 1991by AcademicPress, Inc. All fights of reproduction in any form reserved.
302
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
Codon for residue of interest AGC
[15]
Nonsense codon TAG
Oligonucleotide-directed mutagenesis
(aa)NA dC C pdCpA-aa,
I
(m RNA ) ---".--.-.~'---~ I
G
~
_.~
~, In vitro
transcription
3'12
SuppressortRNA (-CA)
A
In vitro
translation
Mutant enzymewith unnatural amino acid site-specifically incorporated.
FIG. 1. Strategy for biosynthetic incorporation of unnatural amino acids (aa) into proteins. A, Adenosine; C, cytidine; G, guanosine; T, thymidine; U, uridine.
methodology and improving the efficiency of protein synthesis are also discussed. Construction of Suppressor tRNA The suppressor t R N A used to deliver the unnatural amino acid to the growing peptide chain on the ribosome must meet two criteria: it must efficiently insert the amino acid in response to the U A G message, and it must be neither acylated nor deacylated by any of the E s c h e r i c h i a c o l i aminoacyl-tRNA synthetases present in the in v i t r o transcription-translation system. The first condition is necessary for producing q u a n t i t i e s o f protein that can be purified and further studied, and the second condition
[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
303
is required to ensure that only the desired unnatural amino acid and not one or more of the 20 natural amino acids in the in vitro reaction are inserted into the protein. An amber suppressor tRNA derived from yeast tRNA Phe was expected to meet these requirements): Anticodon loop replacement. Two methods have been used for the generation of this suppressor tRNA, runoff transcription and a chemical/ enzymatic anticodon loop replacement procedure. The former has the advantage of simplicity and high yield, while the latter preserves modified bases of the tRNA, the importance of which remains unclear. Anticodon loop replacement was initially used to construct the suppressor tRNA, tRNA~z~A. The protocol reported by Bruce and Uhlenbeck 7,8 was optimized for the production of t R N A ~ A on a 15- to 20-mg scale. Starting with commercially available yeast tRNA Phe, the amber suppressor Phe was prepared according to the synthetic strategy shown in tRNAcuA Fig. 2. Treatment of intact yeast tRNA Phe (1) with HCI at pH 2.9 for 3 hr at 37°, followed by aniline hydrochloride at pH 4.5 for 5 hr at 25°, results in nearly complete cleavage of the tRNA (and concomitant removal of Y-37), yielding intermediate 2 [half-molecules of 36 nucleotides (the 5' half-molecule) and 39 nucleotides (the 3' half-molecule)]. Partial RNase A digestion removes the anticodon GAA (residues 34-36) and the acceptor stem nucleotides C-75 and A-76 (intermediate 3). The four excised anticodon loop nucleotides 34-37 are replaced by ligation of the synthetic tetramer 5'CpUpApA-3' onto the 3' half-molecule to yield intermediate 4.The anticodon loop can be resynthesized with T4 RNA ligase if the phosphorylation states of the 3' and 5' termini of intermediate 4 are first reversed. This can be accomplished by treating the tRNA species 4 with T4 polynucleotide kinase, which, at pH 6.9, possesses 3'-phosphatase activity in addition to 5'-kinase activity. 8 The resulting tRNA (intermediate 5) is not isolated. T4 RNA ligase is added directly to the kinase reaction to yield a "one pot" conversion of 4 to 6. This tRNA species still lacks the two 3'-terminal acceptor stem nucleotides pCpA [tRNA~A(-CA)]. The t R N A ~ A ( - C A ) obtained after the final ligation can be purified, as needed, by preparative gel electrophoresis and recovered from the gel by elution. In our laboratory, 600 /zg of crude 6 yielded 90-300/xg of purified t R N A ~ A ( - C A ) . The yield of gel-purified product varied from batch to batch, such that the overall yield for the synthesis of 5 y . Kwok and J. T. Wong, Can. J. Biochem. 58, 213 (1980). 6 M. Yarus, Science 218, 646 0982). 7 A. G. Bruce, J. F. Atkins, N. Wills, O. Uhlenbeck, and R. F. Gesteland, Proc. Natl. Acad. Sci. U.S.A. 79, 7127 (1982). 8 A. G. Bruce and O. C. Uhlenbeck, Biochemistry 21, 855 (1982).
