Thymidine Kinase Mutants Obtained by Random Sequence Selection (Random Nucleotide Sequences/Evolution/Mutations) KHAN M

Home , Thymidine kinase

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4012-4016, May 1993 Biochemistry Thymidine kinase mutants obtained by random sequence selection (random nucleotide sequences/evolution/mutations) KHAN M. MUNIR, DAVID C. FRENCH, AND LAWRENCE A. LOEB* Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, SM-30, University of Washington, Seattle, WA 98195 Communicated by Earl Benditt, December 30, 1992

ABSTRACT Knowledge of the catalytic properties and have been used to define the sequence specificity of DNA structural information regarding the amino acid residues that binding proteins (5, 6), to generate active ribozymes (7, 8), comprise the active site of an enzyme allows one, in principle, and to pan for new ribo- and deoxyribooligonucleotides that to use site-specific mutagenesis to construct genes that encode bind to specific ligands or cellular receptors that mimic enzymes with altered functions. However, such information polypeptide drugs (9-12). A further advance has been the about most enzymes is not known and the effects of specific insertion of random nucleotide sequences into phage display amino acid substitutions are not generally predictable. An libraries for the identification of new binding proteins (13, alternative approach is to substitute random nudeotides for 14). key codons in a gene and to use genetic selection to identify new We demonstrate here the use ofrandom sequence selection and interesting enzyme variants. We describe here the con- to change the catalytic properties of an enzyme without struction, selection, and characterization of herpes simplex knowledge of its three-dimensional structure or even knowl- virus type 1 thymidine kinase mutants either with different edge ofthe specific amino acid residues involved in catalysis. catalytic properties or with enhanced thermostablity. From a Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) library containing 2 x 106 plasmid-encoded herpes thymidine catalyzes the phosphorylation ofdT to thymidine monophos- kinase genes, each with a different nucleotide sequence at the phate in the presence of ATP (15). HSV-1 TK, unlike the putative nucleoside binding site, we obtained 1540 active mammalian or E. coli TK, can also phosphorylate dTMP, mutants. Using this library and one previously constructed, we deoxycytidine, and a variety of nucleoside analogues such as identified by secondary selection Escherichia coli harboring 3'-azido-3'-deoxythymidine (AZT) and acyclovir (15, 16). thymidine kinase mutant clones that were unable to grow in the This phosphorylation of nucleoside analogues that inhibit or presence of concentrations of 3'-azido-3'-deoxythymidine terminate DNA replication is the basis of drug therapy (AZT) that permits colony formation by E. coi harboring the against herpetic infections. The gene for HSV-1 TK has been wild-type plasmid. Two of the mutant enzymes exhibited a cloned, sequenced (17, 18), and expressed in E. coli (19); a reduced Km for AZT, one of which displayed a higher catalytic crystal structure is not yet available (20). The ATP binding efficiency for AZT over thymidine relative to that of the wild site has been mapped and the nucleoside binding site has been type. We also identified one mutant with enhanced thermosta- putatively identified as encompassing amino acid residues bility. These mutants may have clinical potential as the promise 165-176 (21-23). The informational content of each amino of gene therapy is increasingly becoming a reality. acid residue within this sequence has been assessed by substituting an oligonucleotide that was 20% degenerate for Genetic diversity can be achieved in vitro by inserting codons 165-175 (24). Here we present the results of substi- random nucleotide sequences into cloned genes. By genetic tuting a 100% random nucleotide sequence for 33 nucleotides complementation, new