Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography

Definitions

• the present invention relates generally to the field of protein synthesis. More specifically, the present invention relates to a high efficiency system for cell-free translation of desired proteins or cell-free, coupled transcription/translation of exogenously added genes. Description of the Related Art.
• S30 Escherichia coli
• the S30 extract is essentially free of DNA and devoid of R A due to preincubation under translation conditions but retains most of the soluble proteins and R As of intact cells.
• the use of the S30 system for coupled transcription/translation of linearized phage or plasmid DNA and linearized DNA fragments has been demonstrated (J. M. Pratt et al. (1981) Nucl. Ac. Res. 9:4459-4474; and J. M. Pratt et al. (1984) Transcription and Translation: A practical Approach (Hames, B. D., and Higgins, S. J., eds.), pages 179-209, IRL Press).
• the original system for coupled transcription/translation is based on the crude S30 fraction using the method of Zubay.
• this fraction contains nearly all of the soluble protein that is present in intact E. coli cells. This includes many degradative proteases and nucleases.
• CFCF run apparently due, at least in part, to denatured protein that is formed from the soluble protein in the S30 fraction during the run.
• S30 fraction appears to contain one or more very active soluble exo-deoxyribonucleases that degrade linear DNA from its free end.
• protein and nucleic acid factors required for the cell-free synthesis of enzymatically active proteins are poorly defined and characterized.
• the initial folding of the nascent peptides may occur as they are extended on ribosomes. These folding events may take place in a protected area within a cavity or tunnel in the large riboso al subunit. Chaperones, DnaJ, DnaK, GrpE, GroEL and GroES, may be involved in the folding of some proteins on ribosomes.
• the prior art remains deficient in a high efficiency, coupled transcription/translation system for the cell-free engineering and synthesis of proteins.
• the prior art is also deficient in a high efficiency translation system for the cell-free synthesis of proteins.
• the present invention fulfills a much-needed and longstanding need in the art of protein synthesis for a high efficiency, coupled transcription/translation system for the cell-free engineering and synthesis of proteins.
• the present invention also fulfills the longstanding need in this art for a high efficiency, cell-free translation system for the synthesis of proteins.
• a method for the high efficiency, cell free synthesis of proteins comprising the steps of: preparing a cell free extract; separating out a ribosome fraction from said extract; incubating said ribosome fraction in the presence of a transcription/translation medium; and measuring the amount of protein synthesized.
• a method for the high efficiency, cell free synthesis of proteins comprising the steps of: preparing a cell free extract; separating out a ribosome fraction from said extract; incubating said ribosome fraction in the presence of a transcription/translation medium containing chaperone proteins; and measuring the amount of protein synthesized.
• a method for the high efficiency, cell-free synthesis of proteins comprising the steps of: preparing a cell-free extract from Escherischia coli; separating out a ribosome fraction from said extract by centrifugation; incubating said ribosome fraction in the presence of a medium, said medium comprising 55 mM Tris-acetate (pH 7.8), 12 mM Mg(OAc) 2 , 36 mM NH 4 0Ac, 72 mM KOAc, 2 mM Ca(0Ac) 2 , 0.5 mM EDTA, 2% polyethylene glycol-6000, 2 mM DTT, 1.2 mM ATP, 0.8 mM GTP, 0.8 mM UTP, 0.8 mM CTP, 0.4 mM CAMP, 27 mM phosphoenol pyruvate (monopotassium salt, pH 7.0), 0.35 ⁇ q pyruvate kin
• a and B represent tracks from an autoradiogram derived from a 15% Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis (performed according to Laemmli (1970) Nature 227:680-685) on which identical aliquots after in vitro protein synthesis were analyzed (A, without, and B with the plasmid) . Numbers 1-5 indicate positions of marker proteins on the gel.
• Figure 2 shows the synthesis of dihydrofolate reductase in the continuous-flow cell-free system. Protein synthesis was followed over time by determining 1 C-leucine incorporation into protein. Fractions of 1.5 ml were collected per hour. Insert A: The Coomassie blue-stained gel (15% acrylamide) of individual fractions from the first 7 hours (100 ⁇ l from each) . Insert B: Autoradiogram prepared from a gel similar to the one shown in Insert A with samples collected up to 22 hours. Figure 3 shows the degradation of the linearized in contrast to the circular plasmid.
