Patent Application: US-25164802-A

Abstract:
a method and composition for trna synthetases that activate and aminoacylate nonstandard and noncognate amino acids to trna adaptor molecules is described that can be used to generate custom designed protein products for uses in medicinal , therapeutic , diagnostic , biotechnology , engineering , and spectroscopy applications . some trna synthetases naturally misactivate and misaminoacylate noncognate amino acids . many of these trna synthetases , including but not limited to leucyl -, isoleucyl -, and valyl - trna synthetases , have evolved proofreading and editing mechanisms to correct these mistakes . inactivation of the enzyme &# 39 ; s editing activity allows and facilitates production and accumulation of trnas that are misaminoacylated with nonstandard and noncognate amino acids . these misaminoacylated trnas can be used to introduce novel amino acids into proteins .

Description:
although making and using various embodiments of the present invention are discussed in detail below , it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts . the specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention , and do not limit the scope of the invention . to facilitate the understanding of this invention , a number of terms are defined within . terms defined and used herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention . terms such as “ a ,” “ an ,” and “ the ” are not intended to refer to only a singular entity , but include the general class of which a specific example is used for illustration . the terminology and examples herein are used to describe specific embodiments of the invention , but their usage does not limit the invention , except as outlined in the claims . misaminoacylation of trna by mutant synthetases or derivatives thereof can be used to enzymatically aminoacylate trnas with nonstandard , unusual or novel amino acids for incorporation into custom designed proteins during ribosomal - based protein synthesis . leurs and also ilers and valrs are organized modularly into three domains ( cusack et al ., 2000 , martinis and schimmel , 1996 ). an n - terminal conserved catalytic core is responsible for atp - dependent activation of amino acids and subsequent aminoacylation to trna molecules . a less or unconserved c - terminal domain is typically responsible for interactions with non - acceptor stem parts of the trna molecule such as the anticodon in trna synthetases that are closely related to leurs . in the case of most leurs proteins , the c - terminal domain as well as any other part of the leurs protein does not interact with the anticodon ( asahara et al ., 1993 ; dietrich et al ., 1990 ). the primary sequence of the conserved catalytic core that folds into a rossmann nucleotide binding fold is split by a protein insertion called connective polypeptide 1 ( cp1 ) ( starzyk et al ., 1987 ). the cp1 protein insert folds into a discrete domain and contains the amino acid editing active site ( chen et al ., 2000 ; hendrickson et al ., 2000 ; lin et al ., 1996 ; mursinna et al ., 2001 ; nureki et al ., 1998 ; nureki et al ., 1999 ). to enhance fidelity , the aarss rely on a “ double sieve ” mechanism for amino acid selection and discrimination ( fersht , 1977 ). the first sieve encompasses the aminoacylation active site that binds cognate amino acids but cannot adequately filter out all closely related amino acids . the second sieve is an editing active site that targets those noncognate amino acids that are misactivated or mischarged but , importantly , bars the correctly charged cognate amino acid from hydrolysis . leurs , valrs , and ilers enzymes edit by homologous cp1 domains ( chen et al ., 2000 ; chen et al ., 2001 ; lin et al ., 1996 ; mursinna et al ., 2001 ; mursinna and martinis , 2002 ). an editing active site hydrolytically cleaves the misactivated aminoacyl - adenylate ( called “ pre - transfer editing ”) or the mischarged trna ( called “ post - transfer editing ”) as shown in the reaction in fig1 ( baldwin and berg , 1966 ; fersht , 1977 ; norris and berg , 1964 ; englisch et al ., 1986 ; schimmel and schmidt , 1995 ). it has been unclear whether the editing active sites for pre - and post - transfer editing are separate , overlap , or partially overlap . separate amino acid binding pockets for pre - and post - transfer editing substrates have been postulated based on modeling and comparative analysis of the homologous valrs and ilers co - crystal structures ( fukai et al ., 2000 ; nureki et al ., 1998 ). the two sites are distinct but proximal to each other and rely on a distinct set of amino acids to confer amino acid specificity . the prediction was based on the location of the terminal adenosine of the complexed trna val and modeling of threonyl - adenylate and threonine in the editing site . threonine , but not valine , could be fit into two distinct pockets . leucyl - trna synthetase ( leurs ) misactivates a diverse group of standard amino acids and non - standard metabolic amino acid intermediates ( apostol et al ., 1997 ; englisch et al ., 1986 ; lincecum jr . and martinis , 2000 ; martinis and fox , 1997 ). because of the shape and the larger size of leucine , specific recognition by leurs may be more complex than simply distinguishing isosteric substrates that differ by a single methyl group by size exclusion . its hydrolytic editing activity targets either misactivated aminoacyl - adenylate or mischarged aminoacyl - trna leu ( chen et al ., 2000 ; chen et al ., 2001 ; englisch et al ., 1986 ; mursinna et al ., 2001 ; mursinna and martinis , 2002 ). previous work has suggested that leurs from different organisms may preferentially rely on either pre - or post - transfer editing mechanisms ( englisch et al ., 1986 ) rather than a combination as found with ilers ( baldwin and berg , 1966 ; norris and berg , 1964 ). in particular , it was proposed that s . cerevisiae cytoplasmic leurs used primarily pre - transfer editing although trna was shown to stimulate the reaction . in contrast , e . coli leurs exclusively utilized a post - transfer editing mechanism to prevent the release of mischarged trna leu . as used herein leucyl trna synthetase includes any variation of the enzyme , including any derivative , natural enzyme , synthetic analog , engineered enzyme , mimetic , single mutant , multiple mutant , mutant with deletions , chimeric molecule , versions that are cognate to other amino acids , and combinations thereof and from any eukaryotic , bacterial , and archaebacterial organism . the chimeric molecules may also include different isoacceptors or non - leucine trnas as well as a linker molecule that may be a protein , peptide , antibody , etc . in one embodiment of the present invention , the inventors use alanine - scanning mutagenesis of e . coli leurs to identify a conserved threonine residue ( thr 252 ) in a cp1 - based threonine - rich region ( fig2 ) that is critical to amino acid substrate discrimination within the amino acid editing active site ( mursinna et al ., 2001 ). substitution via site - directed mutagenesis of the gene encoding e . coli leurs changed the thr 252 residue to an alanine ( t252a ) resulting in uncoupled amino acid specificity and facilitated editing of the correctly aminoacylated leucine - trna leu molecule ( fig3 and 5 ). the full homology modeled and minimized structure for e . coli leurs ( lee and briggs , 2002 ) is shown in fig7 b along with that of the x - ray structure for t . thermophilus ( fig7 a ; cusack et al ., 2000 ) subsequent computational homology modeling of e . coli leurs followed by docking studies suggested that the thr 252 is located near the bottom of a depression on the protein surface that could act as an amino acid binding pocket ( fig6 and 8 ). two bulky amino acids , phenylalanine ( t252f ) and tyrosine ( t252y ), were substituted subsequently for thr 252 by site - directed mutagenesis to block the amino acid binding pocket and abolish amino acid editing activity ( mursinna and martinis , 2002 ). the mutant enzymes aminoacylated leucine similar to the wild - type leurs ( fig9 ). while wild - type leurs edits isoleucine - trna leu , these two composition inventions of mutant leurs ( t252f and t252y ) stably misaminoacylate the noncognate isoleucine amino acid to trna leu ( fig1 ). primary sequence alignments of leurs , ilers , and valrs enzymes from prokaryotic and also eukaryotic cytoplasmic and mitochondrial origins identified a completely conserved aspartic acid within the cp1 domain that is also found in ilers and valrs ( fig1 ). in one embodiment of the present invention , this aspartic acid was changed to an alanine in e . coli ( ecd345a ) and s . cerevisiae cytoplasmic leurss ( ycd419a ). the mutant and wild - type enzymes from s . cerevisiae and e . coli were isolated by affinity chromatography via an n - terminal six - histidine tag . each of the purified mutant enzymes aminoacylated trna leu with leucine at a significant , but slightly reduced activity compared to the wild - type enzymes ( fig1 and 18 ). the cp1 - based aspartic acid mutant leurss were tested for isoleucylation activity by isolating trna linked to [ 3 h ]- isoleucine via trichloroacetic acid precipitation followed by washing . while neither the wild - type s . cerevisiae cytoplasmic and e . coli leurss yielded mischarged trna leu , both the composition invention ycd419a and ecd345a leurss stably generated isoleucine misaminoacylated to trna leu ( ile - trna leu ) ( fig1 and 19 ). correctly charged and mischarged trna were separated by acid gel electrophoresis ( varshney et al , 1991 ). fig1 shows that ycwt and ycd419a yielded similar levels of correctly charged trna leu . consistent with the isoleucine - mischarging activity based on tca precipitation of trna ( fig1 ), the acid gel in fig1 shows that the mutant ycd419a enzyme stably produced trna leu linked to isoleucine . the wild - type enzyme failed to yield a detectable band representing the mischarged ile - trna leu . the production levels of ile - trna leu compared to leu - trna leu are decreased as would be expected since leurs misactivates isoleucine less efficiently than leucine . incorporation of methionine into the aminoacylation assay with the mutants ycd419a ( fig1 ) and ecd345a ( fig2 ) followed by either gel analysis or tca - precipitation recovery of the trna showed that both mutants formed met - trna leu . as found with the isoleucine mischarging activity , the level of methionylation activity was significantly lower than that of leu - trna leu production which would correspond to a relative decrease in activation . the combined mischarging phenotypes of methionine and isoleucine by both s . cerevisiae cytoplasmic and e . coli leurss support that the conserved aspartic acid plays a critical role in the amino acid editing active site . the e . coli leucyl - trna synthetase thr 252 residue was shown to be a fine discriminant for molecular recognition that blocks leucine from binding to the editing active site ( mursinna et al ., 2001 ). since e . coli leurs t252a mutation recognizes an additional substrate ( leu - trna leu ), it provided a unique opportunity to test the effects of the conserved aspartic acid in post - transfer editing of an alternate substrate . the e . coli t252a mutant leurs was combined with the ecd345a mutation ( ect252a / d345a ) to further analyze the latter &# 39 ; s effect on catalysis and the overall editing reaction . while the ect252a mutation decreases apparent leucylation of trna leu , ect252a / d345a restores activity to the level of the ecd345a leurs mutant that is also similar to wild - type enzyme ( fig2 ). the e . coli mutants as well as the wild - type enzymes were tested for their ability to hydrolyze correctly charged leu - trna leu ( fig2 ). as would be expected , the ecd345a single mutation failed to edit leu - trna leu . as fig2 illustrates , ecd345a restores leucylation activity of ect252a mutant by preventing the hydrolysis of leu - trna leu . the ecd345a mutation was also efficient in disrupting binding in the altered editing pocket of the ect252a modification to allow mischarging of isoleucine to trna leu ( fig2 ). these combined results support that the universally conserved aspartic acid plays a key role in the overall editing reaction . sequence alignments of prokaryotic and eukaryotic mitochondrial leurss along with the highly homologous valrss identified a highly conserved aspartic acid ( d342 in e . coli leurs ) in valrs and prokaryotic and mitochondrial leurss that is nearby the universally conserved aspartic acid ( fig5 ). both aspartic acids overlap in the tertiary structure of t . thermophilus leurs ( cusack et al ., 2000 ) and valrs ( fukai et al ., 2000 ) ( fig5 ). in one embodiment of the present invention , alanine was substituted at the aspartic acid site in e . coli leurs ( d342a ). a double mutation to substitute each of the two conserved aspartic acids ( d342a / d345a ) was also created . the e . coli wild - type , d342a , d345a , and d342a / d345a mutants were purified to homogeneity via affinity chromatography using an n - terminal six - histidine tag . the leucylation activity for each of the single mutants was comparable to the wild - type leurs . the double mutant retained significant aminoacylation activity although it was slightly decreased ( fig2 ). the composite invention d342a / d345a double mutant leurs was able to produce significant quantities of misaminoacylated isoleucyl - trna leu , valyl - trna leu , and methionyl - trna leu ( fig2 - 27 ). hydrolytic editing activity assays demonstrated that the d342aid345a double mutant leurs editing activity was abolished ( fig2 ). in one embodiment of the present invention , a triple mutation was also created that combined the d342a , d345a , and t252y mutations in e . coli leurs ( t252y / d342a / d345a ). the composite invention lacked amino acid editing activity . combining multiple editing - defective mutations would decrease the possibility of reversion mutations due to natural mutagenesis when the composite invention is used in vivo . in several embodiments of the present invention , editing - defective mutations in s . cerevisiae leurs were created and included substitution of an aspartic acid at position 418 by an alanine ( d418a ), substitution of an asparagine at position 415 by an alanine ( n415a ), and substitution of a threonine at position 319 by a tyrosine ( t319y ). each of the composite single mutant inventions in s . cerevisiae leurs , which included the t319y , n415a , and d418a mutants exhibited decreased amino acid editing activities ( fig3 , 38 , and 44 ). the d419a mutation was combined with each of these single mutations in s . cerevisiae leurs , and also substitution of a serine at position 416 by an alanine ( s416a ), to create the double mutants d418a / d419a , s416a / d419a , n415a / d419a , and t319y / d419a leurss . each of the composite inventions t319y / d419a mutant leurs , n415a / d419a mutant leurs , s416a / d419a mutant leurs , and d418a / d419a mutant leurs had significantly diminished editing activities figs . ( 36 , 40 , 42 , and 46 ). combining multiple editing - defective mutations in these composite inventions would decrease the possibility of reversion mutations due to natural mutagenesis when the composite invention is used in vivo . high expression of recombinant proteins has shown that norvaline is substituted for leucine during protein synthesis ( apostol et al ., 1987 ). identification of a more specific leucyl - trna synthetase , which could discriminate more efficiently leucine from noncognate amino acids , would enable more accurate expression of pure recombinant proteins in e . coli . in one embodiment of the present invention , tyr 330 of e . coli leurs was substituted by an alanine ( y330a ). the composite invention mutant y330a e . coli leurs enzyme was shown to aminoacylate leucine to trna leu at higher levels relative to wild type ( fig3 ). a triple mutation that combined the y330a , d342a , and d345a mutations in e . coli leurs ( y330a / d342a / d345a ) was created . the composite invention e . coli leurs y330a / d342a / d345a mutant was determined to yield significantly higher levels of misaminoacylated isoleucine - trna leu compared to any of the e . coli mutants generated that exhibited amino acid editing defects . combining multiple editing - defective mutations would decrease the possibility of reversion mutations due to natural mutagenesis when the composite invention is used in vivo . the t . thermophilus leurs crystal structure ( cusack et al ., 2000 ) and the e . coli leurs homology model ( lee and briggs , 2002 ) were used to identify active site residues that were in close proximity to the leucine side chain in the aminoacylation active site . in one embodiment of the present invention , the methionine residue at position 40 was substituted by a serine in e . coli leurs ( m40s ). the mutation was combined with multiple other mutations ( t252y / d342a / d345a ) in the cp1 domain of e . coli leurs that resulted in an editing defect . aminoacylation of leucine to trna leu was decreased relative to the wild type enzyme ( fig2 ). amino acid - dependent pyrophosphate exchange activity showed that leucine discrimination relative to norvaline discrimination was increased by the composite invention mutant m40s e . coli leurs ( fig3 and 31 ). table 1 illustrates some of the various mutations obtained from e . coli and s . cerevisiae ( cytoplasmic and mitochondrial ) as further described below , from e . coli and s . cerevisiae ( cytoplasmic and mitochondrial ) as further described below , while table 2 illustrates the wild - type enzyme mutations . plasmids harboring the wild - type or mutant leus gene from a prokaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( martinis and fox , 1997 , mursinna et al ., 2001 ). using the polymerase chain reaction ( pcr ), the single or multiple mutant leus gene is then generated using pcr primers that contain the desired t252y mutation . plasmids harboring the leurs t252y mutation are used to transform protein expression strains for recombinant protein production . the leurs t252y mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs t252y mutant are quantitated by scintillation counting ( fig9 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the e . coli leurs t252y mutant , this editing mutant leucylates trna leu at a similar efficiency to the wild - type protein . isoleucylation of trna leu by leurs t252y is quantitated by scintillation counting and is compared to results obtained from experiments performed in parallel using the wild - type leurs ( fig1 ). in the case of the e . coli leurs t252y mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no detectable levels of isoleucylation activity . the hydrolytic editing activity of the leurs t252y mutant is quantitated by scintillation counting and compared with results obtained from experiments performed in parallel using both the wild - type leurs and the leurs t252a mutant ( fig1 ). in the case of the e . coli leurs proteins and mutants thereof , both the wild - type and t252a leurss deacylated the mischarged trna . in contrast , the leurs t252y mutant did not hydrolyze the misacylated trna . plasmids harboring the wild - type or mutant leus gene from a prokaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( martinis and fox , 1997 , mursinna et al ., 2001 ). using pcr , the single or multiple mutant leus gene is then generated using pcr primers that contain the desired t252f mutation ( mursinna and martinis , 2002 ). plasmids harboring the leurs t252f mutation are used to transform protein expression strains for recombinant protein production . the leurs t252f mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs t252f mutant is quantitated by scintillation counting ( fig9 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the e . coli leurs t252f mutant , this editing mutant leucylates trna leu at a similar efficiency to the wild - type protein . isoleucylation of trna leu by leurs t252f is quantitated by scintillation counting and then is compared to results obtained from experiments performed in parallel using the wild - type leurs ( fig1 ). in the case of the e . coli leurs t252f mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no detectable levels of isoleucylation activity . the hydrolytic editing activity of the leurs t252f mutant is quantitated by scintillation counting and is compared with results obtained from experiments performed in parallel using both the wild - type leurs and the leurs t252a mutant ( fig1 ). in the case of the e . coli leurs proteins and mutants thereof , both the wild - type and t252a leurss deacylate the mischarged trna . in contrast , the leurs t252f mutant does not hydrolyze the misacylated trna . plasmids harboring the wild - type cdc60 gene from a eukaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( lincecum and martinis , 2000 ). using pcr , the mutant cdc60 gene is then generated using pcr primers that contain the desired d419a mutation ( fig1 ). plasmids harboring the leurs d419a mutation are used to transform protein expression strains for recombinant protein production . the leurs d419a mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs d419a mutant is quantitated by scintillation counting ( fig1 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the s . cerevisiae leurs d419a mutant , this editing mutant leucylates trna leu at a similar efficiency to the wild - type protein . isoleucylation of trna leu by leurs d419a is quantitated by scintillation counting ( fig1 ) and also acid gel electrophoresis ( fig1 ) and then compared to results obtained from experiments performed in parallel using the wild - type leurs . in the case of the s . cerevisiae leurs d419a mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity . acid gel electrophoresis is also used to detect misaminoacylation of methionine by the yeast cytoplasmic d419a mutant leurs ( fig1 ). the hydrolytic editing activity of the leurs d419a mutant is quantitated by scintillation counting and is compared with results obtained from experiments performed in parallel using the wild - type yeast cytoplasmic leurs ( fig1 ). the yeast cytoplasmic wild - type enzyme deacylates the mischarged trna . in contrast , the leurs d419a mutant does not hydrolyze the misacylated trna . plasmids harboring the wild - type or mutant leus gene from a prokaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( martinis and fox , 1997 , mursinna et al ., 2001 ). using pcr , the single or multiple mutant leus genes are then generated using pcr primers that contain the desired d342a and / or d345a mutations . plasmids harboring the leurs with the desired d342a and / or d345a mutations are used to transform protein expression strains for recombinant protein production . the leurs d342a , d345a , and d342a / d345a mutants are then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). replacement of the single aspartic acid at position 342 or 345 in e . coli leurs with alanine reduces hydrolytic cleavage of mischarged ile - trna leu ( fig2 ). combining the d342a and d345a mutations appears to completely abolish editing and results in significant increases in the misaminoacylation activity of isoleucine , valine , and methionine yielding isoleucine - trna leu , valine - trna leu , and methionine - trna leu respectively in contrast to the wild - type protein that exhibits no isoleucylation , valylation , and methionylation activities ( fig2 - 27 ). this combined mutation suggest that when the universally conserved aspartic acid is replaced with alanine , it still maintains some low undetectable level of hydrolysis . mutation of just the single aspartic acid at position 342 yielded a decrease in the post - transfer editing . however , this single mutation did not allow for considerable misaminoacylation of isoleucine or valine and no detectable misaminoacylation of methionine ( fig2 - 27 ). using multiple mutations that decrease or abolish amino acid editing activity will reduce the possibility of restoring the mutant leurs editing activity via natural mutagenesis when used in vivo within an organism or cell line . plasmids harboring mutant leus gene from a prokaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( martinis and fox , 1997 , mursinna et al ., 2001 ). using pcr , the multiple mutant leus genes are then generated using pcr primers that contain the desired d342a and / or d345a mutations . plasmids harboring the leurs with the desired d342a and d345a and t252y mutations are used to transform protein expression strains for recombinant protein production . the leurs t252y / d342a / d345a mutants are then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). combining the t252y , d342a and d345a mutations appears to completely abolish editing ( fig4 ) and results in significant increases in the misaminoacylation activity of isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation , valylation , and methionylation activities . using multiple mutations that decrease or abolish amino acid editing activity will reduce the possibility of restoring the mutant leurs editing activity via natural mutagenesis when used in vivo within an organism or cell line . plasmids harboring the wild - type or mutant leus gene from a prokaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( martinis and fox , 1997 , mursinna et al ., 2001 ). using pcr , the single or multiple mutant leus gene is then generated using pcr primers that contain the desired y330a mutation ( mursinna and martinis , 2002 ). plasmids harboring the leurs y330a mutation are used to transform protein expression strains for recombinant protein production . the leurs y330a mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs y330a mutant is quantitated by scintillation counting ( fig3 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the e . coli leurs y330a mutant , this mutant leucylates trna leu at a higher efficiency to the wild - type protein . plasmids harboring the wild - type or mutant leus gene from a prokaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( martinis and fox , 1997 , mursinna et al ., 2001 ). using pcr , the single or multiple mutant leus genes are then generated using pcr primers that contain the desired y330 / d342a / d345a triple mutations . plasmids harboring the leurs with the desired y330 / d342a / d345amutations are used to transform protein expression strains for recombinant protein production . the leurs y330 / d342a / d345a mutants are then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). in the case of the e . coli leurs y330 / d342a / d345a mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity ( fig4 b ). it also generates significantly higher yields of isoleucine - trna leu compared to the e . coli leurs d342a / d345a double mutant . using multiple mutations that decrease or abolish amino acid editing activity will reduce the possibility of restoring the mutant leurs editing activity via natural mutagenesis when used in vivo within an organism or cell line . plasmids harboring the wild - type or mutant leus gene from a prokaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( martinis and fox , 1997 , mursinna et al ., 2001 ). using pcr , the single or multiple mutant leus gene is then generated using pcr primers that contain the desired m40s mutation ( mursinna and martinis , 2002 ). plasmids harboring the leurs m40s mutation are used to transform protein expression strains for recombinant protein production . the leurs m40s mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucine aminoacylation activity for the m40s mutant e . coli leurs that also contained the editing defect mutations ( t252y / d342a / d345a ) was decreased relative to the wild type enzyme ( fig2 ). amino acid - dependent pyrophosphate exchange activity by the leurs m40s mutant is quantitated by tlc separation followed by phosphorimaging ( fig3 and 31 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the e . coli leurs m40s mutant , norvaline activation is decreased relative to leucine activation . plasmids harboring the wild - type cdc60 gene from a eukaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( lincecum and martinis , 2000 ). using pcr , the mutant cdc60 gene is then generated using pcr primers that contain the desired t319y mutation . plasmids harboring the leurs t319y mutation are used to transform protein expression strains for recombinant protein production . the leurs t319y mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs t319y mutant is quantitated by scintillation counting ( fig3 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the s . cerevisiae leurs t319y mutant , this editing mutant leucylates trna leu at a similar efficiency to the wild - type protein . isoleucylation of trna leu by leurs t319y is quantitated by scintillation counting ( fig3 ) and then is compared to results obtained from experiments performed in parallel using the wild - type leurs . in the case of the s . cerevisiae leurs t319y mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity . plasmids harboring the wild - type or mutant cdc60 gene from a eukaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( lincecum and martinis , 2000 ). using pcr , the mutant cdc60 gene is then generated using pcr primers that contain the desired t319y and d419a mutations . plasmids harboring the leurs t319y / d419a mutations are used to transform protein expression strains for recombinant protein production . the leurs t319y / d419a mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs t319y / d419a mutant is quantitated by scintillation counting ( fig3 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the s . cerevisiae leurs t319y / d419a mutant , this editing mutant leucylates trna leu at a slightly lower efficiency compared to the wild - type protein . isoleucylation of trna leu by leurs t319y / d419a is quantitated by scintillation counting ( fig3 ) and then is compared to results obtained from experiments performed in parallel using the wild - type leurs . in the case of the s . cerevisiae leurs t319y / d419a mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity . plasmids harboring the wild - type cdc60 gene from a eukaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( lincecum and martinis , 2000 ). using pcr , the mutant cdc60 gene is then generated using pcr primers that contain the desired n415a mutation . plasmids harboring the leurs n415a mutation are used to transform protein expression strains for recombinant protein production . the leurs n415a mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs n415a mutant is quantitated by scintillation counting ( fig3 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the s . cerevisiae leurs n415a mutant , this editing mutant leucylates trna leu at a slightly lower efficiency compared to the wild - type protein . isoleucylation of trna leu by leurs n415a is quantitated by scintillation counting ( fig3 ) and then is compared to results obtained from experiments performed in parallel using the wild - type leurs . in the case of the s . cerevisiae leurs n415a mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity . plasmids harboring the wild - type or mutant cdc60 gene from a eukaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( lincecum and martinis , 2000 ). using pcr , the mutant cdc60 gene is then generated using pcr primers that contain the desired n415a and d419a mutations . plasmids harboring the leurs n415a / d419a mutations are used to transform protein expression strains for recombinant protein production . the leurs n415a / d419a mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs n415a / d419a mutant is quantitated by scintillation counting ( fig3 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the s . cerevisiae leurs n415a / d419a mutant , this editing mutant leucylates trna leu at a slightly lower efficiency compared to the wild - type protein . isoleucylation of trna leu by leurs n415a / d419a is quantitated by scintillation counting ( fig4 ) and then is compared to results obtained from experiments performed in parallel using the wild - type leurs . in the case of the s . cerevisiae leurs n415a / d419a mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity . plasmids harboring the wild - type or mutant cdc60 gene from a eukaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( lincecum and martinis , 2000 ). using pcr , the mutant cdc60 gene is then generated using pcr primers that contain the desired s416a and d419a mutations . plasmids harboring the leurs s416a / d419a mutations are used to transform protein expression strains for recombinant protein production . the leurs s416a / d419a mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs s416a / d419a mutant is quantitated by scintillation counting ( fig4 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the s . cerevisiae leurs s416a / d419a mutant , this editing mutant leucylates trna leu at a similar efficiency compared to the wild - type protein . isoleucylation of trna leu by leurs s416a / d419a is quantitated by scintillation counting ( fig4 ) and then is compared to results obtained from experiments performed in parallel using the wild - type leurs . in the case of the s . cerevisiae leurs s416a / d419a mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity . plasmids harboring the wild - type cdc60 gene from a eukaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( lincecum and martinis , 2000 ). using pcr , the mutant cdc60 gene is then generated using pcr primers that contain the desired d418a mutation . plasmids harboring the leurs d418a mutation are used to transform protein expression strains for recombinant protein production . the leurs d418a mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs d418a mutant is quantitated by scintillation counting ( fig4 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the s . cerevisiae leurs d41 8a mutant , this editing mutant leucylates trna leu at a similar efficiency compared to the wild - type protein . isoleucylation of trna leu by leurs d418a is quantitated by scintillation counting ( fig4 ) and then is compared to results obtained from experiments performed in parallel using the wild - type leurs . in the case of the s . cerevisiae leurs d418a mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity . plasmids harboring the wild - type or mutant cdc60 gene from a eukaryotic organism are used to create leurs fusion proteins that contain n - terminal six - histidine tags ( lincecum and martinis , 2000 ). using pcr , the mutant cdc60 gene is then generated using pcr primers that contain the desired d418a and d419a mutations . plasmids harboring the leurs d418a / d419a mutations are used to transform protein expression strains for recombinant protein production . the leurs d418a / d419a mutant is then purified using immobilized metal affinity chromatography and identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation activities of trna leu by the leurs d418a / d419a mutant is quantitated by scintillation counting ( fig4 ). its enzymatic activities are compared to wild - type leurs enzymatic activities . in the case of the s . cerevisiae leurs d418a / d419a mutant , this editing mutant leucylates trna leu at a similar efficiency compared to the wild - type protein . isoleucylation of trna leu by leurs d418a / d419a is quantitated by scintillation counting ( fig4 ) and then is compared to results obtained from experiments performed in parallel using the wild - type leurs . in the case of the s . cerevisiae leurs d418a / d419a mutant protein , the editing mutant aminoacylates trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein that exhibits no isoleucylation activity . additional objects , advantages and novel features of the invention will be set forth in part in the description which follows , and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention . the objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims . based on the x - ray crystal structure of thermus thermophilus leurs ( cusack et al ., 2000 ) and its coordinates , minimized homology models for e . coli ( lee and briggs , 2002 ) and yeast leurss have already been developed to aid designing mutants to inactivate the amino acid editing mechanism and also subsequent rational design of the leurs editing and aminoacylation active sites . compared to other closely related editing trna synthetases such as ilers and valrs , the amino acid binding pocket for leucine in the aminoacylation active site would have a larger volume and thus would be expected to accommodate a greater diversity of noncognate amino acids . target remodeling of the leucine binding pocket via iterative computational and mutagenesis experiments is planned and in progress to adapt the active site for productive binding to specific noncognate amino acids that facilitate efficient and stable aminoacylation to trna molecules . in one embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs t252a . in the present example the e . coli leurs t252a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p15ec3 - 1 harboring the wild - type e . coli leus gene to create plasmid pmure10 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng p15ec3 - 1 and 225 pmoles each of a forward and reverse primer [ 5 ′- ctacccgcccggacgcctttatgggttgtacc - 3 ′ ( seq id no . : 1 ) and 5 ′- gatgggcgggcctgcggaaatacccaacatgg - 3 ′ ( seq id no . : 2 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 pmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 16 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( pmure10 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of pmure10 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ccgaaggcgtggagatc - 3 ′) ( seq id no . : 3 ) that annealed upstream of the mutated dna region . plasmid pmure10 harboring e . coli leurs t252a was used to transform e . coli strain bl21 ( de3 ) plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of near 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs t252a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs t252a mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 100 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 100 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs t252a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 μm trna leu , 25 . 4 μm [ 3 h ]- leucine ( 150 μci / ml ) and 15 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 60001c ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the t252a leurs did not leucylate appriciatable amounts of trna leu ( fig3 ). activation of leucine by the e . coli t252a mutant was measured in a pyrophosphate exchange assay . the reaction contained 50 mm n - 2 - hydroxyethylpiperazine - n ′- 2 - ethanesulfonic acid ( hepes ), ph 8 . 0 , 1 mm atp , 10 mm mgcl 2 , 1 mm dtt , and 1 mm [ 32 p ]- pyrophosphate ( 53 . 3 μci / ml ), 50 nm enzyme , and 250 μm leucine . the reaction was initiated with the addition of leucine . tlc cellulose pei plates ( scientific adsorbents inc .) were pre - run in water for 2 hours . 2 μl aliquots of the reaction were removed at 0 , 3 , 6 , 9 , and 12 minutes and placed on the pre - run tlc plates to quench the reaction . the tlc plates were placed in a 75 mm kh 2 p0 4 , ph 3 . 5 , 4 m urea running buffer , and allowed to run for 3 hours . the tlc plates were dried , wrapped in saran wrap , and placed onto a phosphoimager screen in a fujifilm bas 2040 cassette . the phosphorimager screen was imaged in the fujix bas - 1000 phosphorimager and the amount of radiolabel present in the spots on the plates quantified using the quantitation mode of the imageguage v3 . 0 software program . the e . coli leurs t252a mutant activates leucine at a slightly lower efficiency than the wild - type leurs protein ( fig4 a and b ). the hydrolytic editing activity of e . coli leurs t252a was measured using a reaction mixture consisting of 60 mm tris , ph 7 . 