Patent Application: US-26675094-A

Abstract:
a novel method for synthesizing peptides and proteins having a desired amino acid sequence by utilizing an aminoacyl - trna and misaminoacyl - trna as well as an artificial mrna in a system for in vitro protein synthesis is provided . according to the present method , desired peptides and proteins can be synthesized using naturally - occurring and non naturally - occurring amino acids . accordingly , the present invention provides a fundamental technique which is important in the fields of various industries and various fundamental researches such as biochemistry , molecular biology and protein engineering .

Description:
the simplest example in practicing the present invention is the case wherein the number of multiple 10 genetic codons which are different from each other is two . in this case , an artificial mrna wherein these two genetic codons are arranged alternately is used as a template . on the other hand , for each of two trnas corresponding to the above two genetic codons , an aminoacyl - trna ( wherein a corresponding naturally - occurring amino acid binds to trna ) and misaminoacyl - trnas ( wherein a non - corresponding naturally - occurring or non - naturally - occurring amino acid binds to trna ) are prepared . then , a peptide having a desired amino acid sequence can be synthesized by adding only an aminoacyl - trna or misaminoacyl - trna ( wherein an amino acid component to be incorporated into the peptide binds to trna ) to a system for in vitro protein synthesis containing the above mrna and ribosome , and carrying out stepwise extension reaction of the peptide chain . for example , synthesis of tripeptide , alanyl - glycyl - tyrosine , by step reaction is described below . as an artificial mrna , for example , mrna comprising a repeat structure of a base sequence uuugag is prepared utilizing genetic codons uuu and gag coding for phenylalanine and glutamic acid , respectively . the artificial mrna and s - 100 fraction as well as a suitable buffer containing components necessary for in vitro protein synthesis are added to ribosome prepared from e . coli . then , to the mixture is added a misaminoacyl - trna wherein the first amino acid of the desired peptide , alanine , binds to trna phe ( corresponding amino acid is phenylalanine , and the trna forms a complex together with a codon for phenylalanine on mrna ), and the mixture is reacted at about 37 ° c . for an appropriate time ( for example , 15 minutes ). by the reaction , a bond is formed between the phenylalanine codon on mrna , ribosome and alanyl - trna phe to give a complex . at this time , the next codon of the phenylalanine codon on mrna is a codon for glutamic acid and there exists no aminoacyl - trna glu in the reaction system , and therefore , the peptide - forming reaction can not proceed ahead . the complex is separated from other components , for example , by a means such as molecular sieve chromatography . the complex fraction is then concentrated , for example , by a means such as ultrafiltration . the ribosome - mrna - alanyl trna phe complex thus concentrated can be used for binding the second amino acid of the desired peptide . thus , to the concentrated complex fraction are added s - 100 fraction , a suitable buffer containing components necessary for in vitro protein synthesis , and glycyl - trna glu wherein the second amino acid of the desired peptide , glycine , binds to trna glu , and the mixture is again reacted at about 37 ° c . for an appropriate time . as a result , a reaction between the first misaminoacyl - trna ( alanyl - trna phe ) and the second misaminoacyl - trna ( glycyl - trna glu ) on ribosome takes place to give alanyl - glycyl - trna glu . at this time , alanyl - glycyl - trna glu binds to the glutamic acid codon on mrna and the next codon is a codon for phenylalanine . however , there exists no aminoacyl - trna phe in the reaction system , and therefore , the peptide - forming reaction can not proceed ahead . accordingly , only alanyl - glycine is formed . then , the ribosome - mrna - alanyl - glycyl - trna glu complex is again separated from other components by molecular sieve chromatography , and the complex fraction is concentrated by ultrafiltration . to the resulting complex fraction are added s - 100 fraction , a suitable buffer containing components necessary for in vitro protein synthesis , and tyrosyl - trna phe wherein the third amino acid of the desired peptide , tyrosine , binds to trna phe , and the mixture is again reacted at about 37 ° c . for an appropriate time . as a result , a reaction between the alanyl - glysyl - trna glu and the tyrosyl - trna phe on ribosome takes place to give alanyl - glycyl - tyrosyl - trna phe . at this time , alanyl - glycyl - tyrosyl - trna phe binds to the phenylalanine codon on mrna and the next codon is a codon for glutamic acid . however , there exists no aminoacyl - trna glu in the reaction system , and therefore , the peptide - forming reaction can not proceed ahead . accordingly , only