Gene expression using cell-free systems William C. Merrick Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. Current Opinion in Biotechnology 1990, 1:79-81 Introduction Two recent events have indicated that reticulocyte lysate translation systems may be very useful in the analysis of protein structure/function relationships. The first is the ability to directly assay translation products for function; this required the development of appropriate assays. The second is the large-scale synthesis of proteins in a 'continuous flow' lysate system. Both of these findings can now allow for more rapid analysis of proteins by site-directed mutagenesis.
Background Cell-free protein synthesizing systems have traditionally been used for the analysis of those proteins and RNAs required for accurate and efficient synthesis of proteins. In both bacterial and eukaryotic systems, this has led to the elucidation of protein factors that are specific for the initiation, elongation and termination of the polypeptide chain, as well as elements that are involved in secretion and post-translational modifications. The employment of cell-free systems to express a protein of choice used to be feasible only in the few instances where a particular mRNA was extremely abundant in some tissue (e.g. globin, ovalbumin, serum albumin). The availability of several eR]cient systems for the production of mRNAs (SP6, T7, T4) has now changed this and made it possible to generate any given mRNA in a pure form. However, given the short lifespan of a cell-free system and the limited amount of pure mRNA available, it has generally not been possible to produce much protein product (perhaps a microgram or so). Furthermore, this product is part of a mixture of proteins, which makes it dit~cult to purify. This would seem to imply that cell-free expression of proteins is only of interest in relation to polysome size or formation, post-translational modification, protein trafficking, etc. However, cell-free expression in conjunction with appropriate assays or isolation techniques has actually proven to be quite useful. Cell-free systems have primarily been used to characterize eukaryotic proteins, because the ease of manipulation and the genetic capability of Escberichia coli make similar efforts for bacterial proteins easier to accomplish in viva Such systems mainly use rabbit-reticulocyte lysates as they synthesize proteins in vivo at a rate that is approximately 10--100-fold greater than other translation extracts. From studies on the mechanism of translation, it has been possible to define two distinct initiation pathways. The first is the most common eukaryotic scheme--the 'cap-depen-
dent' initiation of translation which involves the eukaryotic unique 7-methylguanosine cap structure connected to the 5' terminal base of mRNAs via a 5' to 5' linkage using a triphosphate bridge. Experimentally, the use of this pathway can be tested in vitro either by omitting the cap structure when the mRNA is made or by inhibiting translation by the addition of 7-methylguanosine diphosphate, an analog of the cap structure. From studies on initiation factor function [1,2 °',3] and the extensive work of Marilyn Kozak on AUG selection [4,5], it is possible to identify those optimal characteristics of an mRNA that aid initiation in a cap-dependent fashion. The characteristics include: a 7-methylguanosine cap structure; a relatively short (less than 50 bases) 5' untranslated region; an optimal context around the initiating AUG codon (A/GXXAUGG); little or no secondary structure in the 5' untranslated region; and no AUG codons in the 5' untranslated region. Absence of G residues in the 5' untranslated region achieves the same effect not only because mRNA secondary structure is minimized and upstream AUG codons are avoided, but also because of the avoidance of potential, if weak, non-AUG initiating codons that can cause reduced fidelity and reduced translational efficiency. The second pathway of initiation is termed 'cap independent' or 'internal' initiation. To date, this pathway of initiation has only been observed in viral mRNAs, the best examples of which are the picornaviral mRNAs. In this scheme, an internal portion of the mRNA is the site for direct binding of the ribosome. The characteristics of such a specific site have not yet been fully characterized; however, segments of the 5'untranslated region of encephalomyocarditis virus mRNAs and poliovirus mRNAs have been shown to act in cL~ to allow for internal initiation of heterologous mRNAs [6,7]. Generally, the use of such elements to enhance expression is discouraged as the biochemical basis for their use in still largely unknown and some of the picomavirus mRNAs, such as poliovirus mRNAs, are poorly translated. On the other hand, the 5' untranslated leader of the encephalomyocarditis virus mRNA is perhaps the most efficient known and is better than all capped mRNAs tested to date in competitive translation assays.