mutants that encode active proteins that span positions 165-175. From -2 x 106 transformants can be identified in these random nucleotide sequence librar- we obtained 1540 new active TK mutants. By screening ies. This approach offers the promise of obtaining enzymes mutants from this and previous studies (24) we identified TKs with different substrate specificities or unique physical prop- that selectively phosphorylate AZT as well as one that resists erties. The underlying premise is that multiple amino acid thermal denaturation. sequences can carry out the same or similar reactions and that during the course of evolution many of these sequences were discarded on the basis of fitness criteria that are no MATERIALS AND METHODS longer relevant or ones that are different from those imposed Bacterial Strain, Plasmids, and Transformations. The E. by the experimenter. These techniques of applied molecular coli K-12 TK-deficient strain KY895 (K12, tdk-, F-, ilv 276) evolution (1) could be used to generate entirely new enzy- and anti-TK antibody were gifts of William Summers (Yale matic activities and could provide insights into pathways that University, New Haven). The TK expression vector, pMCC, governed natural selection. and a "dummy vector," pMDC (which expresses an inactive We and others have used positive genetic selection to TK), were constructed as described (24). This vector also demonstrate that new biologically active molecules can be contains a 3-lactamase gene to render transformed bacteria obtained from random nucleotide sequences. Random se- carbenicillin resistant. Bacterial transformations by plasmid quence substitutions within the -35 region ofthe promoter of DNAs were carried out by electroporation (24). the Escherichia coli tetracycline-resistance gene yielded a Oligonucleotides. A 52-mer with wild-type tk sequence, collection of 190 new active promoters (2). New mutants with 5'-d(TG GGA GCT CAC ATG CCC CGC CCC CGG CCC different specificities toward a series of penicillin analogues TCA CCC TCA TCT TCG ATC GCC AT)-3', and a 56-mer were generated by substituting random sequences within a containing random nucleotides, 5'-d(ATG AGG TAC CGN portion of the active site of 8-lactamase gene (3, 4). Parallel NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN strategies based on screening of random sequence libraries NNA TGG CGA TCG AA)-3', where N = equimolar concen-

The publication costs of this article were defrayed in part by page charge Abbreviations: AZT, 3'-azido-3'-deoxythymidine; HSV-1, herpes payment. This article must therefore be hereby marked "advertisement" simplex virus type 1; TK, thymidine kinase; DTT, dithiothreitol. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4012 Downloaded by guest on October 5, 2021 Biochemistry: Munir et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4013 trations of G, A, T, or C, were synthesized by Operon erate library (TKF) (24) were subjected to secondary negative Technologies (Alameda, CA). The oligonucleotides were selection on medium containing AZT to identify mutants that separated by electrophoresis through a 20% denaturing poly- exhibit enhanced phosphorylation of AZT. This screen is acrylamide gel followed by purification on a reverse-phase based on the premise that mutants with increased ability to mini column (Glen Research, Sterling, VA). phosphorylate AZT relative to dT would be unable to form Random Sequence-Containing Libraries. The random se- colonies on the AZT-selection medium, since the product, quence library was constructed by inserting an oligonucleo- AZT monophosphate, would be further phosphorylated by tide containing a stretch of 33 random nucleotides between the host cell's nonspecific nucleotide kinases or possibly the two unique Kpn I and Sac I restriction sites within the mutant TK and then incorporated into bacterial DNA by host putative nucleoside binding site in the HSV-1 tk gene. A DNA polymerases, terminate DNA synthesis, and prevent