alanyl - glycyl - tyrosine is formed . after the desired peptide is thus synthesized , an rna fraction containing the peptidyl - trna can be obtained from the reaction mixture , for example , by phenol extraction . the desired peptide can be eliminated from trna by decomposing rna , for example , by alkaline hydrolysis of the rna fraction . then , the mixture of the desired peptide obtained and the decomposed products of rna can be applied to a purification procedure such as reverse phase chromatography to obtain only the desired peptide . thus , the above - mentioned reaction for stepwise synthesis of a peptide can be divided into three reaction cycles : ( a ) initiation reaction cycle wherein an aminoacyl - trna or misaminoacyl - trna ( wherein a n - terminal amino acid of the desired peptide or protein binds to trna ) forms a complex together with ribosome and mrna ; ( b ) peptide extension reaction cycle wherein a peptide chain is extended stepwise from the second amino acid residue of the desired peptide or protein on the ribosome - mrna -( mis ) aminoacyl trna complex ; and ( c ) termination reaction cycle wherein the desired peptide or protein is separated from the ribosome - mrna - peptidyl trna complex . initiation reaction cycle ( a ) is composed of the following three steps : ( a - 1 ) reaction of an aminoacyl - trna or misaminoacyl - trna ( wherein a n - terminal amino acid of the desired peptide or protein binds to trna ) with ribosome and mrna to form a ribosome - mrna -( mis ) aminoacyl trna complex ; ( a - 2 ) separation of the ribosome - mrna -( mis ) aminoacyl trna complex by high - performance molecular sieve chromatography ; and ( a - 3 ) concentration of fractions containing the complex by ultrafiltration . peptide extension reaction cycle ( b ) is reaction cycles for binding amino acids stepwise one by one , and composed of the following three steps : ( b - 1 ) peptide formation reaction comprising binding the ( n + 1 ) th amino acid residue on the ribosome - mrna - peptidyl trna complex ( wherein the peptidyl contains n amino acids , and n is an integer of 1 or more ); ( b - 2 ) separation of the resulting ribosome - mrna - peptidyl trna complex by high - performance molecular sieve chromatography ; and ( b - 3 ) concentration of fractions containing the complex by ultrafiltration . by repeating the reaction cycle , a desired number of amino acids can bind stepwise and a peptide having a desired amino acid sequence can be synthesized . termination reaction cycle ( c ) is composed of the following three steps : ( c - 1 ) recovery of the peptidyl - trna from the final ribosome - mrna - peptidyl trna complex by phenol extraction and ethanol precipitation ; ( c - 2 ) elimination of the desired peptide or protein from the peptidyl - trna by hydrolysis of rna ; and ( c - 3 ) purification of the desired peptide or protein by high - performance reverse phase chromatography . in the above description , exemplified is the case 10 wherein the number of multiple genetic codons different from each other is two , that is , the case wherein an artificial mrna wherein these two genetic codons are arranged alternately is used as a template . however , it is also possible to use more genetic codons different from each other . thus , the artificial mrna used in the present method may comprise the following sequence : wherein m is an integer of 2 to 64 , n is an integer of 1 or more , m × n is equal to the number of amino acid residues of a desired peptide or more , and each of a 1 , a 2 , a 3 , . . . and a m represents a genetic codon which differs from the other codons . for example , when an artificial mrna having a repeat structure of five codons different from each other is used as a template , it is possible to use four aminoacyl - trnas or misaminoacyl - trnas per one cycle of peptide forming reaction cycle ( b ), and therefore , it is possible to extend four amino acids per one cycle at one time . since trna can discriminatingly recognize at least twenty codons , it is possible to bind nineteen amino acids per one cycle at one time by using as a template an artificial mrna having a repeat structure composed of twenty codons different from each other . thus , according to the present method , it is possible to synthesize a long peptide in a small cycle number , and therefore , it becomes possible to synthesize a protein which has previously been difficult to prepare by chemical synthesis . in general , e . coli - derived ribosome and s - 100 fraction ( containing various factors for protein synthesis which are necessary for a system for in vitro protein synthesis ) used in practicing the present invention can be prepared according to the method of noll et al . ( reference 18 ). thus , e . coli ( for example , mre600 ) at mid log growth phase is ground together with aluminum oxide in a buffer solution containing 10 mm magnesium ion ( for example , 20 mm tris - hcl , 10 mm ( ch 3 coo ) 2 mg , 100 mm nh 4 cl , 3 mm 2 - mercaptoethanol ( ph 7 . 