0 , 10 mm mgcl 2 , and aminoacylated [ 3 h ]- leucine - trna leu ( 50 μci / ml ). the reaction was initiated by the addition of 100 nm enzyme . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were dried and added to 3 ml of scintisafe econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using both the wild - type e . coli leurs . fig5 demonstrates that leurs t252a edits the cognate charged leu - trna leu . in another embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs t252f . in the present example the e . coli leurs t252f mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p15ec3 - 1 harboring the wild - type e . coli leus gene to create plasmid pmure2 1 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng p15ec3 - 1 and 225 pmoles each of a forward and reverse primer [ 5 ′- ccc gcc cgg act tct tta tgg gtt gta cc - 3 ′ ( seq id no . 4 ) and 5 ′- ggt aca acc cat aaa gaa gtc cgg gcg gg - 3 ′ ( seq id no . 5 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 16 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna for pmure21 was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of pmure2l was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ccgaaggcgtggagatc - 3 ′)( seq id no . : 6 ) that annealed upstream of the mutated dna region . plasmid pmure21 harboring e . coli leurs t252f was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs t252f mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs t252f mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 100 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 7 . 0 ), 100 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs t252f mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 μm trna leu , 25 . 4 μm [ 3 h ]- leucine ( 150 μci / ml ) and 15 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs t252f mutant leucylates trna leu at a similar efficiency as the wild - type protein ( fig9 ). isoleucylation of trna leu by e . coli leurs t252f was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 7 μm in vitro transcribed trna leu , 22 μm [ 3 h ]- isoleucine ( 180 μci / ml ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs t252f protein aminoacylated trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig1 ). a 15 . 4 μl aliquot of 100 μm concentration ( or 1540 pmoles ) of trna leu was aminoacylated with [ 3 h ]- isoleucine in an isoleucylation reaction as described above using e . coli leurs t252f for 30 min at 37 ° c . the ph of the reaction was adjusted to 5 . 0 using acetic acid ( 0 . 1 % v / v ) and the aminoacylated isoleucyl - trna leu was immediately extracted using phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) ( ph 5 . 1 ) ( fisher scientific ; fair lawn , n . j .). the aminoacylated isoleucyl - trna leu was concentrated by ethanol precipitation , and resuspended in 50 mm kh 2 po 4 ( ph 5 . 0 ) at a concentration of 50 μm . the aminoacylated isoleucyl - trna leu was stored at − 20 ° c . the hydrolytic editing activity of e . coli leurs t252f was measured using a reaction mixture consisting of 60 mm tris , ph 7 . 0 , 10 mm mgcl 2 , and aminoacylated [ 3 h ]- isoleucyl - trna leu ( 50 μci / ml ). the reaction was initiated by the addition of 100 nm enzyme . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were dried and added to 3 ml of scintisafe econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those experiments performed in parallel using both the wild - type e . coli leurs and the e . coli leurs t252a mutant . both the wild - type and t252a leurss deacylated the mischarged trna . however , e . coli leurs t252f did not hydrolyze the misacylated trna ( fig1 ). in yet another embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs t252y . in the present example the e . coli leurs t252y mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p 15ec3 - 1 harboring the wild - type e . coli leus gene to create plasmid pmure22 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng p15ec3 - 1 and 225 pmoles each of a forward and reverse primer [ 5 ′- ccc gcc cgg act act tta tgg gtt gta cc - 3 ′ ( seq id no . : 7 ) and 5 ′- ggt aca acc cat aaa gta gtc cgg gcg gg - 3 ′ ( seq id no . : 8 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 16 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min ), annealing ( 60 ° c ., 30 sec ) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna for pmure22 was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of pmure22 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ccgaaggcgtggagatc - 3 ′)( seq id no . : 6 ) that annealed upstream of the mutated dna region . the plasmid pmure22 harboring e . coli leurs t252y was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs t252y mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs t252y mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 100 mm nacl , 100 mm imidazole and 10 % glycerol . the elution was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 7 . 0 ), 100 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs t252y mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 μm trna leu , 25 . 4 μm [ 3 h ]- leucine ( 150 μci / ml ) and 15 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs t252y mutant leucylates trna leu at a similar efficiency as the wild - type protein ( fig9 ). isoleucylation of trna leu by e . coli leurs t252y was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 7 μm in vitro transcribed trna leu , 22 μm [ 3 h ]- isoleucine ( 180 μci / ml ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs t252y protein aminoacylated trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig1 ). a 15 . 4 μl aliquot of 100 μm concentration ( or 1540 pmoles ) of trna leu was aminoacylated with [ 3 h ]- isoleucine in an isoleucylation reaction as described above using e . coli leurs t252y for 30 min at 37 ° c . the ph of the reaction was adjusted to 5 . 0 using acetic acid ( 0 . 1 % v / v ) and the aminoacylated isoleucyl - trna leu immediately extracted using phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) ( ph 5 . 1 ) ( fisher scientific ; fair lawn , n . j .). the aminoacylated isoleucyl - trna leu was concentrated by ethanol precipitation , and resuspended in 50 mm kh 2 po 4 ( ph 5 . 0 ) at a concentration of 50 μm . the aminoacylated isoleucyl - trna leu was stored at − 20 ° c . the hydrolytic editing activity of e . coli leurs t252y was measured using a reaction mixture consisting of 60 mm tris , ph 7 . 0 , 10 mm mgcl 2 , and aminoacylated [ 3 h ]- isoleucyl - trna leu ( 50 μci / ml ). the reaction was initiated by the addition of 100 nm enzyme . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were the washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those from experiments performed in parallel using both the wild - type e . coli leurs and the e . coli leurs t252a mutant . both the wild - type and t252a leurss deacylated the mischarged trna . however , e . coli leurs t252y did not hydrolyze the misacylated trna ( fig1 ). in still another embodiment , leucyl - trna synthetase mutants described herein are made for eukaryotic organisms including , but not limited to s . cerevisiae leurs d419a . in the present example primary sequence alignments using clustalw 1 . 8 sequence alignment analysis identified an aspartic acid residue in the cp1 editing domain that was completely conserved across leurs , ilers , and valrs ( fig1 ). this aspartic acid ( asp 419 ) in yeast cytoplasmic leurs was changed to an alanine residue ( d419a ) by site - directed mutagenesis of the gene encoding yeast cytoplasmic leurs . specifically , the mutant leurs gene was amplified via pcr using the template plasmid p32yl - 2 - 3 annealed to oligonucleotides that contain a mismatch at position 1408 in the nucleotide sequence to convert an adenosine to a cysteine . the plasmid template was isolated using the wizard miniprep kit ( promega ; madison , wis .). the two oligonucleotides were synthesized by sigma ( woodlands , tex .) and include a reverse primer ( 5 ′ ggt ttg tgc aac aag tcc ttg gtg gta atg ta g gc a tct ggt gaa tta gat ggt aca cac gtg 3 ′) ( seq id no . : 9 ) and a forward primer ( 5 ′ cac gtg tgt acc atc taa ttc acc aga t gc c ta cca tac atc caa gga ctt gtt gca caa acc 3 ′) ( seq id no . : 10 ). each was designed to anneal to the region containing the nucleotides that code for asp 419 . ( the underlined nucleotides represent the site of the codon mutation to change asp 419 to an alanine in the expressed protein .) the final reaction contained 125 ng of each of the forward and reverse primers , 0 . 1 μg p32yl - 2 - 3 , 1x pfu dna polymerase buffer ( promega ; madison , wis . ), 200 μm dntps ( promega ; madison , wis . ), and 0 . 06 u / μl pfu dna polymerase ( promega ; madison , wis .). the reaction was carried out by first mixing and heating all of the components , excluding the pfu dna polymerase and dntps , at 95 ° c . for 5 minutes . the pfu dna polymerase and dntps were then added and the reaction was thermocycled 1 time at : denaturation ( 95 ° c ., 30 sec ), annealing ( 58 ° c ., 30 sec ) and extension ( 68 ° c ., 30 s followed by 16 times at : denaturation ( 95 ° c ., 30 sec ), annealing ( 58 ° c ., 30 sec ) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . the amplified pcr product yielding the mutant yeast cytoplasmic leurs gene called p32 - amw1 was analyzed on a 1 % agarose gel . the pcr reaction containing plasmid p32 - amw1 was used to transform competent e . coli dh5α cells ( invitrogen corporation ; carlsbad , calif .) by the heat shock method . transformed cells were incubated at 37 ° c . overnight on luria broth ( lb ) agarose plates that contained 100 μg / μl ampicillin . the p32 - amw1 plasmid was then isolated using the wizard miniprep kit ( promega ; madison , wis .) and the gene insert was sequenced by lone star labs ( houston , tex .) to verify the correct mutation had been made . once verified , competent e . coli bl21 - codonplus cells ( stratagene ; la jolla , calif .) were transformed with the p32 - amw1 plasmid by the heat shock method . two 3 ml cultures of lb containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol were inoculated either with e . coli bl21 (- codon plus ) cells that contain p32yl - 2 - 3 encoding the wild - type yeast cytoplasmic leurs and the other with e . coli bl21 (- codon plus ) cells which contained p32 - amw1 that encoded the d419a mutant yeast cytoplasmic leurs . both genes were fused to a 5 ′ upstream region that encodes a six - histidine tag . the cultures were then incubated overnight at 37 ° c . with shaking and used to inoculate two separate 500 ml cultures that contained 100 μg / ml ampicillin and 34 μg / ml chloramphenicol . the 500 ml cultures were incubated at 37 ° c . with shaking for approximately 3 . 5 hours . when the optical density ( o . d . 600 ) was between 0 . 6 and 0 . 9 , protein expression was induced in both cultures by the addition of 1 mm iptg . the cultures were incubated for another 30 minutes at 37 ° c . with shaking and harvested by centrifugation in a beckman j2 - hs centrifuge at 10 , 000 × g for 15 minutes . the harvested pellets were stored at − 80 ° c . until purification . the pellet containing either the wild - type or mutant d419a yeast cytoplasmic leurs fused to an n - terminal six histidine tag was resuspended in 2 ml of cold buffer a ( 20 mm tris ph 7 . 4 , 100 mm nacl , 5 % glycerol ) at 4 ° c . phenylmethyl sulfonyl fluoride ( pmsf ) was then added to the resuspended pellet to a final concentration of 0 . 