synthetic 52-mer corresponding to the wild-type HSV-1 tk replication ofthe host chromosome. We first determined that sequence containing a Kpn I site at the 5' end was hybridized 1 ,ug ofdT per ml was the lowest concentration that supported with a 56-mer containing random nucleotides corresponding colony formation by E. coli harboring wild-type TK as well to HSV-1 tk codons 165-175 (N = A, C, G, or T) and as most of the TK mutants obtained in the primary TK- containing a Sac I site at the 3' end. The methods for selection protocol. We also determined that wild-type tk extension, PCR amplification, and ligation of this fragment harboring E. coli could form colonies on medium containing into the E. coli expression vector pMDC to replace the 1.0 ,ug of dT per ml plus 0.05 ug of AZT per ml. Of 880 nonfunctional insert have been described (24). The ligated primary selectants that we screened, only two, TKF 105 product was introduced into tk- E. coli strain KY895. The (from the 20% library) and TKI 208 (from the 100% library), total number of transformants was determined by plating on formed colonies on the TK-selection medium at an efficiency LB agar containing 50 ,ug of carbenicillin per ml and the similar to that of E. coli harboring the wild-type plasmid and number of transformants that produced catalytically active yet failed to form colonies on the AZT-selection medium thymidine kinase was determined by plating on TK-selection (Fig. 1A). The nucleotide and deduced amino acid sequences medium [2% BBL peptone, 0.5% NaCl, 0.2% glucose, 0.8% of TKF 105 and TKI 208 are presented in Fig. 1B. Both Gel-Rite (Scott Laboratories, Carson, CA)], 50 ,g of car- mutants contain a single amino acid substitution at the same benicillin per ml, 10 ,ug of fluorodeoxyuridine per ml, 2 ,ug of position: Leu-170 was changed to Ile in TKF 105 and to Val dT per ml, and 20 ug of uridine per ml (24). in TKI 208. No other substitutions were observed in the Selection of AZT-Sensitive TK Mutants. A subset of 690 surrounding 220 nucleotides. To make sure that this differ- mutants from the 100% random library and 190 previously ence was not due to differential expression of TK in E. coli isolated mutants from the 20% degenerate library (24) were harboring mutant and wild-type plasmids, we compared subjected to secondary screening to identify AZT-sensitive Western blots ofextracts from cells containing either TKI 208 clones. The mutants were first grown as individual colonies or wild type. No significant difference was observed in the on TK-selection medium (1.0 ,g of dT per ml) and then amount or electrophoretic mobility of immunoreactive stain- replica plated onto AZT-selection medium (0.05 ,mg of AZT ing protein (data not shown). Also, the rate of dT phosphor- per ml, 1.0 ug of dT per ml). All other components in the ylation per mg of protein was similar in extracts of E. coli AZT-selection medium are the same as the TK-selection harboring TKI 208, TKF 105, and wild-type plasmids (data medium. Those TK mutants that failed to grow on the not shown). AZT-selection medium were picked and retested for growth That the lack of growth ofthese mutants on AZT-selection on TK- and AZT-selection media separately. medium is due to enhanced phosphorylation ofAZT is further Affinity Purification. Purification of wild-type and mutant indicated by two criteria. (i) We determined the rate of TKs was performed by affinity chromatography on CH- [3H]AZT uptake relative to [3H]dT into E. coli harboring Sepharose 4B (Pharmacia) coupled to p-aminophenylthymi- wild-type and mutant plasmids. Studies have indicated that dine 3'-phosphate (25, 26). Crude bacterial extract (24) was dT uptake in E. coli is correlated with the amount of TK passed three times through a 7-ml bed-volume affinity col- activity (27, 28). We found that E. coli harboring the AZT- umn. The column was then washed sequentially using 30 ml sensitive mutants, TKF 105 and TKI 208, exhibited a 4-fold each of buffer A [0.1 M Tris HCl, pH 