• Figure 4 shows the rhodanese polypeptides in the supernatant and ribosome fractions.
• the arrow on the left indicates migration of native rhodanese purified from bovine liver.
• Figure 5 shows the release of full-length rhodanese from the ribosomes by chaperones.
• Resuspended ribosomes bearing rhodanese polypeptides were incubated in the absence or presence of the indicated chaperones (amounts given in TABLE VII) , then centrifuged. Aliquots of 20 ⁇ l of the supernatant (first lane of each set) and 20 ⁇ l of the ribosome fraction that had been resuspended in 30 ⁇ l (each second lane) were analyzed by SDS-PAGE and autoradiography. The fraction of the full-length rhodanese released into the supernatant relative to the total amount of full-length rhodanese originally bound to the ribosomes is given as percentage beneath the tracks. The amount of full-length rhodanese was determined by cutting out and solubilizing the band from the gel, then counting the radioactivity in the presence of Ecolite (ICN) .
• ICN Ecolite
• S30 refers to a cell-free extract from E . coli prepared according to Zubay (1973) .
• the preparation of a cell free extract from E. coli is a well known procedure in the prior art and can readily be accomplished by a person having ordinary skill in this area of research.
• cell free translation refers to in vitro synthesis of a protein.
• continuous flow, cell free or CFCF system refers to synthesis of a protein in a reaction chamber from which product is pumped out and feeding solution is pumped in continuously similar to the CFCF system described by Spirin et al. (1988). Briefly, a continuous flow of feeding buffer, including amino acids and nucleotide triphosphates is used through a reaction mixture. There is continuous removal of the polypeptide product. Both prokaryotic and eukaryotic systems were tested.
• Coupled transcription/translation refers to synthesis of a mRNA from a plasmid i.e., transcription and translation in the same reaction mixture containing this mRNA.
• ribosome fraction refers to a fraction derived from the crude cell extract which contains the ribosomes and certain proteins, tRNA and other cellular components.
• static assay or static system refers to an assay carried out in a test tube and is not a continuous flow system.
• the present invention provides a high efficiency method for the cell free synthesis of proteins. This method comprises the steps of: (1) preparing a cell free extract; (2) separating out a ribosome fraction from the extract; (3) incubating the ribosome fraction in the presence of a medium; (4) and measuring the amount of protein synthesized.
• the cell free extract prepared for use in the method of the present invention may be prepared from many prokaryotic or eukaryotic organisms.
• a representative example of suitable prokaryotic organisms is Escherichia coli .
• suitable eukaryotic organisms include wheat germ and rabbit reticulocytes.
• the ribosome fraction may be separated from the cell free extract by any of the well known methods in the art.
• the ribosome fraction is separated by centrifugation or gel filtration chromatography.
• the ribosome fraction is incubated in the presence of an appropriate medium.
• the ribosome fraction is incubated in the presence of a transcription/translation medium.
• the transcription/translation medium contains buffer, salts (with monovalent and divalent cations) , amino acids, reducing agent, nucleoside triphosphates, cell extract (S30) or ribosomes, a plasmid, RNA polymerase, and a energy regenerating system.
• the transcription/translationmedium contains Hepes or Tris buffer (pH 7.5-7.8), optimal Mg(OAc) 2 concentration, optimal NH 4 + and/or K + salt concentration, 2 mM DTT, 1.2 mM ATP, 0.8 mM each of GTP, UTP, and CTP, 0.5 mM cAMP, energy regenerating system (either phosphoenol pyruvate and pyruvate kinase or creatine phosphate and creatine phosphate kinase) , 1 ⁇ g folinic acid (only for E.
• Mg(OAc) 2 , NH 4 + and K* The optimal concentration of Mg(OAc) 2 , NH 4 + and K* will vary depending on whether prokaryotic or eukaryotic organisms are used. For example, the K + concentration used with bacterial system was 72 mM but was 112 mM in the wheat germ system.