Optimization of cell-free translation A problem associated with efficient translation is catering for the rare codons that are found in mRNA. This can be bothersome in rabbit-reticulocyte lysates, as the tRNA population, which is heavily biased towards the synthesis
(~) Current Biology Ltd ISSN 0958-1669
79
80 Expressionsystems of the a and 13 globin chains, is usually quite different from that of other tissues or species and therefore lacking the necessary anti-codons. Thus, optimal translation of a given mRNA is often only achieved by addition of crude tRNA from the appropriate tissue or species. In addition, efforts have also been made to increase the reticulocyte lysate's capacity for faithful initiation [8"',9 "']; ettbrts have concentrated on examining the effects of ionic strength, Mg2 + concentration and mRNA concentration. Unfortunately, the optimal conditions for protein synthesis are usually not those optimal for faithful initiation. The most common errors are initiation at an incorrect AUG start codon (this can be reduced at elevated, non-optimal ionic strength), and premature chain termination at a rare codon (this can often be overcome by the inclusion of either tRNA from the same tissue as the mRNA is usually translated In or liver tRNA). Even when following guidelines for optimal translation, the investigator should monitor protein synthesis as the source of lysate can influence the quality of the final expression product [ 9 " ] .
Assaying biologic function When even optimal conditions for translation only yield micrograms of protein, why select a cell-free system at all? The main reason is the ease of analysis of site-directed mutants as long as an appropriate assay is also at hand; that is, one need not bother with the effort reqnired to express the cDNAs in a particular host system and then to purify the products before testing function. Clearly the trick is to set up an assay system that will directly test the function of the expressed protein. Such assays have been developed for/3-tubulin and the capbinding subunit of elF-4F, a eukaryotic protein synthesis factor [10,11 " , 1 2 " ] . In the [3-tubulin study, the expressed protein was characterized by its ability to form heterodimers (0t13) and its co-assembly with unlabelled tubulin [10,11 *o]. Furthermore, the [3-tubulin could be tested by its ability to bInd GTP, as quantified by the GTPdependent increased yield of [35S]methionine-labelled [~-tubulin purified from reticulocyte lysates by fast protein liquid chromatography using Mono-Q colunms [ 10]. In the second example [12-.] the function of the 7methylguanosine cap-binding protein subunit of elF-4F was investigated by generating a high-specific-activity labelled form. Under various conditions the expressed protein was or was not associated with ribosomal subparticles and was or was not phosphorylated. Thus the expressed subunit behaved in the same way as the native subunit during protein synthesis. The expressed protein was also able to bind to 7-methylguanosine triphosphate Sepharose, an affinity column used in the purification of elF-4F. Given that assays for the above two proteins have now been established, the next step will be to use site-directed mutagenesis to identify sequences or domains necessary for specific functions. An interesting subject for investigation is the GTP-binding site in [3-tubulin; this site is poorly characterized and is apparently dissimilar to that of most GTP-binding proteins [13]. Can an appropriate
site-directed modification be made that will alter the nucleotide specificity of [~-tubulin as has been accomplished for E. coli EF-Tu, the protein synthesis elongation factor that binds GTP and aminoacyl-tRNA [14]? It remains to be seen whether one can define those residues that are important for the association of tubulin and the cap-binding subunit of elF-4F with other proteins/subunits. The above examples indicate that for at least several proteins conditions can be obtained that allow their relatively easy cell-free synthesis and assay. The range of proteins could be extended by assaying specific binding of the cell-free expressed protein to an appropriate immobilized ligand (nucleotide, sugar, protein, etc). Another approach would be to measure the formation of specific aggregates; for example, one could use the popular gel-shift assay to monitor the interaction between an expressed DNA-binding protein and DNA.