7.5/5 mM dithiothreitol increase in the ratio ofAZT to dT uptake compared to E. coli (DTT)/10% glycerol], buffer B (0.1 M Tris-HCl, pH 7.5/0.5 with the wild-type plasmid (results not shown). (ii) We M KCl/5 mM DTT/10% glycerol), and buffer A. TK was determined the kinetics of AZT phosphorylation (Table 1). eluted using a 60-ml linear gradient of 0-600 ,uM dT in buffer The AZT-sensitive variant TKI 208 (Table 1) exhibits a lower C (0.3 M Tris HCl, pH 7.4/50 mM KCl/10% glycerol). Active Km (4.4 uM) compared to that of the wild type, 8.5 u.M. By fractions were pooled and dialyzed against three changes comparing the kcat/Km between the two substrates (AZT vs. each of 2 liters of 50 mM Tris HCl, pH 7.4/5 mM DTT/10% dT), we find that TKI 208 selectively phosphorylates AZT glycerol. Except in the final dialysis, all the above buffers 2.3-fold more efficiently than dT. Our preliminary observa- contained 50 ,ug of aprotinin per ml and 2 jig each ofpepstatin tion with purified TKF 105 TK also showed lower Km (3.7 and leupeptin per ml. ,M) for AZT but similar values for kcat/Km compared to the wild type (data not shown). Enhanced Thermostability. We analyzed the thermostabil- RESULTS ity of50 TKF mutants. One ofthe mutants, TKF 2, was more Construction and Characterization of Random Sequence- thermostable at 42°C than any ofthe other mutants or than the Containing Library. The scheme for the insertion of a 33- wild type. Except for TKF 2, all of the mutants tested, nucleotide 100% random sequence within the herpes tk is including the wild type, had ratios of residual activity after similar to that previously used in the construction of a library preincubation at 42°C compared to 34°C of 0.05-0.30; TKF with 20% degeneracy (24). Functional tk mutants were iden- 2 had a ratio of 0.7. TKF 2 contains three amino acid tified by colony formation on TK-selection medium based on substitutions: Pro-165 -* His, Ala-167 -- Ser, and Ala-174 -* their ability to phosphorylate dT. We screened 2 x 106 Val (Fig. 1B). We also examined our collection of mutants for transformants from the 100% random library, of which 1540 the corresponding single amino acid substitutions. TKF 75 formed colonies on the TK-selection medium. contained an Ala-167 - S ersubstitution and TKF 56 con- Selection Based on the Enhanced Phosphorylation of AZT. A tained Ala-174 -* Val, whereas TKI 440 with Pro-165 -+ Ala subset of 690 mutants from the 100% random library (TKI) was the closest to the Pro-165 -- His substitution. Analyses and 190 mutants previously identified from the 20% degen- of the thermostability of the unfractionated extracts from Downloaded by guest on October 5, 2021 4014 Biochemistry: Munir et al. Proc. Natl. Acad. Sci. USA 90 (1993) A. TK-selection AZT-selection B. Sequence

.z\ 165 166 167 168 169 170 171 172 173 174 175 CCC ATC GCC GCC CTC CTG TGC TAC CCG GCC GCG Wild-Type Pro lie Ala Ala Leu Leu Cys Tyr Pro Ala Ala

CCC ATC GCC GCC CTC TGC TAC CCG GCC GCG TKF 105 Pro lie Ala Ala Leu lie Cys. Tyr Pro Ala Ala .7.3

:. I CCC ATC GCC GCC CTC [ TGC TAC CCG GCC GCG TKI 208 Pro lie Ala Ala Leu | Cys Tyr Pro Ala Ala - i

I ) ATC E GCC CTC CTG TGC jm pi TAC CCG GCG TKF 2 .... Ile Ala Leu Leu Cys Tyr Pro [aI| Ala

FIG. 1. Selection ofAZT-specific TK mutants. (A) Two AZT-sensitive clones, TKF 105 and TKI 208, along with an AZT-insensitive mutant, TKF 2, and the wild type were grown on TK- and AZT-selection media. The wild type and TKF 2 each formed a similar number of colonies on TK- and AZT-selection media. In contrast, TKF 105 and TKI 208 showed almost no visible colonies in the presence of AZT. In other experiments, TKF 105 and TKI 208 formed colonies on AZT-selection medium but they were 40-60%o fewer in number and smaller in size than those formed on the TK-selection medium. (B) The nucleotide and amino acid sequences within the targeted region are indicated; codon and amino acid substitutions are boxed and the codon numbers are presented above the wild-type nucleotide sequence.