• the Mg 2+ concentration in the bacterial system was 14 mM while it was 4 mM in the wheat germ system.
• concentration of various salts may vary slightly from laboratory to laboratory.
• the ribosome fraction is incubated in the presence of a translation medium.
• the translation medium contains buffer, salts, source of energy e.g., ATP and GTP, an energy regenerating system e.g., phosphoenol pyruvate, pyruvate kinase, amino acids, tRNAs, and isolated mRNA.
• a high efficiency method for the cell free synthesis of proteins comprising the steps of: preparing a cell free extract; separating out a ribosome fraction from said extract; incubating said ribosome fraction in the presence of a transcription/translation medium containing chaperone proteins; and measuring the amount of protein synthesized.
• the chaperones preferably included in the medium are DnaJ, DnaK, GrpE, GroEL and GroES.
• the ribosome fraction of the cell free extract is incubated in the medium at about 37"C for about 20 minutes to about 60 minutes.
• the incubation time is about 30 minutes at 37 * C.
• the protein synthesized by the methods of the present invention may be determined by any of the well known techniques.
• protein is measured by trichloroacetic acid precipitation followed by quantitation of the amount of amino acid incorporated into the protein or analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography.
• biological activity like enzymatic activity is determined where applicable and reaction with specific antibodies (if these are available) may be carried out.
• protein levels may be necessary or desirable to determine protein levels by measuring the biological activity of the synthesized protein.
• the native conformation of the protein may be determined by measuring the biological activity of the protein.0
• Representative examples of proteins that can be measured by examining the protein's biological activity includes rhodanese, chloramphenicol acetyl transferase and dihydrofolate reductase.
• Ribosomes separated from the S30 E . coli extract retain all the components necessary for coupled transcription/translation from a plasmid which contains the coding sequence under an appropriate promoter. A considerably larger amount of synthesis was obtained with the nonlinearized plasmids and the linearized forms most often used in the past were rapidly degraded in the system. The utility of the system was shown for both prokaryotic and eukaryotic proteins. The present invention has important advantages over any prior art system.
• Plasmid preparations were made by standard procedures (J. Sambrook et al. (1989) Molecular Cloning. Cold Spring Harbor Laboratory Press) except that the CsCl centrifugation step was replaced by Q cartridge (Bio-Rad) chromatography. SP6 and T7 RNA polymerases are commercially available. The plasmids used are described in the following references: (1) Coding sequence:
• Coding sequence CAT; plasmid SP65-CAT; provided by A. Spirin and co-workers; (4) Coding sequence: PP1-C; plasmid pGEM-3Z-PPl-C; (Bai et al. (1988) FASEB J . 2:3010-3016) ; (5) Coding sequence: STNV; plasmid pTZ19R-STNV, (Browning et al. (1988) J. Biol . Chem . 263:8380-8383); (6) Coding sequence: rhodanese; plasmid pETlld-rho, (Miller et al. (1991) J. Biol .
• the ribosome fraction used for coupled transcription/translation was isolated from the S30 fraction prepared according to G. Zubay from E. coli K12 (A19) .
• Cells were grown at 37 * C in LB (Sigma) broth to which 20% glucose (10 ml per 2 liter medium) was added.
• Cells were harvested in mid-log phase at 37 * C and lysed in a pressure cell. The cells were then centrifuged for 30 minutes at 30,000 x g and the S30 extract was prepared.
• a protease inhibitor, phenylmethylsulfonyl fluoride, was added to the lysis buffer to give a final concentration of 0.5 mM.
• the system used to carry out coupled transcription/translation contained in a total volume of 30 ⁇ l: 50 mM Tris-acetate (pH 7.8), 14 mM Mg(OAc) 2 , 36 mM NH 4 OAc, 72 mM KOAc, 2 mM Ca(OAc) 2 , 0.5 mM EDTA, 2% polyethylene glycol-6000, 2 mM DTT, 1.2 mM ATP, 0.8 mM each of GTP, UTP, and CTP, 0.5 mM cAMP, 27 mM phosphoenol pyruvate, 0.35 ⁇ g pyruvate kinase, 1 ⁇ g folinic acid, 83 ⁇ M u C-leucine, 330 ⁇ M of each of the other 19 amino acids, 20 ⁇ g E .