Purification and large-scale expression An alternate approach is to purify the expressed protein from the reticulocyte lysate. Given the small amount of material, a one-step purification would be most desirable. A standard method is to use an immobilized monoclonal antibody raised against a portion of the protein or to use a unique peptide epitope fused to the protein in question [ 15]. The function of the protein can then be assessed by detecting a modification in the lysate or assay, or a modification within the immune complex (i.e. by photoatt3nity label binding of radiolabelled substrate, etc). Because of their large size, use of antibodies as described above can cause problems, especially ff the antigenic site is close to the active site (or effector site) of the protein under study. An alternative to using antibodies is to attach a specific 'handle' for purification to the coding region. An example is the attachment of six histidine residues to the amino- or carboxy-terminal ends. The presence of this histidine cluster allows for the specific purification of the expressed protein by metal-chelate-affinity chromatography using a nickel nitrilotriacetate column [16..]. The protein would then be assayed for function as described above. It should be noted that in the 'standard' synthetic reaction, one would expect, at best, the synthesis of about 100 pmol of globin per 100 gl of reaction mixture. Thus, any assay for a specific protein function must be very sensitive. Given the apparent advantages of cell-free translation systems, it would be nice ff they could produce more protein. A single report indicates that this is possible [17], although the work has yet to be confirmed by others. It was based on the fact that during protein synthesis products accumulate (e.g. ADP, GDP, AMP, PO4) that inhibit further synthesis. To avoid this build up, a continuousflow system was developed that allowed for the infusion of fresh reagents and the wash out of inhibitory materials. The results for one test case were spectacular. The quantity of protein obtained from a 20-40 h incubation was essentially 20-40 times as great as from a 1 h reac-
Gene expressionusingcell-free systemsMerrick 81 tion, i.e. the system displayed none of the usual slowing down. If reproducible, this methodology would allow for large-scale production, perhaps even to the 100-1000 ~tg range, and thus become a reasonable alternative to in vivo expression for preparative amounts of proteins. It should be noted that E. coli and wheat-germ extracts were used in this study and that these are significantly less efficient than reticulocyte lysates. If it is possible to use rabbit-reticulocyte lysate in the continuous flow system one would anticipate at least a lO-fold increase in product formation.
Conclusion Overall, the reticulocyte lysate is an ideal extract for expression because the reticulocyte is highly differentiated, which in mammalian species means it has no nucleus and very few organeUes. The extract is nearly always nuclease- and protease free, which allows for efficient use of mRNAs and the accumulation of protein product. While reticulocyte lysate is generally preferable because of its high synthetic rate and commercial availability, wheat-germ extracts have also been used extensively in the past. The advantages of the wheat-germ system are low price, low background levels of translation in the absence of added mRNA and a generally greater 7-methylguanosine cap-dependence than is seen in the reticulocyte system. In this report, cell-free expression systems that produce some biologically active proteins have been identified. In several cases, assay techniques have also been developed to test the products. These systems could now be used in conjunction with site-directed mutagenesis. It is envisaged that similar isolation and assay systems can be generated for a large number of proteins and thus obviate the difficulties often encountered in the expression of proteins in vivo, such as proteolysis, inclusion bodies, toxicity, etc.
Acknowledgements The author wishes to thank Ms Toni Bodnar for her expert editorial assistance. This work was supported in part by a grant from the National Institutes of Health, GM26796.
Annotated references and recommended reading • ••
Of interest Of outstanding interest
1.
MERRICK,In Ribosomes edited by Hill. American Society for Microbiology, 1990, pp292-299. .• HERSHEYJWB: ProteIn phosphorlyation controls translation rates. J Biol Chem 1989, 264:20823-20826. This is a nice, short overview of protein synthesis in eukaryotes. While the emphasis is on the control of protein synthesis by phosphorylation, it is a generally readable review, i.e. not just for the translation specialist. 3.