each of these mutants at 42°C are presented in Fig. 2. The vector with a promoter for T3 RNA polymerase. The RNA thermolability of mutants TKF 56 and TKF 75 with Ala-174 produced by transcription in vitro was translated using a

-* Val and Ala-167 -* Ser substitutions, respectively, was rabbit reticulocyte lysate with [35S]methionine. An autoradi- similar to that of the wild type. Both lost >80% of their ograph ofthe labeled proteins after SDS/PAGE (Fig. 3 Inset) activity after incubation for 5 min at 42°C. TKF 440 with a indicates that the translation products migrate as double Pro-165 -* Ala is more stable but not as stable as TKF 2, the bands corresponding to a protein of 43 kDa, which is in triple mutant. accord with the reported size of HSV-1 TK expressed in E. Two types of experiments were carried out to verify the coli (19, 20). The two bands could be due to the proteolytic thermostability of TKF 2. (i) We purified to near homoge- degradation of a 32-residue fragment at the amino-terminal neity TKs from TKF 2 and the wild-type plasmid harboring end, which does not detectably alter TK activity of the E. coli and determined that the loss of activity is less in TKF HSV-1 TK (19, 20). The loss of TK activity of the in vitro 2 than in the wild type after preincubation at 42°C (Fig. 2E). synthesized proteins from the wild-type and TKF 2 tk genes (ii) We transferred TKF 2 and the wild-type tk genes into a as a function of preincubation at 42°C is shown in Fig. 3. The Table 1. Ability of wild-type and mutant HSV-1 TKs to phosphorylate AZT and dT kcat/Km, kcat/Km (AZT) Phosphorylation Km, ,uM kcat, s-1 s-LM-1 kcat/Km (dT) AZT Wild-type 8.46 ± 1.3 3.6 x 10-2 4.2 X 103 1.7 x 10-3 TKI 208 4.40 ± 0.43* 3.0 x 10-2 6.5 x 103 4.0 x 10-3 dT Wild-type 0.475 ± 0.10 1.21 2.5 x 106 TKI 208 0.35 ± 0.008 0.56 1.57 x 106 Phosphorylation ofAZT: Kinetic analyses ofTK purified from wild-type and AZT-sensitive mutants. Reactions were carried out in a final volume of 100 ,ul containing 50 mM Tris HCl (pH 7.5), 5 mM ATP, 4 mM MgCl2, 2.5 mM DTT, 12 mM KCI, 0.18 mg of bovine serum albumin per ml, 5% glycerol, 0.08 ,Ci of [3H]AZT (Sigma), various concentrations of unlabeled AZT (0-4.0 ,uM), and purified enzymes (4 and 1.2 units, respectively, for wild-type and TKI 208). One unit ofenzyme is defined as that amount that can phosphorylate 1.0 pmol of dT to TMP in 1 min under the conditions described above. Incubation was at 34°C ± 1C for 10 min and reactions were stopped by adding 1.0 mM unlabeled dT and cooling on ice. Half of the reaction mixtures were pipetted onto a DEAE-cellulose disc (25 mm), dipped in distilled water (1 min), followed by four washes in absolute ethanol. The amount of radioactivity adsorbed to the disc was determined by scintillation spectroscopy. Km and Vmax values were determined by using the Cleland SUBIN program (29). The values for kcat were calculated using the equation Vm = kcat4[Elo, where [Elo = total enzyme concentration. Phosphorylation of dT. TK assays were carried out in a final volume of 50 ,ul using 0.3 ,Ci (3H-methyl]dT; 87 Ci/mmol; Amersham) and various concentrations of unlabeled dT (0-4.0 uM) and 1.1 and 0.5 unit of TK for the wild type and TKI 208, respectively. All other components in the reaction mixtures and the incubation conditions were as described for phosphorylation of AZT. *Statistically significant values compared with wild type, P < 0.02. Downloaded by guest on October 5, 2021 Biochemistry: Munir et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4015