• Synthesis was carried out in a reaction chamber with the YM100 (Amicon) membrane on its upper side through which product was pumped bottom to top at 1.5 ml/hr. The eluate was replaced by feeding solution which contained all low molecular weight components of the reaction mixture and tRNA at 3 ⁇ g/ml. Incubation was at 37 * C for the times indicated.
• the product formed was analyzed in one of the following ways.
• the amount of u C-labeled product in the 30- ⁇ l test tube assay or from a 100- ⁇ l aliquot of the fractions collected from the continuous-flow system was determined by trichloroacetic acid precipitation following the procedure described previously ( . Kudlicki et al. (1987) J. Biol. Chem. 262:9695-9701).
• DHFR dihydrofolate reductase
• DHFR activity is defined as the amount of enzyme required to reduce 1 ⁇ mol dihydrofolate/min based on a molar extinction coefficient of 12.3X10 3 for NADPH (B. L. Hillcoat et al (1967) Anal. Biochem. 21:178-189).
• Enzymatic activity of rhodanese was determined in a calorimetric assay according to Sorbo (1953), Acta Chem. Scand. 7:1137-3145. This assay measures the conversion of CN " to SCN " by the enzyme using S 2 0 3 z" as substrate. SCN " formed was detected and quantitated by measuring absorbance at 460 nm of a complex between this product and ferric ions.
• Chloramphenicol acetyl trancferase (CAT) activity was measured by the transfer of [ 3 H]acetyl from acetyl-CoA to chloramphenicol attached to agarose beads. Radioactivity associated with the beads after the enzyme reaction was quantitated.
• One unit was defined as the transfer of 1 pmol 3 H-acetyl to chloamphenicol.
• the ribosome fraction used in the experiments was derived from an E . coli S30 fraction. Either the S30, or the ribosome fraction, was incubated with plasmids which contained a coding sequence under the SP6 or T7 promoter unless otherwise indicated. SP6 or T7 RNA polymerase was added to carry out transcription from the respective promoter. The 30 ⁇ l transcription/translation reaction mixtures also were supplemented with tRNA and the low molecular weight components necessary to carry out RNA and protein synthesis. When the static assay was used, incubations were carried out for 30 minutes at 37 * C.
• Protein synthesis was carried out as follows: Reaction mixtures contained either 10 ⁇ l S30 or 1 ⁇ l ribosome fraction. In plasmids 2, 3 and 4, from which the genes listed in lines 2-4 of Table 1 were transcribed, the respective coding sequence was under the SP6 promoter. In these cases, SP6 RNA polymerase and rifampicin were added. In plasmid 5, the DHFR fol A gene was inserted into a plasmid (pDF34 - (V. I. Baranov et al. (1989) Gene 84:463-466)) . Transcription of this gene as well as of S-lactamase is under the E .
• coli promoter i.e., no RNA polymerase or rifampicin were added.
• the plasmids whose genes are listed in lines 6-8 contain the coding sequence under the T7 promoter. In these cases, T7 RNA polymerase and rifampicin were added. The amount of protein synthesized was analyzed by incorporation of u C-leucine. An aliquot of the reaction mixture containing
• DHFR protein synthesized in the static system 50 ng was used to measure enzymatic activity. An equal aliquot was taken from a parallel incubation mixture from which the plasmid was omitted. To determine enzymatic activity, the assay mixture contained in a total volume of 1.0 ml, 100 mM imidazole-HCl (pH 7.6), 10 mM 2-mercaptoethanol, 75 ⁇ M dihydrofolate, and 80 ⁇ M NADPH. The reaction carried out at 30'c was followed in the cuvette by the decline in absorbance at 340 nm.
• the absorbance at 460 nm of the final reaction mixture was used to determine its activity.
• About 30 ng CAT (determined from the amount of 14 C-leucine incorporated) was added to a mixture containing [ 3 H]acetyl-CoA (3000 Ci/mol) and chloramphenicol bound to agarose beads.