PAIN VM: Initiation of protein synthesis in mammalian cells. Biochem J 1986, 235:625-637.
4. 5.
KoZ~. M: An analysis of 5'-non-coding sequences from 699 verteorate mRNAs. Nucl Acids Res 1987, 15:8125-8148. KOZAKM: Comparison of initiation o f protein synthesis in procaryotes, eucaryotes and organelles. Microbiol Rev 1983, 47:1-45.
PELLETIERJ, KAPLAN G, RACANIELLOVR, SONENBERG N: Capindependent translation of poliovirus mRNA is conferred by s e q u e n c e elements within the 5' non-coding region. Mol Cell Biol 1988, 8:1103-1112. 7. PELLETIERJ, SONENBERG N: Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNK Nature 1988, 334:320-325. 8. DASSOMC, JACKSON RJ: On the fidelity of mRNA transla•• tion in the nuclease-treated rabbit-reticulocyte lysate system. Nucl Acids Res 1989, 17:3129-3144. This is an excellent guide to some of the precautions that must be taken in the setting-up of the conditions for translation of specific mRNAs. As might be anticipated, no two mRNAs are the same. Fortunately, there appear to be only a few variables that must be determined for each mRN& 6.
9. . o
KOZAKM: Context effects and inefficient initiation at nonAUG codons in eukaryotic cell-free translation systems. Mol Cell Biol 1989, 9:5073-5080. In this paper the influence of the bases surrounding the initiating AUG are characterized in both wheat-germ and rabbit-reticulocyte lysates. For individuals who wish to tailor their mRNA to initiate only at the correct AUG codon, helpful hints for site-directed mutagenesis and lysate optimization are provided. YAFFEMB, FARRGW, STERNLICHTH: Translation of f~-tubulin mRNA in vitro generates multiple molecular forms. J Biol (70em 1988, 263:16023-16031. 11. YAFFEMB, FARR GW, STERNI/CHT H: Kinetics of J3-tubulin ~. exchange following translation. Evidence for a slow conformational change in [3-tubulin necessary for incorporation into heterodimers. J Biol Chem 1989, 264:19045-19051. s is a follow up to [ 8 * . ] and indicates the depth of analysis possible with proteins expressed in reticulocyte tysates. For those who feel that little more than sodium dodecyt sulfate polyacrylamide gel dectrophoresis molecular weights can be determined for translation products, this paper should be a real eye-opener. The system described is currently being studied in conjunction with site-directed mutagenesis. 12. HIREMATHLS, HIREMATHST, RYCHLIKW, HOSHI S, DOMIER LIo •, RHOADSRE: In vitro synthesis, phosphorylation and localization on 48S initiation complexes of human protein synthesis initiation factor 4E. J Biol Chem 1989, 264:1132-1138. A study of the participation of the in vitro synthesized 7-methylguanosine cap-binding subunit of eIF-4F (here termed elF-4E) in the process of translation. The translation product behaved normally and is now a candidate for investigating the alterations in function caused by site-directed mutagenesis. This also indicates that multiple types of analysis can be performed on the translation product. 13. DEVERAND MERRICKIn Guanine Nucleotide Binding Proteins edited by Bosch et at Plenum Publishing Corporation, 1989, pp35-48. 14. HWANGY, MILLERDL A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein. J Biol Chem 1987, 262:13081-13085. 15. FIELDJ, NIKAWAJ, BROEKD, MACDONALDB, RODGERSL, WmSON IA, LERNERRA, WIGLER M: Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 1988, 8:2159-2165. 16. LEGRICE SFJ, GRUNINGER-LE1TCH F: Rapid purification of •• homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate aflh~ty chromatography. Eur J Biochem 1990, 187:307-314. The addition of six histidine residues to the amino terminus of (HIV)-I reverse transcriptase facilitates (via site-directed mutagenesis) one-step purification using a 'nickel' column. In spite of the complex reactions catalyzed by the enzyme, the additional amino acids did not appear to alter its function. 10.