5 )), and dnase i was further added to decompose endogenous dna . the sample solution is then centrifuged ( for example , at 30 , 000 × g for 45 minutes ) to obtain a supernatant ( s - 30 fraction ). the s - 30 fraction is overlayered on equal volume of buffer solution b ( for example , 10 mm tris - hcl ( ph 7 . 5 ) containing 1 . 1m sucrose , 10 mm ( ch 3 coo ) 2 mg , 60 mm nh 4 cl , and 3 mm 2 - mercaptoethanol ), and it is centrifuged using ultracentrifuge for separation such as beckman model l8m ( for example , at 100 , 000 × g for 20 hours ). ribosome is recovered as a clear precipitation . on the other hand , the supernatant contains various factors necessary for protein synthesis , and can be used as s - 100 fraction in a system for in vitro protein synthesis . as mrna having a repeat structure of multiple genetic codons used in practicing the present invention , for example , poly ( ag ) encoding poly ( arg - glu ), poly ( ac ) encoding poly ( thr - his ), poly ( au ) encoding poly ( ile - tyr ), and poly ( cu ) encoding poly ( leu - ser ) are commercially available from sigma chemical company . alternatively , mrnas having a repeat structure of desired genetic codons can be chemically synthesized by a solid phase synthesis based on β - cyanoethyl method , utilizing four rna nucleotides wherein functional groups are protected ( for example , dmt - cyanoethyl rna phosphoamidite reagents ) and a solid phase resin to which a protected rna nucleotide corresponding to 3 &# 39 ;- terminus of rna to be synthesized is bound , whereby nucleotides are condensed in the order of the desired base sequence ( references 19 and 20 ). commercially available dna synthesizer ( for example , abi model 380b ; applied biosystems ) can be used for the synthesis . the synthesized rna fragment is cut off from the resin , and deprotection of 2 &# 39 ;- hydroxyl protecting group is performed ( for example , by treating the rna fragment with a tetrahydrofuran solution containing 1m tetrabutylammonium fluoride at room temperature for 6 hours ). the resulting rna fragment is then purified by high - performance reverse phase chromatography ( for example , by using waters μ - bondasphere 5μc18 - 300 column , and eluting the rna fragment with an acetonitrile linear gradient ). the synthetic mrna is used as a template in the present method . an aminoacyl - trna used in practicing the present invention can be prepared by the method of nishimura et al . ( reference 6 ) using aminoacyl - trna synthetase . thus , to an amino acid - specific trna to be aminoacylated are added a desired amino acid , adenosine triphosphate ( atp ) and aminoacyl - trna synthetase , and the mixture is reacted in a suitable buffer solution . proteins are removed from the reaction mixture by phenol extraction and the resulting aminoacyl - trna is recovered by ethanol precipitation . amino acid - specific trnas as a substrate and aminoacyl - trna synthetase ( e . coli - derived ) are commercially available from sigma chemical company . as a synthetic method of a misaminoacyl - trna , the method of s . m . heckler et al . ( reference 21 ) is known . thus , dinucleotide pcpa is first synthesized chemically and an amino acid is chemically ester - bonded to 3 &# 39 ;- terminus of pcpa to obtain aminoacyl - pcpa . on the other hand , trna is partially digested with venom phosphodiesterase to obtain trna - c - oh lacking pcpa at its 3 &# 39 ;- terminus . finally , the aminoacyl - pcpa is enzymatically linked to the 3 &# 39 ;- terminus of trna - c - oh using rna ligase to obtain an aminoacyl - trna . components and reaction conditions used in a standard system for in vitro protein synthesis ( for example , synthesis of polyphenylalanine depending on poly u ) are as follows ( reference 22 ). thus , a reaction mixture ( 100 - 500 μl ) containing 20 - 60 mm tris - hcl ( ph 7 . 0 - 8 . 0 ), 10 - 15 mm magnesium chloride or magnesium acetate , 10 - 100 mm nh 4 cl , 1 - 10 mm 2 - mercaptoethanol or dithiothreitol , 1 - 2 mm adenosine triphosphate ( atp ), 0 . 1 - 0 . 5 mm guanosine triphosphate ( gtp ), 5 - 15 mm sodium phosphoenolpyruvate , 2 - 50 μg / ml pyruvate kinase , 50 - 170 μm non - labeled amino acid ( excepting phenylalanine ), 5 - 100 μm labeled phenylalanine ( for example , 10 - 100 μci / μmol of l -[ u - 14 c ]- phenylalanine ), 0 . 1 - 1 mg / ml phenylalanine - specific trna , 0 . 1 - 0 . 