TFA B C DE TKF56 TKF75 TKF 440 a) AlX>-tyAa174--a174 Ala167-*-Ser167 Pro -.Ala165 cr 10- TKF2 Wild-typeWidTp 0-0 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 40 Preincubation (min) FIG. 2. (A-D) Thermostability of wild-type and TK mutants using crude extracts. Twenty-five micrograms of each extract in 0.3 ml of 28 mM Tris*HCl, pH 7.5/0.28 mg of bovine serum albumin per ml/28 ,ug of aprotinin per ml/2 ug (each) of pepstatin and leupeptin per ml was preincubated at 42°C for 0, 5, 10, 20, 30, or 40 min. At each time point 30-,ul (2.5 ,ug) aliquots were assayed for residual TK activity in a total reaction volume of 50 ,ul containing 50 mM Tris HCl (pH 7.5), 5 mM ATP, 4 mM MgCl2, 2.5 mM DTT, 12 mM KCI, 0.18 mg of bovine serum albumin per ml, 5% glycerol, and 1 ,uM [3H-methyl]dT (60 x 103 dpm/pmol). Incubation was at 34°C for 10 min. The reaction was stopped by cooling on ice, and 25 ,ul was pipetted onto a DEAE-cellulose disc. Washing and assaying radioactivities ofthe discs were performed as described for the AZT assay in Table 1. (E) Thermostabilities ofpurified wild-type and TKF 2 enzymes. Affinity-purified enzymes were concentrated with a Centricon 30 filtration unit prior to use in the assays. The amount of enzyme used was adjusted so that a similar amount ofactivity was present in each sample prior to preincubation. protein encoded by TKF 2 lost <10% of its activity after ning codons 165-175 of the tk gene. Two of the mutants preincubation for 45 min. In contrast, the protein encoded by studied contain amino acid substitutions for Leu-170 and are the wild-type gene lost >80% ofits initial activity. The degree sensitive to AZT. Another mutant, containing three different of thermostability exhibited by the in vitro synthesized TKF amino acid substitutions, encodes a TK that is highly ther- 2 was similar to or greater than that of crude extracts mostable relative to the wild-type enzyme. This method for harboring the original TKF 2 plasmid. random sequence selection can be scaled up and yield even larger collections of mutants with different properties. Based on the efficiency ofDNA transformation by current methods DISCUSSION ('1%) and on procedures for identifying mutants by genetic We have obtained >1700 active TK mutants from two selection, we estimate that it is feasible to select mutants that libraries by selection from -2.5 x 106 recombinant plasmid code for active enzymes from as many as 1011 E. coli clones containing a segment of 33 random nucleotides span- transformed with plasmids containing random sequences. Once information is obtained about permissible substitutions 14 , _ (24), the inserts can be redesigned to randomize only those vvI-I TKF 2 substitutions that are most likely to yield enzymes with desired properties. Many current methods for protein engineering utilize site- 0) c \ V..: specific nucleotide substitutions to change the amino acid sequence of proteins. Specified amino acid substitutions E 4":3 kDa within enzymes have been used to alter substrate specificity, a) to determine functional residues, or to redesign active sites Wild-Type TKF 2 (30-34). However, tailoring enzymes by site-specific mutagenesis is currently limited: (i) it is conveniently guided by .: structural knowledge (only a few are known), (ii) the effects 0en of most single amino acid substitutions are not easily predictable, and (iii) the rules to analyze the effects of multiple Wild-1rype amino acid substitutions are only beginning to be developed (35), even though multiple substitutions are more likely to 0 10 20 30 40 50 yield mutants with unusual properties (1, 36). We have shown here that random sequence selection offers an attractive Preincubation (min) alternative to site-specific mutagenesis for the generation of enzymes with improved properties and that random sequence FIG. 3. Heat-inactivation profiles of in vitro translated wild-type selection does not require a detailed knowledge of the three- and TKF 2 TK. The full-length Bgl II-Pvu II fragments of tk genes dimensional structure of the enzyme. from wild-type and TKF 2 plasmids were isolated and subcloned into the pBluescript SK+ (Stratagene) vector between the Spe I and The secondary screening procedure for AZT-specific mu- EcoRI sites with the use