1 mm . the cells were lysed by sonication ( vibracell sonics and materials ) at 50 % duty for two periods of 90 seconds each . the cell lysate was cleared by centrifugation at 13 , 800 × g for 15 minutes at 4 ° c . the supernatant was applied to 2 ml of talon metal affinity resin that had been pre - equilibrated with buffer a . the resin was incubated with the lysate via gentle shaking at 4 ° c . for 30 minutes to allow binding of the wild - type or mutant d419a yeast cytoplasmic leurs with the fused n - terminal affinity six histidine tag . the resin was then centrifuged at very low speed in a clinical centrifuge ( international equipment company ) to pellet the resin . the supernatant was removed and stored at 4 ° c . the resin was then washed 3 times with 10 ml of cold buffer a . these washes were quick washes in that the resin was resuspended and then centrifuged immediately in a clinical centrifuge . the resin was then washed three more times with 10 ml of cold buffer a . these washes included a 10 minute incubation period with gentle shaking at 4 ° c . the wash supernatants were removed by centrifugation . the wild - type and mutant yeast cytoplasmic leurs was eluted using six 500 μl aliquots of 100 mm imidazole in buffer a . the resin was resuspended and then incubated at 4 ° c . for 8 - 10 minutes . the elution fractions were then collected and stored at 4 ° c . all fractions from the affinity batch purification of leurs were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ). elution fractions that contained a band at about 150 kda molecular weight were combined and concentrated in a ym - 50 centricon ( amicon ; bedford , mass .). the centricons were also used to remove excess imidazole by replacing the original elution buffer with buffer a . the concentrated protein was stored in 50 % glycerol at − 20 ° c . leucylation of trna leu was carried out at a constant temperature of 35 ° c . using an mj research peltier thermal cyler - 200 dna engine with 26 nm enzyme . the reaction mixture contained 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dtt , 10 μm leucine , 100 μci / ml of [ 3 h ]- leucine ( amersham life science ; piscataway , n . j . ), and 5 mg / ml crude yeast trna ( sigma ; st . louis , mo .). the reaction mixture was pre - incubated for 5 minutes with enzyme and initiated by the addition of 4 mm atp ( amersham life science ; piscataway , n . j .). at specific time points , 5 μl of the reaction were removed and transferred to whatman filter pads that were pre - wetted with 5 % tca . about 10 seconds after transferring the aliquot to the pad , the filter pad was then placed in 5 % tca ( chilled on ice ). at the end of the reaction time course , the pads in 5 % tca ( chilled on ice ) were placed on a shaker at 65 rpm . the pads were allowed to wash for 10 minutes and then recovered for subsequent washes in 5 % tca . the pads were washed three times in cold 5 % tca followed by one wash in 70 % ethanol , and once in anhydrous ether . the pads were dried for 1 hour and then placed in a 20 ml glass scintillation vial with 3 ml of scintsafe econo 1 cocktail ( fisher scientific ; pittsburgh , pa .). the radioactivity was measured using an ls 6000sc scintillation counter . results showed that wild - type leurs and the d419a mutant leurs aminoacylated trna leu with leucine at similar rates ( fig1 ). isoleucylation of trna leu was carried out at a constant temperature of 35 ° c . using an mj research peltier thermal cyler - 200 dna engine with 300 nm wild - type or d419a mutant yeast cytoplasmic yeast leurs . the reaction mixture included 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dtt , 20 μm [ 3 h ]- isoleucine ( 250 μci / ml ; amersham life science ; piscataway , n . j . ), and 5 mg / ml crude yeast trna ( sigma ; st . louis , mo .). the reaction and washes were carried out as described above . results showed that the d419a leurs mutant could carry out isoleucylation of trna leu whereas the wild - type enzyme did not at detectable levels ( fig1 ). misaminoacylated isoleucyl - trna leu was produced enzymatically using the yeast cytoplasmic d419a leurs mutant . the reaction conditions were carried out as described above in a 240 μl reaction . the reaction was allowed to proceed for 120 minutes and then stopped by quenching with the addition of acetic acid to a final concentration of 0 . 15 %. the protein was removed by extraction with two equal volumes of phenol : chloroform : isoamyl alcohol ( ph 5 . 2 ). the aqueous phase was removed and ½ volume of 4 . 6 m nh 3 oac ( ph 5 . 0 ) was added followed by 0 . 23 mg / ml glycogen ( sigma ; st . louis , mo .). the reaction was mixed and 2 . 5 volumes of 100 % cold ethanol were added and incubated at − 80 ° c . for 1 hour . the precipiated isoleucyl - trna leu was recovered by centrifugation at 20 , 000 g for 30 minutes . the rna - containing pellet was then washed twice with cold 70 % ethanol and dried in a speed vac . mischarged trna was stored at − 20 ° c . pelleted misaminoacyalated [ 3 h ]- isoleucyl - trna leu was resuspended in 50 mm kpi ( ph 5 . 0 ) yielding approximately a 40 mg / ml stock of total rna . a hydrolytic editing reaction mixture included 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 7 . 2 mg / ml [ 3 h ]- isoleucine - trna leu and either 100 nm wild - type or d419a mutant yeast cytoplasmic leurs . the reaction was carried out at room temperature . at specific time points , a 5 μl aliquot of the reaction mixture was spotted on a filter pad pre - wetted with 5 % tca and washed as described above . the results showed that the d419a mutant leurs did not hydrolyze isoleucyl - trna leu and that the wild - type leurs edited the mischarged trna ( fig1 ). misaminoacylated trna leu was analyzed by electrophoresis on a denaturing gel under acidic conditions . reactions were carried out as described above for leucylation and isoleucylation of trna leu with the exception of using [ 14 c ]- leucine ( 0 . 3 ci / mole ) and [ 14 c ]- isoleucine ( 0 . 3 ci / mole ) at a concentration of 40 μm each . methionylation of trna leu was carried out at 35 ° c . using an mj research peltier thermal cyler - 200 dna engine with 300 nm enzyme . the reaction mixture included 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dtt , 20 μm [ 35 s ]- methionine ( 303 μci / ml ; amersham life sciences ; piscataway , n . j . ), and 5 mg / ml crude yeast trna ( sigma ; st . louis , mo .). a 5 μl aliquot of each reaction was stopped at specific time points in 5 μl of quench buffer [ 100 mm naoac ( ph 5 . 0 ), 8 m urea , 0 . 05 m edta , 0 . 05 % bromophenol blue , and 0 . 05 % xylene cyanol ] on ice . the sample was analyzed via acid gel electrophoresis by loading 5 μl of the quenched reaction onto a 6 . 5 % acrylamide : bis - acrylamide solution [ 19 : 1 ] ( biorad , hercules , calif . ), 8 m urea , 25 mm naoac ( ph 5 . 0 ), 0 . 08 % ammonium persulfate , 0 . 05 % n , n , n ′, n ′- tetramethylethylenediamine ( promega , madison , wis .). the gel was electrophoresed at 500 v until dye front reached the end of gel . the gel was dried using a model 583 gel dryer ( biorad , hercules , calif .) and imaged using a fujix bas 1000 phosphorimager ( fuji , stamford , conn .). results showed detectable bands of leucine - trna leu for wt leurs ( fig1 ). no detectable bands were seen for mischarging isoleucine to trna leu by the wild - type leurs ( fig1 ). in contrast to the wild - type enzyme , the d419a leurs mutant yielded detectable bands for leucyl -, isoleucyl -, and methionyl - trna leu ( fig1 and 16 ) in yet another embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs d342a . in the present example the e . coli leurs d342a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p15ec3 - 1 harboring the wild - type e . coli leus gene to create plasmid pmure15 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng p15ec3 - 1 and 225 pmoles each of a forward and reverse primer [ 5 ′ ggt acc ggg gca cg c cca gcg cga cta cg 3 ′ ( seq id no . : 11 ) and 5 ′ cca tgg ccc cgt gcg ggt cgc gct gat gc 3 ′ ( seq id no . : 12 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( pmure15 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of pmure15 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ccgaaggcgtggagatc - 3 ′) ( seq id no . : 6 ) that annealed upstream of the mutated dna region . plasmid pmure15 harboring e . coli leurs d342a was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs d342a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs d342a mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs d342a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude e . coli trna , 21 . 5 μm [ 3 h ]- leucine ( 150 μci / ml ) and 5 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d342a mutant leucylates trna leu at a similar efficiency as the wild - type protein ( fig2 ). isoleucylation of trna leu by e . coli leurs d342a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 22 μm [ 3 h ]- isoleucine ( 180 μci / ml ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d342a protein aminoacylated very low yields of trna leu with isoleucine in contrast to the wild - type protein ( fig2 ). valylation of trna leu by e . coli leurs d342a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 22 μm [ 3 h ]- valine ( 200 μci / ml ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d342a protein aminoacylated low yields of trna leu with valine in contrast to the wild - type protein ( fig2 ). a 15 . 4 μl aliquot of 100 μm concentration ( or 1540 pmoles ) of trna leu was aminoacylated with [ 3 h ]- isoleucine in an isoleucylation reaction as described above using e . coli leurs d342a / d345a for 30 min at 37 ° c . the ph of the reaction was adjusted to 5 . 0 using acetic acid ( 0 . 1 % v / v ) and the aminoacylated isoleucyl - trna leu immediately extracted using phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) ( ph 5 . 1 ) ( fisher scientific ; fair lawn , n . j .). the aminoacylated isoleucyl - trna leu was concentrated by ethanol precipitation , and resuspended in 50 mm kh 2 po 4 ( ph 5 . 0 ) at a concentration of 50 μm . the aminoacylated isoleucyl - trna leu was stored at − 20 ° c . the hydrolytic editing activity of e . coli leurs d342a was measured using a reaction mixture consisting of 60 mm tris , ph 7 . 0 , 10 mm mgcl 2 , and aminoacylated [ 3 h ]- isoleucyl - trna leu ( 50 μci / ml ). the reaction was initiated by the addition of 100 nm enzyme . the reaction was quenched by placing a 5 μt aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were the washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the wild - type leurs deacylated the mischarged trna . however , e . coli leurs d342a only deacylated at low levels ( fig2 ). in another embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs d345a . in the present example the e . coli leurs d345a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p15ec3 - 1 harboring the wild - type e . coli leus gene to create plasmid phappy - 1 - 1 - 28 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng p15ec3 - 1 and 225 pmoles each of a forward and reverse primer [ 5 ′- gggcacgaccagcgcgcctacgagtttgcc 3 ′ ( seq id no . : 13 ) and 5 ′ ggcaaactcgtaggcgcgctggtcgtgccc 3 ′ ( seq id no . : 14 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy - 1 - 1 - 28 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy - 1 - 1 - 28was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ccgaaggcgtggagatc - 3 ′) ( seq id no . : 6 ) that annealed upstream of the mutated dna region . plasmid phappy - 1 - 1 - 28 harboring e . coli leurs d345a was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs d345a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs d345a mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs d345a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude e . coli trna , 21 . 5 μm [ 3 h ]- leucine ( 150 μci / ml ) and 5 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d345a mutant leucylates trna leu at a similar efficiency as the wild - type protein ( fig1 , 21 , and 24 ). isoleucylation of trna leu by e . coli leurs d345a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 22 μm [ 3 h ]- isoleucine ( 180 μci / ml ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d345a protein aminoacylated trna leu with isoleucine in contrast to the wild - type protein ( fig1 , 23 , 25 ). valylation of trna leu by e . coli leurs d345a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 72 μm [ 3 h ]- valine ( 1250 μci / ml ) and 800 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d345a protein aminoacylated low yields of trna leu with valine in contrast to the wild - type protein ( fig2 ). methionylation of trna leu by e . coli leurs d345a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 22 μm [ 35 s ]- methionine ( 303 μci / ml ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ) with 1 mm methionine . the pads were slowly shaken on ice in 5 % tca with 1 mm methionine with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d345a protein aminoacylated low yields of trna leu with methionine in contrast to the wild - type protein ( fig2 and 27 ). a 15 . 4 μl aliquot of 100 μm concentration ( or 1540 pmoles ) of trna leu was aminoacylated with [ 3 h ]- isoleucine in an isoleucylation reaction as described above using e . coli leurs d342a / d345a for 30 min at 37 ° c . the ph of the reaction was adjusted to 5 . 0 using acetic acid ( 0 . 1 % v / v ) and the aminoacylated isoleucyl - trna leu immediately extracted using phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) ( ph 5 . 1 ) ( fisher scientific ; fair lawn , n . j .). the aminoacylated isoleucyl - trna leu was concentrated by ethanol precipitation , and resuspended in 50 mm kh 2 po 4 ( ph 5 . 0 ) at a concentration of 50 μm . the aminoacylated isoleucyl - trna leu was stored at − 20 ° c . the hydrolytic editing activity of e . coli leurs d345a was measured using a reaction mixture consisting of 60 mm tris , ph 7 . 0 , 10 mm mgcl 2 , and aminoacylated [ 3 h ]- isoleucyl - trna leu ( 50 μci / ml ). the reaction was initiated by the addition of 100 nm enzyme . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were the washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the wild - type leurs deacylated the mischarged trna . however , e . coli leurs d345a did not deacylate the mischarged trna ( fig2 ). in yet another embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs d342a / d345a . in the present example the e . coli leurs d342a / d345a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p15ec3 - 1 harboring the wild - type e . coli leus gene to create plasmid phappy - 1 - 1 - 29 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng p15ec3 - land 225 pmoles each of a forward and reverse primer [ 5 ′ ggtaccggggcacgcccagcgcgcctacgagtttgc 3 ′ ( seq id no . : 15 ) and 5 ′ ggcaaactcgtaggcgcgctgggcgtgccccggtac 3 ′ seq id no . 