17.
SPIRINAS, BARANOVVI, RYABOVALA, OVODOV SY, ALAKHOV YB: A continuous cell-free translation system capable of producing, polypeptides in high yields. Science 1988, 242:1162-1164.
Background Cell-free protein synthesizing systems have traditionally been used for the analysis of those proteins and RNAs required for accurate and efficient synthesis of proteins. In both bacterial and eukaryotic systems, this has led to the elucidation of protein factors that are specific for the initiation, elongation and termination of the polypeptide chain, as well as elements that are involved in secretion and post-translational modifications. The employment of cell-free systems to express a protein of choice used to be feasible only in the few instances where a particular mRNA was extremely abundant in some tissue (e.g. globin, ovalbumin, serum albumin). The availability of several eR]cient systems for the production of mRNAs (SP6, T7, T4) has now changed this and made it possible to generate any given mRNA in a pure form. However, given the short lifespan of a cell-free system and the limited amount of pure mRNA available, it has generally not been possible to produce much protein product (perhaps a microgram or so). Furthermore, this product is part of a mixture of proteins, which makes it dit~cult to purify. This would seem to imply that cell-free expression of proteins is only of interest in relation to polysome size or formation, post-translational modification, protein trafficking, etc. However, cell-free expression in conjunction with appropriate assays or isolation techniques has actually proven to be quite useful. Cell-free systems have primarily been used to characterize eukaryotic proteins, because the ease of manipulation and the genetic capability of Escberichia coli make similar efforts for bacterial proteins easier to accomplish in viva Such systems mainly use rabbit-reticulocyte lysates as they synthesize proteins in vivo at a rate that is approximately 10--100-fold greater than other translation extracts. From studies on the mechanism of translation, it has been possible to define two distinct initiation pathways. The first is the most common eukaryotic scheme--the 'cap-depen-
dent' initiation of translation which involves the eukaryotic unique 7-methylguanosine cap structure connected to the 5' terminal base of mRNAs via a 5' to 5' linkage using a triphosphate bridge. Experimentally, the use of this pathway can be tested in vitro either by omitting the cap structure when the mRNA is made or by inhibiting translation by the addition of 7-methylguanosine diphosphate, an analog of the cap structure. From studies on initiation factor function [1,2 °',3] and the extensive work of Marilyn Kozak on AUG selection [4,5], it is possible to identify those optimal characteristics of an mRNA that aid initiation in a cap-dependent fashion. The characteristics include: a 7-methylguanosine cap structure; a relatively short (less than 50 bases) 5' untranslated region; an optimal context around the initiating AUG codon (A/GXXAUGG); little or no secondary structure in the 5' untranslated region; and no AUG codons in the 5' untranslated region. Absence of G residues in the 5' untranslated region achieves the same effect not only because mRNA secondary structure is minimized and upstream AUG codons are avoided, but also because of the avoidance of potential, if weak, non-AUG initiating codons that can cause reduced fidelity and reduced translational efficiency. The second pathway of initiation is termed 'cap independent' or 'internal' initiation. To date, this pathway of initiation has only been observed in viral mRNAs, the best examples of which are the picornaviral mRNAs. In this scheme, an internal portion of the mRNA is the site for direct binding of the ribosome. The characteristics of such a specific site have not yet been fully characterized; however, segments of the 5'untranslated region of encephalomyocarditis virus mRNAs and poliovirus mRNAs have been shown to act in cL~ to allow for internal initiation of heterologous mRNAs [6,7]. Generally, the use of such elements to enhance expression is discouraged as the biochemical basis for their use in still largely unknown and some of the picomavirus mRNAs, such as poliovirus mRNAs, are poorly translated. On the other hand, the 5' untranslated leader of the encephalomyocarditis virus mRNA is perhaps the most efficient known and is better than all capped mRNAs tested to date in competitive translation assays.