of synthetic linkers. In vitro transcription tants that we used relies on negative genetic selection. The using the T3 promoter was carried out using the Promega transcrip- assumption is that the enhanced phosphorylation of AZT tion system. In vitro translation was carried out using a reticulocyte relative to dT results in the incorporation ofthis analogue into lysate system (Promega) following the supplier's protocol. For DNA and the termination of DNA replication. Presumably, SDS/PAGE analysis, the translated products were labeled with an analogous protocol can be designed to search for mutants [35S]methionine. (Inset) Autoradiograph of the SDS/PAGE- that preferentially phosphorylate other nucleoside ana- fractionated in vitro translated products. The arrow indicates the logues. Only 2 ofthe 880 active tk mutants that we screened, expected size of translated TKs as judged by molecular mass TKF 105 and TKI did not form colonies on AZT- standards (Bio-Rad). For thermostability studies, TKs were synthe- 208, sized in the presence of unlabeled methionine. Three experiments selection medium containing 0.05 ,g of AZT per ml and 1 ,ug were carried out on different days using different amounts of crude ofdT per ml. E. coli harboring either ofthese mutants exhibit preparations and, without exception, TKF 2 was more stable than the a >3.5-fold increase in the uptake of AZT relative to that of wild type. dT when compared to either the wild type or to two other Downloaded by guest on October 5, 2021 4016 Biochemistry: Munir et al. Proc. Natl. Acad. Sci. USA 90 (1993)

mutants that were tested (not shown). Studies on purified TK 1. Eigen, M., McCaskill, J. & Schuster, P. (1988) J. Phys. Chem. from TKI 208 and TKF 105 indicate that the enhanced 92, 6881-6885. catalytic efficiency with AZT as a substrate is predominantly 2. Horwitz, M. S. Z. & Loeb, L. A. (1986) Proc. Natl. Acad. Sci. due to a reduction in the Km. The substituted amino acids, USA 83, 7405-7409. valine and isoleucine, in the two AZT-sensitive mutants are 3. Dube, D. K. & Loeb, L. A. (1989) Biochemistry 28, 5703-5707. not structurally very different from that of the wild-type 4. Oliphant, A. R. & Struhl, K. (1989) Proc. Natl. Acad. Sci. USA Leu-170. These results indicate that an isosteric change with 86, 9094-9098. 5. Blackwell, T. K. & Weintraub, H. (1990) Science 250, 1104- no addition or removal of polar groups and no major confor- 1110. mational alteration is likely to have taken place in producing 6. Tuerk, C. & Gold, L. (1990) Science 249, 505-510. the mutant enzymes. The finding that the two mutants 7. Robertson, D. L. & Joyce, G. F. (1990) Nature (London) 344, identified as AZT-sensitive contain a single amino acid sub- 467-468. stitution at Leu-170 highlights the importance of this residue 8. Beudry, A. A. & Joyce, G. F. (1992) Science 257, 635-641. in substrate binding. 9. Ellington, A. D. & Szostak, J. W. (1990) Nature (London) 346, Random sequence selection of P-lactamase mutants in our 818-822. laboratory (3) and Struhl's laboratory (4) as well as studies on 10. Ellington, A. D. & Szostak, J. W. (1992) Nature (London) 355, HSV-1 TK (37) indicate that the many mutant enzymes 850-852. obtained by random sequence selection are thermolabile. 11. Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kaz- Only 1 of the 50 mutants that we have analyzed was signif- enierski, W. M. & Knapp, R. J. (1991) Nature (London) 354, icantly more thermostable than the wild type. The loss of 82-84. 12. Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., activity at 42°C for up to 40 min in extracts from either TKF Dooley, C. T. & Curevo, J. H. (1991) Nature (London) 354, 2 or the wild type was not the result of proteolysis since there 84-86. was no degradation in immunoreactive protein upon electro- 13. Devlin, J. J., Panganiban, L. C. & Devlin, P. E. (1990) Science phoresis after incubation at 42°C. The thermostability of TKF 249, 404-406. 