16 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c , 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy - 1 - 1 - 29 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy - 1 - 1 - 29 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ccgaaggcgtggagatc - 3 ′) ( seq id no . : 6 ) that annealed upstream of the mutated dna region . plasmid phappy - 1 - 1 - 29 harboring e . coli leurs d342a / d345a was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs d342a / d345a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs d342a / d345a mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs d342a / d345a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude e . coli trna , 21 . 5 μm [ 3 h ]- leucine ( 150 μci / ml ) and 5 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d342a / d345a mutant leucylates trna leu at a similar efficiency as the wild - type protein ( fig2 ). isoleucylation of trna leu by e . coli leurs d342a / d345a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 22 μm [ 3 h ]- isoleucine ( 180 μci / mi ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d342a ! d345a protein aminoacylated trna leu with isoleucine at high levels in contrast to the wild - type protein ( fig2 ). valylation of trna leu by e . coli leurs d342a / d345a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 72 μm [ 3 h ]- valine ( 1250 μci / ml ) and 800 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d342a / d345a protein aminoacylated high yields of trna leu with valine in contrast to the wild - type protein ( fig2 ). methionylation of trna leu by e . coli leurs d342a / d345a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 22 μm [ 35 s ]- methionine ( 303 μci / ml ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ) with 1 mm methionine . the pads were slowly shaken on ice in 5 % tca with 1 mm methionine with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs d342a / d345a protein aminoacylated high yields of trna leu with methionine in contrast to the wild - type protein ( fig2 ). a 15 . 4 μl aliquot of 100 μm concentration ( or 1540 pmoles ) of trna leu was aminoacylated with [ 3 h ]- isoleucine in an isoleucylation reaction as described above using e . coli leurs d342a / d345a for 30 min at 37 ° c . the ph of the reaction was adjusted to 5 . 0 using acetic acid ( 0 . 1 % v / v ) and the aminoacylated isoleucyl - trna leu immediately extracted using phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) ( ph 5 . 1 ) ( fisher scientific ; fair lawn , n . j .). the aminoacylated isoleucyl - trna leu was concentrated by ethanol precipitation , and resuspended in 50 mm kh 2 po 4 ( ph 5 . 0 ) at a concentration of 50 μm . the aminoacylated isoleucyl - trna leu was stored at − 20 ° c . the hydrolytic editing activity of e . coli leurs d342a / d345a was measured using a reaction mixture consisting of 60 mm tris , ph 7 . 0 , 10 mm mgcl 2 , and aminoacylated [ 3 h ]- isoleucyl - trna leu ( 50 μci / ml ). the reaction was initiated by the addition of 100 nm enzyme . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were the washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the wild - type leurs deacylated the mischarged trna . however , e . coli leurs d342a / d345a did not deacylate the mischarged trna ( fig2 ). in another embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs t252a1d345a . in the present example the e . coli leurs t252a / d345a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid pmure10 harboring the t252a e . coli leus gene to create plasmid phappy2 - 1 - 1 - 19 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng pmure10 and 225 pmoles each of a forward and reverse primer [ 5 ′ gggcacgaccagcgcgcctacgagtttgcc 3 ′ ( seq id no . : 17 ) and 5 ′ ggcaaactcgtaggcgcgctggtcgtgccc 3 ′ ( seq id no . : 18 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy2 - 1 - 1 - 19 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy2 - 1 - 1 - 19 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ccgaaggcgtggagatc - 3 ′) ( seq id no . : 6 ) that annealed upstream of the mutated dna region . plasmid phappy2 - 1 - 1 - 19 harboring e . coli leurs t252a / d345a was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 1g / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs t252a / d345a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs t252a / d345a mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs t252a / d345a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude e . coli trna , 21 . 5 μm [ 3 h ]- leucine ( 150 μci / ml ) and 5 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs t252aid345a mutant leucylates trna leu at a slightly lower efficiency as the wild - type protein ( fig2 ). isoleucylation of trna leu by e . coli leurs t252a / d345a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 22 μm [ 3 h ]- isoleucine ( 180 μci / ml ) and 1 μm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs t252a / d345a protein aminoacylated trna leu with isoleucine at high levels in contrast to the wild - type protein ( fig2 ). a 15 . 4 μl aliquot of 100 μm concentration ( or 1540 pmoles ) of trna leu was aminoacylated with [ 3 h ]- leucine in a leucylation reaction as described above using the wild - type e . coli leurs for 1 hour at 37 ° c . the ph of the reaction was adjusted to 5 . 0 using acetic acid ( 0 . 1 % v / v ) and the aminoacylated isoleucyl - trna leu immediately extracted using phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) ( ph 5 . 1 ) ( fisher scientific ; fair lawn , n . j .). the aminoacylated isoleucyl - trna leu was concentrated by ethanol precipitation , and resuspended in 50 mm kh 2 po 4 ( ph 5 . 0 ) at a concentration of 50 μm . the aminoacylated isoleucyl - trna leu was stored at − 20 ° c . the hydrolytic editing activity of e . coli leurs t252a / d345a was measured using a reaction mixture consisting of 60 mm tris , ph 7 . 0 , 10 mm mgcl 2 , and aminoacylated [ 3 h ]- leucyl - trna leu ( 50 μci / ml ). the reaction was initiated by the addition of 100 nm enzyme . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were the washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the wild - type leurs as well as the e . coli leurs t252a / d345a did not deacylate the leu - trna ( fig2 ). in still another embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs t252y / d342a / d345a . in the present example the e . coli leurs t252y / d342a / d345a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid phappy2 - 1 - 1 - 29 harboring the mutant d342a / d345a e . coli leus gene to create plasmid phappy2 - 1 - 1 - 23 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng phappy2 - 1 - 1 - 29 and 225 pmoles each of a forward and reverse primer [ 5 ′- ccc gcc cgg act act tta tgg gtt gta cc - 3 ′ ( seq id no . : 19 ) and 5 ′- ggt aca acc cat aaa gta gtc cgg gcg gg - 3 ′ ( seq id no . : 8 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 pmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min ), annealing ( 60 ° c ., 30 sec ) and extension ( 68 ° c ., 20 min ). the pcr products may be stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna for phappy2 - 1 - 1 - 23 was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy2 - 1 - 1 - 23 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- gaaggcgtggagat - 3 ′) ( seq id no . : 20 ) that annealed upstream of the mutated dna region . the plasmid phappy2 - 1 - 1 - 23 harboring e . coli leurs t252y / d342a / d345a was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs t252y / d342a / d345a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs t252y / d342a / d345a mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs t252y / d342a / d345a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 8 mg / ml crude e . coli trna , 10 . 93 μm [ 3 h ]- leucine ( 150 μci / ml ) and 10 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs t252y / d342a / d345a mutant leucylates trna leu at a lower efficiency than the wild - type protein ( fig4 ). a 44 μl aliquot of 40 mg / ml crude e . coli trna was aminoacylated with [ 3 h ]- isoleucine in an isoleucylation reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude trna leu , 22 μm [ 3 h ]- isoleucine ( 180 μci / ml ) and 1 μm enzyme using e . coli leurs d342a / d345a for 60 min at 37 ° c . the ph of the reaction was adjusted to 5 . 0 using acetic acid ( 0 . 1 % v / v ) and the aminoacylated isoleucyl - trna leu immediately extracted using phenol / chloroform / isoamyl alcohol ( 25 : 24 : 1 ) ( ph 5 . 1 ) ( fisher scientific ; fair lawn , n . j .). the aminoacylated isoleucyl - trna leu was concentrated by ethanol precipitation , and resuspended in 50 mm kh 2 po 4 ( ph 5 . 0 ) at a concentration of 50 μm . the aminoacylated isoleucyl - trna leu was stored at − 20 ° c . the hydrolytic editing activity of e . coli leurs t252y / d342a / d345a was measured using a reaction mixture consisting of 60 mm tris , ph 7 . 0 , 10 mm mgcl 2 , and 25 % aminoacylated [ 3 h ]- isoleucyl - trna leu . the reaction was initiated by the addition of 200 nm enzyme . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 10 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were the washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the wild - type leurs deacylated the mischarged trna . however , e . coli leurs t252y / d342a / d345a did not hydrolyze the misacylated trna . ( fig4 ). another embodiment of the present invention include leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs m40s / t252y / d342a / d345a . in the present example the e . coli leurs m40s / t252y / d342a / d345a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid phappy2 - 1 - 1 - 23 harboring mutant e . coli leus t252y / d342aid345a gene to create plasmid phappy2 - 1 - 1 / 3 - 46 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng phappy2 - 1 - 1 - 23 and 225 pmoles each of a forward and reverse primer [ 5 ′- gaa gta tta ctg cct gtc tag cct tcc cta tcc ttc tgg tcg - 3 ′ ( seq id no . : 21 ) and 5 ′- cga cca gaa gga tag gga agg cta gac agg cag taa tac ttc - 3 ′ ( seq id no . 22 ), respectively ] that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c , 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy2 - 1 - 1 / 3 - 46 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy2 - 1 - 1 / 3 - 46 was performed by lone star labs ( houston , tex .) using a primer ecr 785 ( 5 ′- gtt gtc gta cgt cca cgg - 3 ′) ( seq id no . : 23 ) that annealed upstream of the mutated dna region . plasmid phappy2 - 1 - 1 / 3 - 46 harboring e . coli leurs m40s / t252y / d342a / d345a was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs m40s / t252y / d342a / d345a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs m40s / t252y / d342a / d345a mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs m40s / t252y / d342a / d345a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude e . coli trna , 10 . 76 μm [ 3 h ]- leucine ( 150 μci / ml ) and 5 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs m40s / t252y / d342a / d345a mutant leucylates trna leu at a lower rate compared to the wild - type protein ( fig2 ). activation of leucine by the e . coli leurs m40s / t252y / d342a / d345a mutant was measured in an amino acid - dependent pyrophosphate exchange assay . the reaction contained 50 mm n - 2 - hydroxyethylpiperazine - n ′- 2 - ethanesulfonic acid ( hepes ), ph 8 . 0 , 1 mm atp , 10 mm mgcl 2 , 1 mm dtt , and 1 mm [ 32 p ]- pyrophosphate ( 53 . 3 μci / ml ), 50 nm enzyme , and 250 μm leucine . the reaction was initiated with the addition of leucine . tlc cellulose pei plates ( scientific adsorbents inc .) were pre - run in water for 2 hours . 2 μl aliquots of the pyrophosphate reaction were removed at 5 , 10 , 20 , 40 , and 80 minutes and placed on the pre - run tlc plates to quench the reaction . the tlc plates were placed in a 75 mm kh 2 po 4 , ph 3 . 5 , 4 m urea running buffer , and incubated for until the buffer front reached the top of the tlc plate . the tlc plates were dried , wrapped in saran wrap , and placed onto a phosphorimaging screen in a fujifilm bas 2040 cassette . the phosphorimaging screen was imaged in the fujix bas - 1000 phosphorimager and the amount of radiolabel present in the spots on the plates quantified using the quantitation mode of the imageguage v3 . 0 software program . the e . coli leurs m40s / t252y / d342a / d345a mutant activates leucine at a slightly lower efficiency than the wild - type leurs protein ( fig3 ). activation of norvaline by the e . coli leurs m40s / t252y / d342a / d345a mutant was measured in an amino acid - dependent pyrophosphate exchange assay . the reaction contained 50 mm n - 2 - hydroxyethylpiperazine - n ′- 2 - ethanesulfonic acid ( hepes ), ph 8 . 0 , 1 mm atp , 10 mm mgcl 2 , 1 mm dtt , and 1 mm [ 32 p ]- pyrophosphate ( 53 . 3 μci / ml ), 50 nm enzyme , and 250 μm norvaline . the reaction was initiated with the addition of norvaline . tlc cellulose pei plates ( scientific adsorbents inc .) were pre - run in water for 2 hours . 2 μl aliquots of the reaction were removed at 5 , 10 , 20 , 40 , and 80 minutes and placed on the pre - run tlc plates to quench the reaction . the tlc plates were placed in a 75 mm kh 2 po 4 , ph 3 . 5 , 4 m urea running buffer , and allowed to run for 3 hours . the tlc plates were dried , wrapped in saran wrap , and placed on a phosphoimager screen in a fujifilm bas 2040 cassette . the phosphoimaging screen was imaged in the phosphorimager and the amount of radiolabel present in the spots on the plates quantified using the quantitation mode of the imageguage v3 . 