• the assay was carried out at 25 * C, the beads were washed and their radioactivity determined.
• the method of Sleigh et al., Analyt . Biochem . 156:251-256 (1986) was used which measures 14 C acetyl-CoA transfer to chloramphenicol after one hour incubation at 37' C under the conditions described by Sleigh et al.
• E. coli dihydrofolate reductase DHFR
• E. coli chloramphenicol acetyl transferase CAT
• rabbit skeletal muscle phosphatase the catalytic subunit of type 1 phosphoprotein phosphatase, PPl-C
• the coding sequences for these three proteins had been cloned into different plasmids but all are under the SP6 promoter. With these plasmids, SP6 RNA polymerase was added for transcription. In these cases, rifampicin was added to the reaction mixture to inhibit the endogenous E . coli RNA polymerase.
• results obtained with a plasmid into which the DHFR gene was inserted under the __.. coli promoter were also included in TABLE I.
• transcription was performed without added RNA polymerase.
• Two products were synthesized, DHFR and a polypeptide of 30,000 dalton ⁇ , which appears to be 3-lactamase. The latter product was also obtained when the other SP65 plasmids were transcribed and translated in the absence of rifampicin.
• the plasmids listed in TABLE I that have the inserted coding sequence under the T7 promoter are: bovine rhodanese, a truncated form of the human TATA box binding protein (TFIID-180C) and a plant viral RNA from Satellite Tobacco Necrosis Virus (STNV) . With these plasmids, T7 RNA polymerase and rifampicin were added for transcription.
• the plasmids used for the experiments in TABLE I were not linearized for transcription by either added SP6 or T7 polymerase or endogenous E. coli RNA polymerase. A lower, in some cases very much lower level of translation was obtained provided the plasmid had been cut 3' of the coding sequence by an appropriate restriction enzyme (TABLE II) . Degradation of the linearized plasmid (in contrast to the uncut plasmid) was observed during the incubation time as analyzed by agarose gel electrophoresis (as is shown in Figure 1) .
• DHFR protein was synthesized in the fractionated system.
• PPl-C was linearized with Not I (restriction site was located about 240 base pairs downstream from the stop codon as described by Bai et al (1988) .
• Both DHFR (short) and DHFR (long) were linearized with Hind III. The influence of the 3' untranslated region of the transcribed sequence was also seen.
• the DHFR "short” plasmid contains only 165 base pairs between the stop codon and the restriction site (Hind III) used for linearization.
• the DHFR "long” plasmid has an additional 350 base pairs (515 total) in this 3' untranslated region. The lower level of translation may be due to 3' exonucleolytic activity that degrades the plasmid and is present even in the ribosome fraction.
• EXAMPLE 7 The methods of the present invention may also be carried out using a cell-free extract obtained from eukaryotic organisms. Protein synthesized from either an S30 or the ribosome fraction of an S30 extract from wheat germ (TABLE III) or rabbit reticulocyte lysate (TABLE IV) .
• the wheat germ system used to carry out coupled transcription/translation contained in a total volume at 50 ⁇ l having 25 mM Hepes-KOH (pH 7.6) , 2.4 mM DTT, 0.1 mM spermine, 1.2 mM ATP, 1.0 mM GTP, 0.8 mM CTP, 0.8 mM UTP, 1.0 mM GMP, 6 mM creatine phosphate, 28 U creatine phosphokinase, 215 ⁇ M each amino acid (excluding leucine) , 15 ⁇ g wheat germ tRNA's, 112 mM KOAc, 4 mM Mg(0Ac) 2 , 50 U human placenta ribonuclease inhibitor, 0.1 ⁇ g each protease inhibitor (aprotinin, leupetin and pepstatin) , 2 - 2.2 ⁇ g plasmid, 27 U( ⁇ 0.5 ⁇ g) T7 RNA polymerase, 25 ⁇ M[ 14
• Rabbit reticulocyte lysate treated with micrococcal nuclease was prepared as described by Pelham and Jackson (1976) Eur. J. Biochem. 67:247-256. After a 4 hour ultracentrifugation, ribosomes were resuspended in 20 mM Tris-HCl (pH 7.5), 1.2 mM MgCl 2 , 100 mM KCl, and 1 mM DTE.