8 mg / ml poly u , 2 - 50 a 260 units / ml ribosome , and 2 mg / ml s - 100 fraction is incubated at about 37 ° c . for 15 - 30 minutes . identification of reaction products is generally carried out as follows . thus , 1 ml of 10 % trichloroacetic acid ( tca ) solution was added to the mixture and it was heated to 90 ° c . for 20 minutes to eliminate the amino acid and peptide from the aminoacyl - trna and peptidyl - trna . next , the precipitation is collected onto a glass microfiber filter ( for example , gf / c , whatman international ltd . ), and washed four times with 5 % tca and four times with 99 % ethanol . after drying , the filter was soaked in 5 ml of liquid scintillation cocktail ( for example , econofluor - 2 , nen research products ) and the radioactivity of the precipitation on the filter is measured by a liquid scintillation counter ( for example , beckman model ls5000td ) to estimate an amount of the formed product . for purifying the product from the reaction mixture , various methods used for purifying a protein can be generally utilized . thus , purification methods such as polyacrylamide gel electrophoresis , isoelectric focusing , ion exchange chromatography , molecular sieve chromatography , and affinity chromatography can be used for purifying the product . the present invention is further illustrated by the following examples , but should not be construed to be limited thereto . synthesis of peptides by a step reaction using naturally - occurring aminoacyl - trnas and a system for in vitro protein synthesis a tripeptide was synthesized by a step reaction using naturally - occurring aminoacyl - trnas in a system for in vitro protein synthesis containing escherichia coli ribosome and s - 100 fraction as well as an artificial mrna . a . preparation of e . coli ribosome and s - 100 fraction as well as properties and activity of purified ribosome e . coli mre600 ( atcc no . 29417 ) was obtained from american type culture collection . l - broth medium was prepared by dissolving 10 g of bacto - tryptone ( difco laboratories ), 5 g of bacto - yeast extract ( difco laboratories ), 5 g of nacl and 1 g of glucose in 1 l of super purified water and then adjusting at ph 7 . 5 , and was used after autoclaving . super purified water was prepared by milli - q sp system ( japan millipore ltd .). e . coli mre600 cells were cultured overnight in 3 ml of l - broth . the culture was then used to inoculate 1 l of l - broth and cultivation was further continued at 37 ° c . for 2 . 0 to 1 . 5 hours ( a 600 = 0 . 4 - 0 . 6 ). the e . coli cells were harvested by centrifuging the culture at 10 , 000 × g for 15 minutes and stored at - 20 ° c . until they were used as a starting material for preparing ribosome . e . coil 70s ribosome and s - 100 fraction were prepared by somewhat modifying the method of noll et al . ( reference 18 ). all procedures were carried out below 4 ° c . to frozen e . coli cells was added two volumes of aluminum oxide , and the mixture was ground in a mortar . when the e . coli cells and aluminum oxide were well mixed and the mixture showed a very high viscosity and crackled upon grinding it in the mortar , then equal volume of buffer a ( 20 mm tris - hcl , 10 mm ( ch 3 coo ) 2 mg , 100 mm nh 4 cl , 3 mm 2 - mercaptoethanol ( ph 7 . 5 )) was added to the mixture , and dnase i ( takara shuzo k . k .) was further added to obtain 3 μg / ml of final concentration . the suspension was again ground for about 10 minutes . when viscosity of the suspension well lowered , it was centrifuged at 30 , 000 × g for 45 minutes to obtain supernatant ( s - 30 fraction ). the s - 30 fraction was overlayered on equal volume of buffer b ( 10 mm tris - hcl ( ph 7 . 5 ) containing 1 . 1m sucrose , 10 mm ( ch 3 coo ) 2 mg , 60 mm nh 4 cl , and 3 mm 2 - mercaptoethanol ), and it was centrifuged using ultracentrifuge for separation beckman model l8m at 100 , 000 × g ( type 45 ti rotor , 30 , 000 × rpm ) for 20 hours . clear precipitation was recovered as ribosome and upper layer ( 2 / 3 of supernatant ) as crude s - 100 fraction . the clear precipitation of ribosome was gently suspended in buffer a and the suspension was gently stirred for about 2 hours to disperse ribosome . the resulting ribosome solution was again overlayered on buffer b and it was centrifuged at 100 , 000 × g ( type 45 ti rotor , 30 , 000 × rpm ) for 20 hours . the resulting precipitation of ribosome was again suspended in buffer a and the suspension was then dialyzed against buffer a . the dialyzed suspension was used as ribosome solution in later experiments . concentration of ribosome was determined by measuring absorbance of the resulting ribosome solution at 260 nm . the solution showing 1 . 0 of absorbance at 260 nm was defined as 1 . 0 u / ml ribosome solution . the ribosome solution was stored at - 80 ° c . until use . the crude s - 100 fraction was further fractionated using 70 % saturated ammonium sulfate and the precipitated fraction was dialyzed against buffer a . the dialyzed fraction was used as s - 100 fraction in later experiments . protein concentration of the s - 100 fraction was determined by measuring absorbance of the resulting fraction at 280 nm . the s - 100 fraction was stored at - 80 ° c . until use . about 8 , 000 u of ribosome ( 1u = 1a 260 ) was obtained from 3 l of e . coli cultured medium . for the purpose of assaying the purity of the resulting purified ribosome , it was analyzed by sucrose density - gradient ultracentrifugation in the presence of 10 mm and 1 mm magnesium ion . the purified ribosome was analyzed by ultrafugation using 10 % to 30 % of sucrose density - gradient in the presence of 10 mm mg 2 + and 1 mm mg 2 + . the sucrose density - gradient was prepared as follows . solutions of 3 mm 2 - mercaptoethanol , 60 mm nh 4 cl , 1 mm or 10 mm ( ch 3 coo ) 2 mg , 10 mm tris - hcl ( ph 7 . 