304
[15]
PROTEINS AND PEPTIDES; PRINCIPLES AND METHODS
cAoH C
AOH
3t
C
A
A
.5'
I1 pH2.9
RNoseA
2 ) Aniline
-4> Cm Ap Up 5~ 3~
c.,p GmAA
G,aAApX
!
Cp A C
2
3 COH A
Cp
A
RNALiqose
T4 Kinase-Phosphatase CmA A
Cm A UoH A pCU
Up A
U COH
4
CA oH
RNA LJgasei r
pG C
~
~
AOH C C A
ATP CTP
Nucleotidyltransferose P
C~ A U A CuA
Cm A U A CuA
6
7
FIG. 2. S u p p r e s s o r construction by anticodon loop r e p l a c e m e n t o f y e a s t t R N A Phe. Modified from Bruce and U h l e n b e c k ?
Phe - CA) starting from yeast tRNA Phe was 12-20%. The procedure tRNA~tjA( can be carried out on a 15-mg scale (starting yeast tRNA Phe) and typically requires 1 to 2 weeks. A 1 mg/ml solution of suppressor tRNA in doubly distilled water (dd H20) can be kept at - 80° for months without noticeable loss of potency as measured by in vitro assay for/3-1actamase activity. The crude 6 is always stored as a lyophilized solid at - 8 0 °. Runoff transcription. 4 More recently, we have prepared Phe tRNAcuA(-CA) in vitro by T7 RNA polymerase-directed runoff transcription9']° of plasmid pYPhe2. 4 Although this method produces 9 0 . C. U h l e n b e c k and R. I. G u m p o r t , The Enzymes 15, 31 (1982). 10 R. Silber, V. G. Malathi, and J. Hurwitz, Proc. Natl. Acad. Sci. U.S.A. 69, 3009 (1972).
[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
305
Phe - CA) lacking modified bases of the natural product, the unmodtRNA~tJA( ified transcript stringently meets the criteria for efficient and accurate suppression. Runoff transcription may be considered the method of choice because it can produce tens of milligrams of suppressor tRNA within a few days and is less costly and less labor-intensive than the anticodon loop replacement procedure. However, as a note of caution, it has recently been demonstrated that tRNA base modifications help prevent misacylation by noncognate tRNA synthetases in the case of yeast tRNAAsp, l~ Though the E. coli-derived transcription-translation system we employ performs equally with native and unmodified suppressor tRNAs, it is possible that other expression systems may require modified suppressor to avoid editing or misacylation by endogenous aminoacyl tRNA synthetases.
General Methods
Enzymes are stored at - 20° in buffered glycerol solutions unless otherwise noted. RNase A is obtained as a powder from Sigma (St. Louis, MO) and stored at - 20° as a stock solution of 10 mg/ml in doubly distilled water. T4 RNA ligase is either obtained from Takara Shuzo Inc. or prepared as described by Heaphy et al. 12 T4 polynucleotide kinase is either obtained from New England Biolabs (Beverly, MA) or prepared as described by Richardson 13 with the modifications of Wu and Kaiser, 14 H~inggi et al., 15 and Cameron and Uhlenbeck.16 Yeast phenylalanine-specific tRNA (yeast tRNA Phe) is obtained from Boehringer Mannheim (Indianapolis, IN). All enzyme reaction buffers are made up with sterile diethylpyrocarbonate (DEPC) treated doubly distilled water. The pH values of the buffers are adjusted at room temperature, and the buffers are filtered through 0.22-/zm sterile filters prior to use. Polypropylene SS-34 centrifuge tubes (Sorvall, Norwalk, CT) are soaked in 0.1% (v/v) diethyl pyrocarbonate in ethanol for several hours, then autoclaved prior to use. Phenol (Bethesda Research Laboratories, Gaithersburg, MD) used in phenol extractions is prepared as described. ~7Etha-
11 V. Perret, A. Garcia, H. Grosjean, J.-P. Ebel, C. Florentz, and R. Geige, Nature (London) 344, 787 (1990). 12 S. Heaphy, M. Singh, M. J. Gait, Biochemistry 26, 1688 (1987). 13 C. C. Richardson, Proc. Natl. Acad. Sci. U.S.A. 54, 158 (1965). 14 R. Wu and A. D. Kaiser, Proc. Natl. Acad. Sci. U.S.A. 57, 170 (1967). 15 U. J. Hanggi, R. E. Streeck, H. P. Voigt, and H. G. Zachau, Biochim. Biophys. Acta 217, 278 (1970). 16 V. Cameron and O. C. Uhlenbeck, Biochemistry 16, 5120 (1977). 17 E. F. Sambrook, E. F. Fritsch, and T. Maniatis, in "Molecular Cloning: A Laboratory Manual, 2nd E d . " Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.