0 software program . the e . coli leurs m40s / t252y / d342a / d345a mutant activates norvaline at a significantly lower efficiency than the wild - type leurs protein ( fig3 ). kinetic measurements demonstrated that the e . coli leurs m40s / t252y / d342a / d345a mutant discriminates leucine versus norvaline more efficiently than the wild type leurs enzyme . in another embodiment , leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs y330a . in the present example the e . coli leurs y330a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p15ec3 - 1 harboring the wild - type e . coli leus gene to create plasmid pmure13 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng p15ec3 - 1 and 225 pmoles each of a forward and reverse primer ( 5 ′- cttcgtattgatggaggccggcacgggcgcag - 3 ′ ( seq id no . : 24 ) and 5 ′- gaagcataactacctccggccgtgcccgcgtc - 3 ′ ( seq id no . : 25 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( pmure13 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of pmure13 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- cggctctgagccagatc - 3 ′) ( seq id no . : 26 ) that annealed upstream of the mutated dna region . plasmid pmure13 harboring e . coli leurs y330a was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs y330a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs y330a mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). leucylation of trna leu by the e . coli leurs y330a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 4 mg / ml crude e . coli trna , 21 . 5 μm [ 3 h ]- leucine ( 150 μci / ml ) and 5 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type e . coli leurs . the e . coli leurs y330a mutant leucylates trna leu at a higher efficiency than the wild - type protein ( fig3 ). another embodiment of the present invention include leucyl - trna synthetase mutants described herein are made for eukaryotic organisms including , but not limited to yeast cytoplasmic leurs t319y . in the present example the s . cerevisiae cytoplasmic leurs t319y mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p32yl - 2 - 3 harboring yeast cdc60 gene to create plasmid phappy1 - 1 - 1 - 8 . expression of this gene from either plasmid yields a s . cerevisiae leurs fusion protein that contains an n - terminal six - histidine tag . the mutant cdc60 gene was amplified using 100 ng p32yl - 2 - 3 and 125 ng each of a forward and reverse primer ( 5 ′- gcc acg ttg aga ccg gaa tat atg tat gga caa ac - 3 ′ ( seq id no . : 27 ) and 5 ′- gtt tgt cca tac at a ta t tcc ggt ctc aac gtg gc - 3 ′ ( seq id no . : 28 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 00 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy1 - 1 - 1 - 8 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy1 - 1 - 1 - 8 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ggt gtt aca cca caa gaa tat att ggt - 3 ′) ( seq id no . : 29 ) that annealed upstream of the mutated dna region . plasmid phappy1 - 1 - 1 - 8 harboring yeast cytoplasmic leurs t319y was used to transform competent e . coli bl21 - codonplus cells ( stratagene ; la jolla , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin and 34 μg / ml chloramphenicol was grown at 37 ° c to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 30 minutes at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 2 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the yeast cytoplasmic leurs t319y mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the yeast cytoplasmic leurs t319y mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) at about 150 kda . leucylation of trna leu by the yeast cytoplasmic leurs t319y mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 30 μm [ 14 c ]- leucine ( 9 . 21 μci / ml ) and 100 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast leurs . the yeast cytoplasmic leurs t319y mutant leucylates trna leu at a slightly lower efficiency than the wild - type protein ( fig3 ). isoleucylation of trna leu by yeast cytoplasmic leurs t319y was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 22 . 25 μm [ 3 h ]- isoleucine ( 200 μci / ml ) and 750 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast cytoplasmic leurs . the yeast cytoplasmic leurs t319y protein aminoacylated trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig3 ). in another embodiment , leucyl - trna synthetase mutants described herein are made for eukaryotic organisms including , but not limited to yeast cytoplasmic leurs t319y / d419a . in the present example the s . cerevisiae cytoplasmic leurs t319y / d419a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p32 - amw1 harboring yeast mutant cdc60 gene encoding the d419a substitution to create plasmid phappy1 - 1 - 1 - 9 . expression of this gene from either plasmid yields a s . cerevisiae leurs fusion protein that contains an n - terminal six - histidine tag . the mutant cdc60 gene was amplified using 100 ng p32 - amw1 and 125 ng each of a forward and reverse primer ( 5 ′- gcc acg ttg aga ccg gaa tat atg tat gga caa ac - 3 ′ ( seq id no . : 27 ) and 5 ′- gtt tgt cca tac at a ta t tcc ggt ctc aac gtg gc - 3 ′ ( seq id no . : 28 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy1 - 1 - 1 - 9 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy1 - 1 - 1 - 9 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- ggt gtt aca cca caa gaa tat att ggt - 3 ′) ( seq id no . : 29 ) that annealed upstream of the mutated dna region . plasmid phappy1 - 1 - 1 - 9 harboring yeast cytoplasmic leurs t319y / d419a was used to transform competent e . coli bl21 - codonplus cells ( stratagene ; la jolla , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin and 34 μg / ml chloramphenicol was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 30 minutes at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 2 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the yeast cytoplasmic leurs t319y / d419a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the yeast cytoplasmic leurs t319y / d419a mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) at about 150 kda . leucylation of trna leu by the yeast cytoplasmic leurs t319y / d419a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 30 μm [ 1 4 c ]- leucine ( 9 . 21 μci / ml ) and 100 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast leurs . the yeast cytoplasmic leurs t319y / d419a mutant leucylates trna leu at a slightly lower efficiency than the wild - type protein ( fig3 ). isoleucylation of trna leu by yeast cytoplasmic leurs t319y / d419a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 22 . 25 μm [ 3 h ]- isoleucine ( 200 μci / ml ) and 750 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast cytoplasmic leurs . the yeast cytoplasmic leurs t319y / d419a protein aminoacylated trna leu at high levels with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig3 ). in another embodiment , leucyl - trna synthetase mutants described herein are made for eukaryotic organisms including , but not limited to yeast cytoplasmic leurs n415a . in the present example the s . cerevisiae cytoplasmic leurs n415a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p32yl - 2 - 3 harboring yeast cdc60 gene to create plasmid phappy1 - 1 - 1 - 55 . expression of this gene from either plasmid yields a s . cerevisiae leurs fusion protein that contains an n - terminal six - histidine tag . the mutant cdc60 gene was amplified using 100 ng p32yl - 2 - 3 and 125 ng each of a forward and reverse primer ( 5 ′- cac gtg tgt acc atc tgc ttc acc aga tga cta c - 3 ′ ( seq id no . : 30 ) and 5 ′- gta gtc atc tgg tga a gc aga tgg tac aca cgt g - 3 ′ ( seq id no . : 31 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy1 - 1 - 1 - 55 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy1 - 1 - 1 - 55 was performed by lone star labs ( houston , tex .) using a primer ( 5 ′- gct acc aag ggt act ggt gtc gtc - 3 ′) ( seq id no . : 32 ) that annealed upstream of the mutated dna region . plasmid phappy1 - 1 - 1 - 55 harboring yeast cytoplasmic leurs n415a was used to transform competent e . coli bl21 - codonplus cells ( stratagene ; la jolla , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin and 34 μg / ml chloramphenicol was grown at 37 ° c to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 30 minutes at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 2 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the yeast cytoplasmic leurs n415a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the yeast cytoplasmic leurs n415a mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) at about 150 kda . leucylation of trna leu by the yeast cytoplasmic leurs n415a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 30 μm [ 14 c ]- leucine ( 9 . 21 μci / ml ) and 100 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast leurs . the yeast cytoplasmic e leurs n415a mutant leucylates trna leu at a slightly lower efficiency than the wild - type protein ( fig3 ). isoleucylation of trna leu by yeast cytoplasmic leurs n415a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 22 . 25 μm [ 3 h ]- isoleucine ( 200 μci / ml ) and 750 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast cytoplasmic leurs . the yeast cytoplasmic leurs n415a protein aminoacylated trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig3 ). other embodiments of the present invention include leucyl - trna synthetase mutants described herein are made for eukaryotic organisms including , but not limited to yeast cytoplasmic leurs n415a / d419a . in the present example the s . cerevisiae cytoplasmic leurs n415a / d419a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p32 - amw1 harboring the yeast mutant cdc60 gene encoding the d419a substitution to create plasmid phappy1 - 1 - 1 - 59 . expression of this gene from either plasmid yields a s . cerevisiae leurs fusion protein that contains an n - terminal six - histidine tag . the mutant cdc60 gene was amplified using 100 ng p32 - amw1 and 125 ng each of a forward and reverse primer ( 5 ′- cac gtg tgt acc atc t gc ttc acc aga tg c cta c - 3 ′ ( seq id no . : 30 ) and 5 ′- gta g g c atc tgg tga a gc aga tgg tac aca cgt g - 3 ′ ( seq id no . : 31 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy1 - 1 - 1 - 59 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy1 - 1 - 1 - 59 was performed by lone star labs ( houston , tex .) using a primer ycf 1356 ( 5 ′- gct acc aag ggt act ggt gtc gtc - 3 ′) ( seq id no . : 32 ) that annealed upstream of the mutated dna region . plasmid phappy1 - 1 - 1 - 59 harboring yeast cytoplasmic leurs n415a / d419a was used to transform competent e . coli bl21 - codonplus cells ( stratagene ; la jolla , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin and 34 μg / ml chloramphenicol was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 30 minutes at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 2 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the yeast cytoplasmic leurs n415a / d419a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the yeast cytoplasmic leurs n415a / d419a mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) at about 150 kda . leucylation of trna leu by the yeast cytoplasmic leurs n415a / d419a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 30 μm [ 14 c ]- leucine ( 9 . 21 μci / ml ) and 100 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast leurs . the yeast cytoplasmic leurs n415a / d419a mutant leucylates trna leu at a slightly lower efficiency than the wild - type protein ( fig3 ). isoleucylation of trna leu by yeast cytoplasmic leurs n415a / d419a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 22 . 25 μm [ 3 h ]- isoleucine ( 200 μci / ml ) and 750 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast cytoplasmic leurs . the yeast cytoplasmic leurs n415a / d419a protein aminoacylated trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig4 ). still other embodiments of the present invention include leucyl - trna synthetase mutants described herein are made for eukaryotic organisms including , but not limited to yeast cytoplasmic leurs s416aid419a . in the present example the s . cerevisiae cytoplasmic leurs s416a / d419a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p32 - amw1 harboring the yeast mutant cdc60 gene encoding the d419a substitution to create plasmid phappy1 - 1 - 1 - 57 . expression of this gene from either plasmid yields a s . cerevisiae leurs fusion protein that contains an n - terminal six - histidine tag . the mutant cdc60 gene was amplified using 100 ng p32 - amw1 and 125 ng each of a forward and reverse primer ( 5 ′- cgt gtg tac cat cta at g cac cag atg c ct ac - 3 ′ ( seq id no . : 33 ) and 5 ′- gta g g c atc tgg tg c att aga tgg tac aca cg - 3 ′ ( seq id no . : 34 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 pg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 [ μg / ml ampicillin . the plasmid dna ( phappy1 - 1 - 1 - 57 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy1 - 1 - 1 - 57 was performed by lone star labs ( houston , tex .) using a primer ycf 1356 ( 5 ′- gct acc aag ggt act ggt gtc gtc - 3 ′) ( seq id no . : 32 ) that annealed upstream of the mutated dna region . plasmid phappy1 - 1 - 1 - 57 harboring yeast cytoplasmic leurs s416a / d419a was used to transform competent e . coli bl21 - codonplus cells ( stratagene ; la jolla , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin and 34 μg / ml chloramphenicol was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 30 minutes at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 2 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the yeast cytoplasmic leurs s416a / d419a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the yeast cytoplasmic leurs s416a / d419a mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) at about 150 kda . leucylation of trna leu by the yeast cytoplasmic leurs s416a / d419a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 30 μm [ 14 c ]- leucine ( 9 . 