5 ) each containing 10 %, 15 %, 20 %, 25 %, and 30 % of sucrose were prepared , and 7 . 4 ml portions of the solutions were overlayered in order of the sucrose density using a syringe in a centrifugation tube ( total volume 37 ml ), charging the solution having the lowest sucrose density at the bottom of the tube . the tube was then allowed to stand overnight at 4 ° c . and sucrose density - gradient was formed . each of solutions ( 0 . 1 ml ) of 1 mm or 10 mm ( ch 3 coo ) 2 mg , 3 mm 2 - mercaptoethanol , 60 mm nh 4 cl , 10 mm tris - hcl ( ph 7 . 5 ) containing 20 u of ribosome was overlayered onto the sucrose density - gradient solution having the same mg 2 + concentration , and the tube was centrifuged at 160 , 000 × g ( sw28 , 20 , 000 rpm ) for 15 hours . after the centrifugation , the sucrose density - gradient solution was taken in 1 ml portions from the bottom of the tube . as a result , in the presence of 10 mm magnesium , only 70s ribosome which is an active form was detected among all ribosomes and 50s and 30s ribosome subunits were little detected ( fig1 ). on the other hand , in the presence of 1 mm magnesium , 70s ribosome was dissociated into 50s and 30s subunits in near completeness . these results indicated that the ribosome prepared retained a sufficient purity . for the purpose of examining a protein synthesis activity of the purified ribosome , an activity for polyphenylalanine synthesis was then determined in a system for in vitro protein synthesis using the purified ribosome and polyuridylic acid as a template . a reaction for in vitro protein synthesis utilizing polyuridylic acid , ribosome and s - 100 fraction was carried out by somewhat modifying the method of spedding et al . ( reference 22 ). thus , to 10 μl of 10 - fold concentrated buffer ( 0 . 5m tris - hcl buffer ( ph 8 . 0 ) containing 0 . 1m mgcl 2 , 0 . 5m nh 4 cl , 10 mm dithiothreitol ( dtt ), 10 mm adenosine triphosphate ( atp ), 10 mm guanosine triphosphate ( gtp ), and 50 mm sodium phosphoenolpyruvate ) were added 1 μl of 3 , 500 u / ml pyruvate kinase ( oriental yeast kogyo k . k . ), 10 μl of 20 u / ml phenylalanine - specific trna ( sigma chemical company ), 1 μl of 140 u / μl ribonuclease inhibitor ( takara shuzo k . k . ), 10 μl of 24 mg / ml s - 100 fraction , 10 - 200 u of purified ribosome , - 20 μg of sodium polyuridylate ( yamasa shoyu k . k . ), and 1 μci of l -[ 2 , 3 , 4 , 5 , 6 - 3 h ]- phenylalanine ( 130 ci / mmol ; amersham japan k . k .). then , water was added to make 100 μl of final volume and the mixture was reacted at 37 ° c . for 15 minutes . after the reaction , 1 ml of 10 % trichloroacetic acid ( tca ) solution was added to the mixture and it was heated to 90 ° c . for 20 minutes . the precipitation was collected onto a glass microfiber filter ( gf / c , whatman international ltd . ), and washed four times with 5 % tca and four times with 99 % ethanol . after drying , the filter was soaked in 5 ml of liquid scintillation cocktail econofluor - 2 ( nen research products ) and the radioactivity of the precipitation on the filter was measured by a liquid scintillation counter ( beckman model ls5000td ). as a result , it was found that the rate of a radioactive amino acid incorporated into the tca - insoluble fraction was 0 . 4 pmol / minute / nmol ribosome and depended on an amount of polyuridylic acid in the reaction solution ( fig2 ). these results indicated that the ribosome prepared retained a sufficient activity . the template mrnas ( auguuugaguuugag : seq id no . 1 and uuugaguuugag : seq id no . 2 ) used in this example were chemically synthesized by dna synthesizer abi model 380b ( applied biosystems ), utilizing β - cyanoethyl method using 0 . 2 μmol of dmt - rg ( ibu )- 2 &# 39 ;- tbusi - cpg ( peninsula laboratories , inc .) as a solid phase as well as dmt - ra ( bz )- 2 &# 39 ;- tbusi - cep , dmt - rg ( ibu )- 2 &# 39 ;- tbusi - cep , and dmt - u - 2 &# 39 ;- tbusi - cep ( peninsula laboratories , inc .). after completing the synthesis , the resin was treated with 2 ml of ammonia / ethanol ( 3 / 1 ) solution for 2 hours to cut off a rna fragment from the resin . the sample was then heated to 55 ° c . for 8 hours in the ammonia / ethanol ( 3 / 1 ) solution to perform deprotection . the sample was then dried using a centrifugal concentrator under a reduced pressure to dryness . tetrahydrofuran solution containing 1m tetrabutylammonium fluoride was added to the dry sample at the ratio of 10 μl per 10 a 260 units of rna to dissolve the sample . the mixture was maintained at room temperature for hours to perform deprotection of 2 &# 39 ;- hydroxyl protecting group . the resulting reaction solution was desalted by gel filtration on nap - 25 column ( pharmacia ) eluting with 50 mm triethylamine acetate ( teaa ). the resulting rna fragment was purified by high - performance reverse phase chromatography on waters μ - bondasphere 5μc18 - 300 ( 3 . 