2 TK was verified by analyzing the purified enzyme and the 14. Scott, J. K. & Smith, G. P. (1990) Science 249, 386-390. enzyme synthesized in vitro. 15. Chen, M. S., Walker, J. & Prusoff, W. H. (1979) J. Biol. Chem. We analyzed the contribution of each of the three amino 254, 10747-10753. acid substitutions in TKF 2 to its enhanced thermostability by 16. Elion, G. B., Furman, P. A., Fyfe, J. A., Miranda, P. D., studying extracts of mutants that contained only single amino Beuchamp, L. & Schaeffer, H. J. (1977) Proc. Natl. Acad. Sci. acid substitutions at the same positions. Only one mutant USA 74, 5716-5720. 17. Wagner, M., Sharp, J. A. & W. C. Proc. with Pro-165 -- Ala is more thermostable than the wild type Summers, (1981) Natl. but is not as thermostable as TKF 2 (Fig. 2). These results Acad. Sci. USA 78, 1441-1445. 18. McKnight, S. L. (1980) Nucleic Acids Res. 8, 5949-5964. suggest that none of the single substitutions is adequate to 19. Waldman, A. S., Haeusslein, E. & Millman, G. (1983) J. Biol. account for the large enhancement in thermostability exhib- Chem. 258, 11571-11575. ited by TKF 2. The identification of a thermostable mutant 20. Sanderson, M. R., Freemont, P. S., Murthy, H. M. K., suggests that a systematic approach to selecting thermostable Krane, J. F., Summers, W. C. & Steitz, T. A. (1988) J. Mol. enzymes from random sequence-containing libraries might Biol. 202, 917-919. be fruitful. Mutants could be selected directly based on the 21. Liu, Q. & Summers, W. C. (1988) Virology 163, 638-642. thermostability of the transformed gene products in thermo- 22. Darby, G., Larder, B. A. & Inglis, M. M. (1986) J. Gen. Virol. philic bacteria (38, 39). The production of enzymes that are 67, 753-758. stable at elevated temperatures and presumably resistant to 23. Balasubramanium, N. K., Veeisetty, V. & Gentry, G. A. other denaturing agents could have useful industrial and (1990) J. Gen. Virol. 71, 2979-2987. 24. Munir, K. M., French, D. C., Dube, D. K. & Loeb, L. A. clinical applications. (1992) J. Biol. Chem. 267, 6584-6589. The broad substrate specificity of HSV-1 TK renders it a 25. Kowal, E. P. & Markus, G. (1976) Prep. Biochem. 6, 369-385. target for nucleoside analogues that abate herpetic infections. 26. Lee, L. E. & Cheng, Y. C. (1976) J. Biol. Chem. 251, 103-138. The success of treating herpetic infections with nucleoside 27. Mckeown, M., Kahn, M. & Hanawalt, P. J. (1976) Bacteriology analogues has brought into the forefront the possibility of 126, 814-822. inserting the HSV-1 tk gene into target cells and then selec- 28. Dube, D. K., Horwitz, M. S. Z. & Loeb, L. A. (1991) Gene 99, tively killing them by growth on specific nucleoside ana- 25-29. logues. Our present study demonstrates the efficacy of ran- 29. Cleland, W. W. (1979) Methods Enzymol. 63, 103-138. 30. Shortle, D., Di Maio, D. & Nathans, D. (1981) Annu. Rev. dom sequence selection to obtain mutant TKs that preferen- Genet. 15, 265-294. tially phosphorylate specific nucleoside analogues such as 31. Knowles, J. R. (1987) Science 236, 1252-1258. AZT. This methodology can also be extended to produce 32. Carter, P. & Wells, J. A. (1988) Nature (London) 332,564-568. TKs that preferentially phosphorylate gancyclovir and other 33. Wells, J. A., Cunningham, B. C., Graycar, T. P. & Estell, nucleoside analogues. The introduction of these tk mutant D. A. (1987) Proc. Natl. Acad. Sci. 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Horwitz, A; Blank, and A. Mildvan for 39. Alber, T. & Wozniak, J. A. (1985) Proc. Natl. Acad. Sci. USA critical comments. This work was supported by Grants OIG R35- 82, 747-750. CA-39903 (L.A.L.) and AG-00057 (K.M.M.) from the National 40. Culver, K. W., Ram, Z., Wallbridge, S. H., Ishi, E., Oldfield, Institutes of Health. H. & Blaese, R. M. (1992) Science 256, 1550-1552. Downloaded by guest on October 5, 2021