21 μci / ml ) and 100 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 1l aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared those from experiments performed in parallel using the wild - type yeast leurs . the yeast cytoplasmic leurs s416a / d419a mutant leucylates trna leu similar to the wild - type protein ( fig4 ). isoleucylation of trna leu by yeast cytoplasmic leurs s416a / d419a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 22 . 25 μm [ 3 h ]- isoleucine ( 200 μci / ml ) and 750 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast cytoplasmic leurs . the yeast cytoplasmic leurs s416a / d419a protein aminoacylated trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig4 ). still other embodiments of the present invention include leucyl - trna synthetase mutants described herein are made for eukaryotic organisms including , but not limited to yeast cytoplasmic leurs d418a . in the present example the s . cerevisiae cytoplasmic leurs d418a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p32yl - 2 - 3 harboring yeast cdc60 gene to create plasmid phappy1 - 1 - 1 - 56 . expression of this gene from either plasmid yields a s . cerevisiae leurs fusion protein that contains an n - terminal six - histidine tag . the mutant cdc60 gene was amplified using 100 ng p32yl - 2 - 3 and 125 ng each of a forward and reverse primer ( 5 ′- cca tct aat tca cca g c t gac tac att acc acc - 3 ′ ( seq id no . : 35 ) and 5 ′- ggt ggt aat gta gtc a g c tgg tga att aga tgg - 3 ′ ( seq id no . : 36 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy1 - 1 - 1 - 56 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy1 - 1 - 1 - 56 was performed by lone star labs ( houston , tex .) using a primer ycf 1356 ( 5 ′- gct acc aag ggt act ggt gtc gtc - 3 ′) ( seq id no . : 32 ) that annealed upstream of the mutated dna region . plasmid phappy1 - 1 - 1 - 56 harboring yeast cytoplasmic leurs d418a was used to transform competent e . coli bl21 - codonplus cells ( stratagene ; la jolla , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin and 34 μg / ml chloramphenicol was grown at 37 ° c to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 30 minutes at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 2 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the yeast cytoplasmic leurs d418a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the yeast cytoplasmic leurs d418a mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) at about 150 kda . leucylation of trna leu by the yeast cytoplasmic leurs d418a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 30 μm [ 14 c ]- leucine ( 9 . 21 [ μci / ml ) and 100 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast leurs . the yeast cytoplasmic leurs d418a mutant leucylates trna leu similar to the wild - type protein ( fig4 ). isoleucylation of trna leu by yeast cytoplasmic leurs d418a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 22 . 25 μm [ 3 h ]- isoleucine ( 200 μci / ml ) and 750 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast cytoplasmic leurs . the yeast cytoplasmic leurs d418a protein aminoacylated trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig4 ). in the present example compositions of leucyl - trna synthetase mutants described herein are made for eukaryotic organisms including , but not limited to yeast cytoplasmic leurs d418a / d419a . in the present example the s . cerevisiae cytoplasmic leurs d418a / d419a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid p32 - amw1 harboring yeast mutant cdc60 gene encoding the d419a mutation to create plasmid phappy1 - 1 - 1 - 58 . expression of this gene from either plasmid yields a s . cerevisiae leurs fusion protein that contains an n - terminal six - histidine tag . the mutant cdc60 gene was amplified using 100 ng p32 - amw1 and 125 ng each of a forward and reverse primer ( 5 ′- cgt tct aat tca cca g c t g c c tac att acc acc - 3 ′ ( seq id no . : 37 ) and 5 ′- ggt ggt aat gta g g c a g c tgg tga att aga tgg - 3 ′ ( seq id no . : 38 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( phappy1 - 1 - 1 - 58 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of phappy1 - 1 - 1 - 58 was performed by lone star labs ( houston , tex .) using a primer ycf 1356 ( 5 ′- gct acc aag ggt act ggt gtc gtc - 3 ′) ( seq id no . : 32 ) that annealed upstream of the mutated dna region . plasmid phappy1 - 1 - 1 - 58 harboring yeast cytoplasmic leurs d418a / d419a was used to transform competent e . coli bl21 - codonplus cells ( stratagene ; la jolla , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin and 34 μg / ml chloramphenicol was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 30 minutes at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 2 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the yeast cytoplasmic leurs d418a / d419a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the yeast cytoplasmic leurs d418a / d419a mutant containing a six histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) at about 150 kda . leucylation of trna leu by the yeast cytoplasmic leurs d418a / d419a mutant was measured in a reaction containing 60 mm tris , ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 30 μm [ 14 c ]- leucine ( 9 . 21 μci / ml ) and 100 nm enzyme . the reaction was initiated by the addition of atp . the reaction was quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the washed pads were dried and combined with 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast leurs . the yeast cytoplasmic leurs d418a / d419a mutant leucylates trna leu similar to the wild - type protein ( fig4 ). isoleucylation of trna leu by yeast cytoplasmic leurs d418a / d419a was measured in a reaction containing 60 mm tris ph 7 . 5 , 10 mm mgcl 2 , 1 mm dithiothreitol ( dtt ), 4 mm atp , 5 mg / ml crude yeast trna , 22 . 25 μm [ 3 h ]- isoleucine ( 200 μci / ml ) and 750 nm enzyme . the reaction was initiated by the addition of atp and quenched by placing a 5 μl aliquot on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from experiments performed in parallel using the wild - type yeast cytoplasmic leurs . the yeast cytoplasmic leurs d418a / d419a protein aminoacylated trna leu with isoleucine yielding isoleucine - trna leu in contrast to the wild - type protein ( fig4 ). other embodiments of the present invention include leucyl - trna synthetase mutants described herein are made for prokaryotic organisms including , but not limited to e . coli leurs y330a / d342a / d345a . in the present example the e . coli leurs y330a / d342a / d345a mutation was introduced via the polymerase chain reaction ( pcr ) into the plasmid phappy2 - 1 - 1 - 29 harboring the d342a / d345a mutant e . coli leus gene to create plasmid pmuret1 . expression of this gene from either plasmid yields an e . coli leurs fusion protein that contains an n - terminal six - histidine tag . the mutant leus gene was amplified using 100 ng pmuret1 and 225 pmoles each of a forward and reverse primer ( 5 ′- cttcgtattgatggaggccggcacgggcgcag - 3 ′ ( seq id no . : 24 ) and 5 ′- gaagcataactacctccggccgtgcccgcgtc - 3 ′ ( seq id no . : 25 ), respectively ) that encoded the gene mutation . the pcr reaction mixture also contained 10 nmoles each of datp , dttp , dgtp and dctp , 3 units of pfu dna polymerase ( promega ; madison , wis .) in 50 μl of 20 mm tris ( hydroxymethyl ) aminomethane ( tris ), ph 8 . 8 , 10 mm kcl , 10 mm ( nh 4 ) 2 so 4 , 2 mm mgso 4 , 0 . 1 % triton x - 100 and 100 μg / ml bovine serum albumen ( bsa ). the pcr reaction was thermocycled 18 times . each cycle was as follows : denaturation ( 95 ° c ., 1 min . ), annealing ( 60 ° c ., 30 sec .) and extension ( 68 ° c ., 20 min ). the pcr products were stored at 4 ° c . until removal from thermocycler . following pcr amplification , the parental plasmid was digested with 10 units of dpn i ( new england biolabs , inc . ; beverly , mass .). the pcr product was then used to transform e . coli strain dh5α ( invitrogen corporation ; carlsbad , calif .). colonies that contained the plasmid were grown and selected on lb media that contained 100 μg / ml ampicillin . the plasmid dna ( pmuret1 ) was isolated using wizard miniprep kits ( promega ; madison , wis .). sequencing of pmuret1 was performed by lone star labs ( houston , tex .) using a primer ecr 1582 ( 5 ′- cggctctgagccagatc - 3 ′) ( seq id no . : 26 ) that annealed upstream of the mutated dna region . plasmid pmuret1 harboring e . coli leurs y330a / d342a / d345a was used to transform e . coli strain bl21 plyss ( invitrogen corporation ; carlsbad , calif .). a 500 ml lb culture that included 100 μg / ml ampicillin was grown at 37 ° c . to an a 600 of approximately 0 . 6 . protein expression was induced by the addition of 1 mm isopropyl - thiogalactopyranoside ( iptg ) for 2 hours at 25 ° c . the cells were recovered by centrifugation and were resuspended in 10 ml of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , 300 mm nacl and 5 % glycerol called hereafter resuspension buffer . the cells were lysed by sonication twice in a vibra cell ( sonics & amp ; materials , inc . ; danbury , conn .) at 50 % power for 1 . 5 minutes on ice . cell debris was pelleted by centrifugation and discarded . the supernatant or lysate was added to a 1 ml bed volume of talon affinity resin ( clontech ; palo alto , calif .) that had been pre - equilibrated in the cell resuspension buffer . the batch preparation for affinity purification of the e . coli leurs y330a / d342a / d345a mutant was mixed gently at 4 ° c . for 30 minutes . the resin was separated from the supernatant in a clinical centrifuge ( international equipment co . ; needham heights , mass .). the resin was washed four times with 10 ml of 20 mm na 2 hpo 4 ( ph 7 . 0 ), 10 mm tris , 500 mm nacl and 10 % glycerol . the e . coli leurs y330a / d342a / d345a mutant containing a six - histidine tag was eluted with 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris ( ph 8 . 0 ), 300 mm nacl , 100 mm imidazole and 10 % glycerol . the elution fraction was concentrated and dialyzed into its final storage buffer of 20 mm na 2 hpo 4 ( ph 8 . 0 ), 10 mm tris , ph 7 . 0 , 200 mm nacl , & lt ; 10 mm imidazole and 50 % glycerol . the purified 98 . 5 kda leurs fusion protein was identified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ). preliminary studies involve the complete isolation of charged trna with either leucine or isoleucine . aminoacylated trna leu linked to leucine or isoleucine was generated in a reaction that contained 60 mm tris ( ph 7 . 5 ), 10 mm mgcl 2 , 4 mm atp , 1 mm dtt , 10 μm trna uaa leu , 20 . 5 μm [ 3 h ]- leucine ( 200 μci / ml ) or [ 3 h ]- isoleucine ( 550 μci / ml ), and 200 nm enzyme . the reaction mixture was incubated at room temperature for 2 hours and then quenched by lowering the ph to approximately 6 . 0 with 0 . 2 % acetic acid to stabilize the labile aminoacyl bond ( schreier and schimmel , 1972 ; mursinna et al ., 2001 ; mursinna and martinis , 2002 ). the mischarged trna was concentrated by butanol extraction and then immediately extracted using phenol / chloroform / isoamyl alcohol ( 125 : 24 : 1 ) ( ph 4 . 3 ) ( fisher biotech , fair lawn , n . j .) and eppendorf phase lock gel ( brinkmann instruments , inc ., westbury , n . y . ), followed by ethanol precipitation . the rna pellet was washed three times with 70 % ethanol , resuspended in 20 mm potassium phosphate ( ph 5 . 0 ), and stored at − 20 ° c . to quantitate the percentage yield of charged trna , an aliquot of the resuspended charged trna was placed on a whatman 1003323 filter pad pre - soaked in 5 % ( v / v ) trichloroacetic acid ( tca ). the pads were slowly shaken on ice in 5 % tca with four changes over a 1 hour time period . the pads were then washed with 70 % ethanol and subsequently with ether ( anhydrous ). the pads were then dried and added to 3 ml of scintisafe ™ econo f scintillation solution ( fisher scientific ; fair lawn , n . j .). this mixture was quantitated by scintillation counting in a beckman ls 6000ic ( beckman instruments , inc . ; fullerton , calif .). results were compared with those obtained from similar experiments performed with other mutant leurss ( fig4 a and b ). the leurs y330a / d342a / d345a aminoacylates isoleucine to trna leu at high levels , particularly compared to the levels yielded by the e . coli leurs d342a / d345a mutant . although making and using various embodiments of the present invention are discussed in detail below , it will be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts . the specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention , and do not delimit the scope of the invention . apostol , i ., j . levine , j . lippincott , j . leach , e . hess , c . b . glascock , m . weickert , and r . blackmore . 1997 . incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in escherichia coli . j biol chem 272 : 28980 - 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