306
PROTEINS AND PEPTIDES" PRINCIPLES AND METHODS
[15]
nol precipitations are carried out by the addition of 0. I volumes of sterile aqueous 2.5 M sodium acetate, pH 4.5, to an aqueous solution of tRNA, followed by addition of 2.5-3 volumes of ice-cold ethanol. Transfer RNA suspensions precipitated in Eppendorf tubes are chilled on dry ice for 10 min prior to microcentrifugation (10 min); tRNA suspensions precipitated in Sorvall SS-34 tubes are chilled at - 2 0 ° for 2-10 hr then spun at 15,000 rpm at 4 ° for 10-30 min in an SS-34 rotor. Transfer RNA is quantitated by measuring the optical density of an aqueous solution at 260 nm (OD260), where 1 OD260 unit corresponds to a concentration of 40 txg tRNA/ml. Denaturing polyacrylamide gel electrophoresis (DPAGE) of tRNA is carried out on analytical (0.75 mm) and preparative (1.5 mm) gels. Analytical DPAGE gels contain 10% (w/v) acrylamide, 19 : 1 (w/w) acrylamide : bisacrylamide, 7 M urea, 50 mM Tris-borate, pH 8.3, and 1 mM EDTA. DPAGE gels are preelectrophoresed at 600 V for 0.5-2 hr prior to loading the samples. Analytical gels are run at 15 or 20 W constant power, and 8% preparative gels are run at 25 or 30 W constant power. Analytical gels are visualized by staining with ethidium bromide followed by UV transillumination. Preparative gels are visualized by staining with 0.02% aqueous toluidine blue O (Aldrich, Milwaukee, WI) or by UV shadowing over an activated TLC plate. Nucleic acid is recovered from preparative gels by excising the desired band with a razor blade, mincing the gel slice, and soaking overnight at room temperature in 5 volumes of elution buffer [50 mM sodium acetate, pH 4.5, 1 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS)]. The supernatant is removed and saved, and an additional 5 volumes of elution buffer is added. After 6 hr of soaking, the supernatant is removed. The combined supernatants are filtered through a 0.45-tzm sterile filter, extracted once with phenol then once with chloroform, and then precipitated twice with ethanol (note that for the first ethanol precipitation it is not necessary to add sodium acetate prior to addition of the ethanol since the elution buffer already contains 50 mM sodium acetate). Alternatively, nucleic acid may be electroeluted from the excised gel slice and rapidly desalted on a sizing chromatography column. R u n o f f transcription o f t R N A ~ A ( - CA) 4 R u n o f f Transcription. Plasmid pYPhe2 is digested with F o k I and used as template for runoff transcription according to Noren et al. 4 Transcription reactions (300/.d) contain 30/zg FokI-cleaved pYPhe2, 40 mM Tris hydrochloride pH 8.1, 20 mM MgClz, 5 mM dithiothreitol (DTT); 50/xg/ ml bovine serum albumin, 1 mM spermidine, 4 mM each neutralized nucleoside triphosphate (NTP), 0.25 units inorganic pyrophosphatase, 0.08
[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
307