9 mm × 15 cm ) column . thus , not more than 500 μl of the rna solution was poured on the column , and the desired rna fragment was eluted with 0 % to 20 % acetonitrile linear gradient containing 50 mm teaa . fractions containing the rna fragment were pooled and dried using a centrifugal concentrator under a reduced pressure to dryness . the dried sample was then dissolved in water and absorbance at 260 nm was measured . standard tripeptide ( phe - glu - phe ) and tetrapeptide ( phe - glu - phe - glu : seq id no . 3 ) were chemically synthesized by automated peptide synthesizer abi model 430a ( applied biosystems ), utilizing t - boc method using p - methyl bha resin as a solid phase resin . a synthetic peptide cut off from the resin was purified by a high - performance reverse phase chromatography on waters μ - bondasphere 5μc18 - 300 ( 3 . 9 mm × 15 cm ) column . an amino acid sequence of the purified peptide was analyzed by protein sequencer model 477a ( applied biosystems ). c . separation of ribosome - mrna - peptidyl trna complex by high - performance molecular sieve chromatography a process for synthesizing a tripeptide by an in vitro step reaction system utilizing a synthetic mrna and a naturally - occurring aminoacyl - trna was constructed by initiation reaction cycle , peptide extension reaction cycle , and termination reaction cycle . firstly , the initiation reaction cycle was defined as a process composed of the following three steps : ( a - 1 ) reaction of an aminoacyl - trna or misaminoacyl - trna ( wherein a n - terminal amino acid of a desired peptide binds to trna ) with ribosome and mrna to form a ribosome - mrna -( mis ) aminoacyl trna complex ; ( a - 2 ) separation of the ribosome - mrna -( mis ) aminoacyl trna complex by high - performance molecular sieve chromatography ; and ( a - 3 ) concentration of fractions containing the complex by ultrafiltration . next , the peptide extension reaction cycle was defined as a process composed of the following three steps : ( b - 1 ) peptide formation reaction comprising binding a next amino acid residue on the previous ribosome - mrna - peptidyl trna complex ; ( b - 2 ) separation of the resulting ribosome - mrna - peptidyl trna complex by high - performance molecular sieve chromatography ; and ( b - 3 ) concentration of fractions containing the complex by ultrafiltration . finally , the termination reaction cycle was defined as a process composed of the following three steps : ( c - 1 ) recovery of the peptidyl - trna from the final ribosome - mrna - peptidyl trna complex by phenol extraction and ethanol precipitation ; ( c - 2 ) elimination of the desired peptide from the peptidyl - trna by hydrolysis of rna ; and ( c - 3 ) purification of the desired peptide by high - performance reverse phase chromatography . synthesis of a tripeptide by a step reaction was accomplished by carrying out the initiation reaction cycle , then repeating twice the peptide extension reaction cycle , and finally performing the termination reaction cycle . however , for the realization of the above - constructed step reaction system by a system for in vitro protein synthesis , the ribosome - mrna - aminoacyl trna complex and the ribosome - mrna - peptidyl trna complex must be separated from trna and other amino acid components by a molecular sieve chromatography , and furthermore , they must be stably retained during the course of separation and / or concentration . accordingly , a complex was prepared using the above - prepared ribosome , synthetic mrna and an aminoacyl - trna , separated by the following molecular sieve chromatography , and further analyzed by sucrose density - gradient ultracentrifugation ( carrying out in a similar manner as described in the above a - 3 ). the ribosome - mrna - peptidyl trna complex was separated by high - performance molecular sieve chromatography on shodex protein kw - 803 columns ( 8 mm × 30 cm , paired ). waters 600e multisolvent delivery system was used as a solvent delivery system . the columns were previously equilibrated using 10 mm tris - cl buffer ( ph 7 . 5 ) containing 60 mm nh 4 cl and 10 mm ( ch 3 coo ) 2 mg . not more than 200 μl of the sample solution containing the ribosome - mrna - peptidyl trna complex was applied to the columns and developed at a rate of 1 . 0 ml / min . absorbance at 260 nm of the eluted solution was monitored by waters 484 tunable absorbance detector , and the results were recorded by waters 741 data module . fractions were collected in 0 . 5 ml portions . to 0 . 5 ml of each fraction was added 2 ml of liquid scintillation cocktail ready safe ( beckman instrument , inc .) and the radioactivity of the mixture was measured by a liquid scintillation counter ( beckman model ls5000td ). protein markers for molecular weight determination on hplc were purchased from oriental yeast kogyo k . k . the markers used were glutamic dehydrogenase ( mw 290 , 000 ), lactate dehydrogenase ( mw 142 , 000 ), enolase ( mw 67 , 000 ), adenylate kinase ( mw 32 , 000 ), and cytochrome c ( mw 12 , 400 ). when radioactive methionine was allowed to incorporate into ribosome in a system for in vitro protein synthesis utilizing the template mrna ( auguuugaguuugag : seq id no . 