• the reaction mixtures of 25 ⁇ l contained about 50% (v/v) nuclease-treated lysate or 1.5 A 260 -units of ribosomes isolated from the lysate and additions to give the following concentrations: 10 mM Tris-HCl (pH 7.5), 1.0 mM ATP, 0.8 mM GTP, CTP and UTP, 5 mM creatine phosphate, 75 ⁇ g/ml creatine phosphokinase, 0.05 mM each unlabeled amino acid (excluding leucine), 3.3 mM MgCl 2 , 80 mM KCl, 500 units/ml T7 RNA polymerase, 15 ⁇ g/ml plasmid DNA, [ 14 C]leucine (10-40 Ci/mol), 50 U human placenta ribonuclease inhibitor. Incubation was at 35'C for 30-60 minutes. TABLE IV
• EXAMPLE 8 The plasmid containing the "short" DHFR was used in the uncut form for the following experiments. Coupled transcription/translation was carried out in the presence of 14 C-leucine in the static system. An aliquot of the reaction mixture was analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Another aliquot was used to determine enzymatic activity of the translation product. The results are shown in Figure 2. The insert indicates a full-length product as the only radioactive band seen in the autoradiogram. Enzymatic activity was determined by the decrease in absorbance at 340 nm due to oxidation of NADPH.
• Enzyme activity of the translation product over time is shown in Figure 2. From other experiments, the specific activity of the in vitro-synthesized DHFR was calculated to be about 55 units/mg protein which compares favorably with isolated homogeneous DHFR. Background DHFR activity was low in the fractionated transcription/translation system, whereas it was at least ten fold higher in the original S30 extract. At this level, it was difficult to measure the enzymatic activity of the in vitro synthesized DHFR. These experiments were carried out in a 30- ⁇ l static transcription/translation system.
• TABLE V presents data concerning the enzymatic activity of in vitro synthesized rhodanese and CAT. In both cases, background values are provided to indicate that the protein-synthesizing system does not contain activities that would interfere with the respective assay.
• the present invention demonstrates that the riboso al synthesis of proteins can be maintained at a linear rate during many hours of incubation in the CFCF system.
• the modifications of the classical E . coli S30 system of Zubay in the present invention have two major advantages for expression of genes by coupled transcription/translation in either the static or CFCF system.
• Fractionation of the S30 removes most of the soluble components of the E . coli extract that may complicate subsequent purification or assays of the product. Also, they may directly inhibit transcription or translation.
• the fractionated system is much less viscous than the S30 extract. Turbidity due to denatured protein in the reaction mixture is greatly reduced with the fractionated system even after many hours of incubation in the CFCF reaction chamber. In the CFCF system, clogging of the membrane due to denatured protein and degraded nucleic acids is greatly reduced.
• the reaction mixture in a total volume of 50 ul contains 25 mM Hepes-KOH (pH 7.6); 2.4 mM DTT; 0.1 mM spermine; 1.2 mM ATP; 1.0 mM GTP; 6 mM creatine phosphate; 28 U creatine phosphokinase; 25 uM each amino acid (excluding leucine) ; 15 ⁇ g wheat germ tRNA; 112 mM KCl; 2 mM Mg(OAc) 2 ; 2-2.5 ⁇ g mRNA; and 12 ⁇ l wheat germ S30 or 1.5-1.7 A 260 units of ribosomes. Incubation was for 30 minutes at 27'C.
• the reaction mixture (25 ⁇ l) contains 10 mM Tris-HCl (pH 7.5); 1.2 mM MgCL 2 ; 90 mM KCl; 5 mM DTT; 0.5 mM ATP; 0.2 mM GTP; 50 ⁇ M each amino acid (except leucine); 3 mM creatine phosphate; 0.2 mg/ml creatine phosphokinase; 8.5 pmol mRNA; 12.5 ⁇ l reticulocyte lysate or 1.7 A 260 units of ribosomes. Incubation was for 30 minutes at 35 * C. TABLE VI
• the factors that are required for the initiation, elongation, and termination of peptides as well as the aminoacyl-tRNA synthetases are generally associated with ribosomes.