units/ml RNase inhibitor and 25-30 units//zl T7 RNA polymerase. Reactions are incubated for 150 min at 42 °, then supplemented with 200/xl of the same solution except containing 10 mM each NTP, 30 mM MgCI2,5-6 units/txl T7 polymerase and lacking template. Reactions are then further incubated overnight at 38°, quenched with 50 tzl of 0.5 M EDTA pH 8, extracted twice with (1 : 1 v/v) phenol : chloroform, once with chloroform and then precipitated with ethanol. Runoff tRNA prepared in this manner is purified on an 8% denaturing gel and yields - 1 I00/xg per 500 tzl reaction mixture. It is important for the T7 RNA polymerase to be sufficiently concentrated and active to produce accurate transcripts. We have noticed that older preparations of polymerase may "stutter" near the template 3' terminus resulting in a heterogeneous population of transcripts. T7 R N A polymerase purification. E. coli strain BL21, containing the plasmid pAR1219 is used for this preparation. Plasmid pAR1219 (amp l) contains the gene for T7 RNA polymerase under transcriptional control of the lac promoter. This strain is grown at 37° and maintained on plates containing ampicillin. Six 4-liter culture flasks are loaded with one liter each of autoclaved growth media containing 1 g NH4CI, 3 g KH2PO 4, 6 g Na2HPO 4, 0.5 g NaCI, 1% tryptone, 2 mM MgSO4, 0.1 mM CaCI2,0.2% glucose, and 100 mg ampicillin. The medium is equilibrated at 37° with vigorous shaking and inoculated with overnight starter culture (12 ml/liter) grown in the same medium. Cells are grown until A590 = 0.9 (2 hrs) after which is added 119 mg of isopropylthio-fl-D-galactoside (IPTG) dissolved in 5 ml of the same medium. Cells are then incubated for an additional 4 hrs, cooled, harvested (GS-3 rotor, 6000 rpm, 10 min, 4°), washed with 500 ml of buffer W (50 mM Tris hydrochloride, pH 8.0 and 25% sucrose) and frozen at - 80°. All further operations are carried out at 4 °. Cells (20.0 g) are thawed and suspended in 270 ml of Buffer L (1.18 g NH4Cl/liter, 20 mM Tris hydrochloride, 1 mM EDTA, 1 mM DTT, 2/xg/ ml phenylmethylsulfonyl fluoride (PMSF), 1/xg/ml leupeptin, and 5% (v/ v) glycerol), then lysed with hen egg white lysozyme solution. After a 30 min incubation, 5 ml of sodium deoxycholate solution (50 mg/ml) is added and the mixture is incubated for 30 min with shaking. The viscous lysate is spun at 40,000 rpm for 4 hrs in a Dupont T-865 rotor at 4 ° and supernatant is then carefully transferred to a GS-3 rotor bottle. Solid ammonium sulfate (80 g) is added slowly to the supernatant on ice (300 ml), and dissolved completely with gentle stirring for 30 min. After sitting an additional 20 min on ice, the white precipitate is collected by centrifugation in a GS-3 rotor at 9000 rpm for 1 hr. The precipitate is then dissolved in 20
308
PROTEINS AND PEPTIDES: PRINCIPLES AND METHODS
[15]
ml of Buffer I (10 mM KH2PO4, pH 8.0, 0.I mM EDTA, 2 ~g/ml PMSF, 0.07% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, and 0.15 M NaCI) and dialyzed against 1 liter of Buffer I. The dialyzed ammonium sulfate fraction is spun down in an SS-34 rotor at 10,000 rpm for 20 min to remove particulates and the clear fraction is applied, at a flow rate of 0.5 ml/min, to a column (2.5 × 20 cm) of P11 pre-equilibrated in Buffer I. After collecting the loading fractions, the column is washed with 10 column volumes of Buffer I until the OD280 is less than 0.1. Bound proteins are then eluted with a 500 ml linear gradient