1 ) encoding the amino acid sequence of met - phe - glu - phe - glu ( seq id no . 4 ) and the reaction mixture was subsequently fractionated by high - performance molecular sieve chromatography on shodex kw - 803 column , then ribosome was eluted in the void fraction , trna eluted in fractions corresponding to a molecular weight of about 60 , 000 , and the free radioactive amino acid eluted in fractions corresponding to a low molecular weight of not more than 10 , 000 , respectively . incorporation of the radioactive amino acid was observed both in the void fraction and in the trna fractions ( fig3 ). when the void fraction containing the radioactivity was analyzed by sucrose density - gradient ultracentrifugation , it was found that most of the radioactivity was recovered in fractions containing 70s ribosome and the radioactive amino acid bound to 70s ribosome ( fig4 ). on the other hand , when radioactive methionine was similarly allowed to incorporate into ribosome in a system for in vitro protein synthesis utilizing the template mrna ( uuugaguuugag : seq id no . 2 ) encoding the amino acid sequence of phe - glu - phe - glu ( seq id no . 3 ) and the reaction mixture was subsequently fractionated by high - performance molecular sieve chromatography , then similar incorporation of the radioactive amino acid into the trna fractions was observed as in the case of using auguuugaguuugag ( seq id no . 1 ) as a template but incorporation of the radioactive amino acid into the ribosome fractions was little observed . accordingly , it is possible to conclude that the radioactive amino acid bound to ribosome only when aug codon existed in the template mrna . next , when the ribosome fractions containing the radioactivity were treated with phenol and the rna fractions were recovered and again analyzed by high - performance molecular sieve chromatography , then it was found that most of the radioactivity was eluted in trna fractions and the aminoacyl - trna bound to ribosome ( fig5 ). in addition , when the ribosome fractions containing the radioactivity were again analyzed by high - performance molecular sieve chromatography , most of the radioactivity was recovered in 70s ribosome fractions ( data not shown ). the above results indicate that the ribosome - mrna - aminoacyl trna complex formed by in vitro protein synthesis was separated from trna and other components by high - performance molecular sieve chromatography , and that the complex stably existed even in the course of high - performance molecular sieve chromatography and sucrose density - gradient ultracentrifugation . a peptide was synthesized in a system for in vitro protein synthesis utilizing as a template an artificial mrna encoding the amino acid sequence of met - phe - glu - phe - glu ( seq id no . 3 ). as a control , 3 h - phe and 14 c - glu were first added to a reaction system for in vitro protein synthesis to serially synthesize a peptide for 15 minutes . ribosome was then recovered by high - performance molecular sieve chromatography and the formed peptide was eliminated from the peptidyl - trna bound to ribosome by phenol treatment and alkaline hydrolysis . the recovered product was analyzed by high - performance reverse phase chromatography . as a result , peaks regarded as those of free 3 h - phe and 14 c - glu were detected at elution positions of 3 h - phe and 14 c - glu , and both radioactivities of 3 h - phe and 14 c - glu were detected at an elution position of standard phe - glu - phe - glu ( seq id no . 3 ) chemically synthesized ( fig6 ). these results indicate that when peptide synthesis was carried out in a system for in vitro protein synthesis utilizing as a template an artificial mrna encoding the amino acid sequence of met - phe - glu - phe - glu ( seq id no . 4 ), then the amino acids added were incorporated into aminoacyl - trnas , which in turn formed a complex together with ribosome and mrna . the results also indicate that part of peptidyl of the complex further grew to a peptide chain by extension reaction and consequently tetrapeptide phe - glu - phe - glu ( seq . id no . 3 ) encoded by the mrna was synthesized . next , a stepwise synthesis of phe - glu - phe was attempted by using a step reaction system using the same mrna as a template and carrying out two extension reaction cycles . to 20 μl of 10 - fold concentrated buffer for protein synthesis were added 2 μl of 3 , 500 u / ml pyruvate kinase , 20 μl of 20 u / ml phenylalanine - specific trna ( sigma chemical company ), 2 μl of 140 u / μl ribonuclease inhibitor , 20 μl of 24 mg / ml s - 100 fraction , 10 ˜ 200 u of purified ribosome , 40 μl of 40 μm synthetic mrna , and 20 μl of 1 mm phenylalanine . water was then added to make 200 μl of final volume and the mixture was reacted at 37 ° c . for 15 10 minutes . after the reaction , the reaction mixture was fractionated by high - performance molecular sieve chromatography on shodex