• the present invention indicates that the E . coli RNA polymerase also sediments with the E. coli ribosomes obtained from the DNA-free S30 fraction.
• tRNAs with modified amino acids for example, amino acids with covalently attached fluorophores
• synthetic tRNA can be added whose anticodon/amino acid relationship has been changed to allow in vitro protein engineering.
• EXAMPLE 10 Propagation of the plasmid, isolation of SP6 RNA polymerase, preparation of the E. coli cell-free extract (S30) and isolation of the ribosome fraction from the S30 were carried out as described above.
• the rhodanese coding sequence was removed from the pETlld vector and ligated into the Xbal-BamHI sites of pSP65. This positions the rhodanese coding sequence under the promoter for SPG RNA polymerase. Under the conditions used, translation was limited to rhodanese polypeptides. No ⁇ -lactamase can be synthesized from this plasmid when it was used in non-linearized form for coupled transcription/translation.
• reaction mixtures containing 5 mM Na 2 S 2 0 3 .
• reaction mixtures were enlarged to 0.9 ml.
• These reaction mixtures contained about 60 A 260 of crude E. coli ribosomes and about 10-15 ⁇ g non-linearized plasmid.
• [ 14 C]Leucine was used at 40 Ci/mol or 160 Ci/mol.
• EXAMPLE 11 The resuspended ribosome fraction was tested for activation and/or release of bound rhodanese in the following way.
• Reaction mixtures of 30 ⁇ l contained 55 mM Tris-acetate (pH 7.8), 2 mM DTT, 1.2 mM ATP, 0.8 mM GTP, 36 mM NH ⁇ OAC, 72 mM KOAc, 12 mM Mg(OAc) 2 , 2 mM Ca(OAc) 2 , 5 mM Na 2 S 2 0 3 , 1.9% polyethylene glycol (M r 6,000), 27 mM phospho(enol) pyruvate (monopotassium salt), 0.33 mM glucose-6-phosphate, 0.5 mM EDTA, 0.3 ⁇ g pyruvate kinase, and 3 ⁇ l resuspended ribosomes.
• Chaperones or antibiotics were added as follows: GroEL - 2.1 ⁇ g, GroES - 0.8 ⁇ g, DnaK - 1.5 ⁇ g, DnaJ - 0.5 ⁇ g, GrpE - 1 ⁇ g.
• the specific enzymatic activity of native rhodanese isolated from bovine liver was 684 units/mg protein. Incubation was for 30 minutes at 37'C, then the sample was centrifuges in an airfuge (Beckman) for 30 minutes at 104,000 rpm. After centrifugation, the supernatant was carefully removed, the ribosomal pellet rinsed then resuspended in 30 ⁇ l of solution A.
• the phenylalanine analog of puromycin has been prepared previously by reacting N-carbobenzoxyphenylalanine with puromycin aminonucleoside using the mixed anhydride method of activation.
• the fluorenylmethyl-oxycarbonyl (FMOC) moiety was used rather than the carbobenzoxy moiety to protect the amino group of phenylalanine and activated the carboxyl group by converting it into the succinimidyl ester using the dicyclohexylcarbodiimide method.
• ( 3 H)-L-Phenylalanine was converted into the FMOC derivative as previously described (Carpino and Han, 1972, J .Org . Chem . 37:3404-3409).
• the final product was estimated to be about 95% pure by thin layer chromatography on silica gel in chloroform : methanol (9:1).
• the R f of the main product in this solvent was about 0.55, almost identical to that of puromycin and about twice that of puromycin aminonucleoside.
• the product had an absorbance maximum at 274 nm, identical to that found for puromycin.
• the overall yield of product was about 50%, based on phenylalanine.
• Ribosome-bound rhodanese is enzymatically inactive .
• Rhodanese was synthesized on __. . coli ribosomes i n a c e l l - f r e e s y s t e m b y c o u p l e d transcription/translation as described above . The results are given in TABLE VII. After a 30 minute incubation at 37'C, aliquots of the reaction mixture were withdrawn to quantitate the amount of protein that was synthesized and to determine its enzymatic activity. The latter parameter is expressed as enzyme units, ⁇ mol of thiocyanate formed per minute.