from 0.25 to 1.0 M NaCI in Buffer I at a flow rate of 1 ml/min and fractions of 7 ml are collected. The absorbance at 280 nm is monitored, and the peak fractions again are analyzed by 7.5% SDS-PAGE. Peak fractions containing T7 RNA polymerase are pooled and concentrated with an Amicon concentrator (PM30 membrane; Lexington, MA) to a protein concentration of 1 mg/ml. The concentrated fraction is dialyzed against 2 × 1 liter changes of Buffer II (5.4 g NH4C1/liter, 0.05 mM EDTA, 10 mM Tris hydrochloride pH 8.0, 1 mM DTT, and 50% (v/v) glycerol. After the activity assay, 200/zl aliquots (specific activity: 256,000 units/mg, recovery yield: 21%) are prepared and stored at - 20°. Assay reactions (100/.rL) for T7 RNA polymerase contain: 40 mM Tris hydrochloride, pH 8.0, 20 mM MgCI2, 5 mM DTT, 0.4 mM each NTP, 50 /zg/ml BSA, 36 gtg/ml plasmid pSG1, and diluted enzyme. Reactions are supplemented with [3H]-UTP (13.7 Ci/mmol) to a final specific activity of 18 mCi/mmol UTP. Enzyme fractions are diluted 1 : 100 to 1 : 1000 with 10 mM Tris hydrochloride, pH 7.5, I0 mM 2-mercaptoethanol, 1 mg/ml BSA such that each assay reaction ultimately contains 10-100 ng enzyme. Reactions are incubated at 37° for 15 min, and acid-insoluble radioactivity is determined by diluting the reactions with I ml of cold 5% (w/v) trichloroacetic acid (TCA) and allowed to remain on ice for 30 min. Precipitated material is collected on Whatman GF-C glass fiber filters (premoistened with 5% TCA). Filters are washed successively with 5 ml each of 5% TCA, a 9 : 1 mixture of 5% TCA: saturated sodium pyrophosphate, aqueous 1 N HC1, and 95% ethanol. Filters are then dried and counted. One unit of enzyme activity is defined as the amount of enzyme required to convert 1 nmol [3H]-UTP to acid-insoluble form in 60 min.
Chemical~Enzymatic Generation 0pf t R N A cPhe uA Y-37 Depurination and Backbone Cleavage. Yeast tRNA ehe (16 rag) is dissolved in 15.2 ml of sterile ddH20. Addition of 0.1 M HC1 (0.4 ml) brings the pH to 3.5; addition of 10 mM HC1 ( - 1 . 2 ml) brings the pH to
[15]
INTRODUCING UNNATURAL AMINO ACIDS INTO PROTEINS
309
2.9. It is important to titrate the pH of the solution slowly so as not to bring the pH below the desired value of 2.9. Readjustment of an overly acidic tRNA solution to pH 2.9 with dilute base (10 mM NaOH) results in nonspecific cleavage of the tRNA (as judged by analytical DPAGE). The acidified tRNA solution is incubated at 37° for 3 hr, then divided into two SS-34 tubes and precipitated with ethanol. Each pellet is resuspended in 4.5 ml doubly distilled water (added directly to the SS-34 tube), and 4.5 ml of 0.9 M aniline hydrochloride (pH 4.5) is added. After 5 hr at room temperature with occasional vortexing, the tRNA is recovered by ethanol precipitation. Cleaved tRNA is separated from intact tRNA by preparative DPAGE (0.15 x 16 x 42 cm; 5 mg crude tRNA is loaded onto each gel). The gel is stained with toluidine blue, and the bands are excised and eluted as described in the general methods section. The two tRNA half-molecules (intermediate 2) (271 OD260, 10.8 mg, 67%) are recovered by ethanol precipitation. Complete separation of uncleaved material (