protein kw - 803 columns ( 8 mm × 30 cm , paired ) and fractions containing ribosome were pooled . the ribosome fractions were then combined and concentrated to about 50 μl of volume by centrifugal ultrafiltration using centricon - 30 (## grease japan k . k .). the resulting concentrated solution was used as a ribosome - mrna - phenylalanyl trna phe complex fraction . to the ribosome - mrna - phenylalanyl trna phe complex fraction obtained in cycle - 0 were added 20 μl of 10 - fold concentrated buffer for protein synthesis , 2 μl of 3 , 500 u / ml pyruvate kinase , 20 μl of 20 u / ml glutamic acid - specific trna ( sigma chemical company ), 2 μof 140 u / μl ribonuclease inhibitor , 20 μl of 24 mg / ml s - 100 fraction , and 1 μci of l -[ u - 14 c ]- glutamic acid ( 270 mci / mmol ; amersham japan k . k .). water was then added to make 200 μl of final volume and the mixture was reacted at 37 ° c . for 15 minutes . after the reaction , the reaction mixture was fractionated by high - performance molecular sieve chromatography as described in cycle - 0 and fractions containing ribosome were collected . the ribosome fractions were then combined and concentrated to about 50 μl of volume by centricon - 30 . the resulting concentrated solution was used as a ribosome - mrna - phenylalanyl glutamic acidyl trna glu complex fraction . to the ribosome - mrna - phenylalanyl glutamic acidyl trna glu complex fraction obtained in cycle - 1 were added 20 μl of 10 - fold concentrated buffer for protein synthesis , 2 μl of 3 , 500 u / ml pyruvate kinase , 20 μof 20 u / ml phenylalanine - specific trna ( sigma chemical company ), 2 μl of 140 u / i ribonuclease inhibitor , 20 μl of 24 mg / ml s - 100 fraction , and 1 μl ci of l -[ 2 , 3 , 4 , 5 , 6 - 3 h ]- phenylalanine ( 130 ci / mmol ; amersham japan k . k .). water was then added to make 200 μl of final volume and the mixture was reacted at 37 ° c . for 15 minutes . after the reaction , the reaction mixture was fractionated by high - performance molecular sieve chromatography . the resulting ribosome fractions were then combined and used as a ribosome - mrna - phenylalanyl glutamic acidyl phenylalanyl trna phe complex fraction . to the ribosome - mrna - phenylalanyl glutamic acidyl phenylalanyl trna phe complex fraction obtained in cycle - 2 was added equal volume of te - saturated phenol solution . the resulting suspension was centrifuged at 10 , 000 × g for 10 minutes . the resulting water phase was extracted with chloroform / isoamylalcohol ( 24 : 1 ). to the water phase was added two volumes of 99 % ethanol and the mixture was allowed to stand for 20 minutes at - 80 ° c . the mixture was then centrifuged at 10 , 000 × g for 10 minutes to precipitate rna . the resulting rna precipitation was dissolved in 100 μl of 0 . 1n koh and rna was hydrolyzed at 37 ° c . for 30 minutes . after the reaction , 100 μl of 0 . 1n hcl was added to the reaction mixture to neutralize it . the neutralized reaction mixture was poured onto μ - bondasphere 5μc18 - 300 ( 3 . 9 mm × 15 cm ) column , and the desired phenylalanyl - glutamic acidyl - phenylalanine was eluted with 12 % to 51 % acetonitrile linear gradient containing 0 . 1 % trifluoroacetic acid ( tfa ). fractions were collected in 0 . 5 ml portions . to 0 . 5 ml of each fraction was added 2 ml of liquid scintillation cocktail ready safe ( beckman instrument , inc .) and the radioactivity of the mixture was measured by a liquid scintillation counter ( bec / an model ls5000td ). the product recovered in the above termination reaction cycle was analyzed by high - performance reverse phase chromatography . as is similar to the case in the serial reaction system , peaks regarded as those of free 3 h - phe and 14 c - glu were detected at elution positions of 3 h - phe and 14 c - glu , and both radioactivities of 3 h - phe and 14 c - glu were detected at an elution position of standard phe - glu - phe chemically synthesized ( fig7 ). these results indicate that in the stepwise reaction system , similar to the case in the serial reaction system , the amino acid added in the initiation reaction was incorporated into aminoacyl - trnas , which in turn formed a complex together with ribosome and mrna . the results also indicate that part of peptidyl of the complex further grew to a peptide chain in the extension reactions and consequently tripeptide phe - glu - phe encoded by the mrna was synthesized . as described above , the results obtained in this example indicate that a peptide having a desired amino acid sequence can be synthesized by a step reaction system for in vitro protein synthesis utilizing ribosome , an aminoacyl - trna or misaminoacyl - trna , and an artificial mrna having a predetermined sequence . 1 . f . c . mckay and w . f . albertson , j . am . chem . soc ., 79 , 4686 ( 1957 ). 2 . l . a . carpino and g . y . han , j . am . chem . soc ., 92 , 5748 ( 1970 ). 4 . r . b . merrifield , j . am . chem . soc ., 85 , 2149 ( 1963 ). 5 . m . bergmann and h . fraenkel - 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