• the reaction mixture was centrifuged after incubation to separate the ribosomes from the soluble fraction. About 50% of the [ 14 C]leucine that was incorporated into polypeptides was recovered in the ribosome fraction. All of the enzymatic activity was found in the supernatant fraction.
• the specific enzymatic activity for the supernatant fraction 564 units per mg of rhodanese (TABLE VII A, No chaperones added) , was about 82 per cent of the value (specific activity of 684 units/mg) determined under identical conditions for the native enzyme isolated from bovine liver by others previously.
• TABLE VII A and Figure 4 illustrate that about half of the total newly formed polypeptides remains associated with ribosomes after 30 minutes of incubation.
• the faster migrating form of active rhodanese in the supernatant retains [ 35 S]methionine at its N-terminus. It has the same electrophoretic mobility and was indistinguishable on this basis from native rhodanese isolated from bovine liver.
• a possible explanation for the size difference shown in Figure 4 was that UGA, a weak terminator in E. coli , was read through.
• the coding sequence of rhodanese mRNA contains a UGA termination codon which is followed by 13 codons then an in-phase UAG termination codon. This explanation assumes proteolytic removal of sixteen amino acids as the polypeptide is released from the E . coli ribosome.
• Ribosomes isolated by centrifugation from a reaction mixture in which rhodanese had been synthesized in the absence of added chaperones were incubated in a second reaction mixture which lacked components required for protein synthesis with the objective of characterizing the activation process. After incubation, these reaction mixtures were again subjected to centrifugation to separate ribosomes from the soluble fraction. All enzymatic activity was found in the supernatant fraction as observed after the first incubation.
• the clear but surprising results shown in TABLE VIII demonstrate that the chaperones promote release of ribosome-bound polypeptides and that active enzyme can be generated from these inactive rhodanese polypeptides.
• the present invention raises several questions such as why the slow migrating, apparently full-length rhodanese polypeptides accumulate on the ribosomes and how they are bound to the ribosomes? Does their release require conventional termination? If translation is stalled at a pause site during peptide elongation that immediately precedes the release codon, they should exist as peptidyl-tRNA. If the peptides exist as peptidyl-tRNA in the ribosomal P site, then they should react with puromycin, an antibiotic that mimics A-site aminoacyl-tRNA resulting in a covalent bond to the nascent peptide during the peptidyl transferase reaction.
• a radioactively labeled analogue of puromycin was chemically synthesized (see above) and used to determine directly the reactivity of the ribosome-bound peptides. Incorporation of 3 H-labeled puromycin into peptides was determined and compared with the molar amount of peptide that was present, as calculated from incorporated radioactive leucine in a parallel sample by the procedure described above. 89% of the full-length polypeptides that were bound to the ribosomes after the initial incubation and centrifugation were reactive with puromycin and this material was present on the ribosomes as peptidyl-tRNA.
• the present invention demonstrates that at least part of the process by which newly formed rhodanese polypeptides are folded and converted into an enzymatically active state occurs on ribosomes.
• the process requires the five chaperones, DnaK, DnaJ, GrpE, GroEL and GroES, plus ATP and is temporally associated with release of the nascent protein from peptidyl-tRNA on the ribosome on which it was formed.
• Reaction with puromycin shows that the ribosome-bound newly formed peptides exist at the P site within the translational apparatus of the ribosome as peptidyl-tRNA.
• the present invention shows that accumulation of full-length polypeptides on the ribosome results from a failure in some part of the termination and release mechanism which is coupled to final folding and activation of the enzyme. Termination and release of the peptides was inhibited by the addition of purified DnaJ to the reaction system which contains ATP, whereas either GroES or DnaK promoted release. Considered together, there is an intimate association between a late stage of folding mediated by chaperones and termination-release of a nascent protein from the ribosome on which it was formed.

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• 1994
  • 1994-04-08 AU AU66288/94A patent/AU693443B2/en not_active Ceased
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