Optimization of cell-free translation A problem associated with efficient translation is catering for the rare codons that are found in mRNA. This can be bothersome in rabbit-reticulocyte lysates, as the tRNA population, which is heavily biased towards the synthesis
(~) Current Biology Ltd ISSN 0958-1669
79
80 Expressionsystems of the a and 13 globin chains, is usually quite different from that of other tissues or species and therefore lacking the necessary anti-codons. Thus, optimal translation of a given mRNA is often only achieved by addition of crude tRNA from the appropriate tissue or species. In addition, efforts have also been made to increase the reticulocyte lysate's capacity for faithful initiation [8"',9 "']; ettbrts have concentrated on examining the effects of ionic strength, Mg2 + concentration and mRNA concentration. Unfortunately, the optimal conditions for protein synthesis are usually not those optimal for faithful initiation. The most common errors are initiation at an incorrect AUG start codon (this can be reduced at elevated, non-optimal ionic strength), and premature chain termination at a rare codon (this can often be overcome by the inclusion of either tRNA from the same tissue as the mRNA is usually translated In or liver tRNA). Even when following guidelines for optimal translation, the investigator should monitor protein synthesis as the source of lysate can influence the quality of the final expression product [ 9 " ] .
Assaying biologic function When even optimal conditions for translation only yield micrograms of protein, why select a cell-free system at all? The main reason is the ease of analysis of site-directed mutants as long as an appropriate assay is also at hand; that is, one need not bother with the effort reqnired to express the cDNAs in a particular host system and then to purify the products before testing function. Clearly the trick is to set up an assay system that will directly test the function of the expressed protein. Such assays have been developed for/3-tubulin and the capbinding subunit of elF-4F, a eukaryotic protein synthesis factor [10,11 " , 1 2 " ] . In the [3-tubulin study, the expressed protein was characterized by its ability to form heterodimers (0t13) and its co-assembly with unlabelled tubulin [10,11 *o]. Furthermore, the [3-tubulin could be tested by its ability to bInd GTP, as quantified by the GTPdependent increased yield of [35S]methionine-labelled [~-tubulin purified from reticulocyte lysates by fast protein liquid chromatography using Mono-Q colunms [ 10]. In the second example [12-.] the function of the 7methylguanosine cap-binding protein subunit of elF-4F was investigated by generating a high-specific-activity labelled form. Under various conditions the expressed protein was or was not associated with ribosomal subparticles and was or was not phosphorylated. Thus the expressed subunit behaved in the same way as the native subunit during protein synthesis. The expressed protein was also able to bind to 7-methylguanosine triphosphate Sepharose, an affinity column used in the purification of elF-4F. Given that assays for the above two proteins have now been established, the next step will be to use site-directed mutagenesis to identify sequences or domains necessary for specific functions. An interesting subject for investigation is the GTP-binding site in [3-tubulin; this site is poorly characterized and is apparently dissimilar to that of most GTP-binding proteins [13]. Can an appropriate
site-directed modification be made that will alter the nucleotide specificity of [~-tubulin as has been accomplished for E. coli EF-Tu, the protein synthesis elongation factor that binds GTP and aminoacyl-tRNA [14]? It remains to be seen whether one can define those residues that are important for the association of tubulin and the cap-binding subunit of elF-4F with other proteins/subunits. The above examples indicate that for at least several proteins conditions can be obtained that allow their relatively easy cell-free synthesis and assay. The range of proteins could be extended by assaying specific binding of the cell-free expressed protein to an appropriate immobilized ligand (nucleotide, sugar, protein, etc). Another approach would be to measure the formation of specific aggregates; for example, one could use the popular gel-shift assay to monitor the interaction between an expressed DNA-binding protein and DNA.
Purification and large-scale expression An alternate approach is to purify the expressed protein from the reticulocyte lysate. Given the small amount of material, a one-step purification would be most desirable. A standard method is to use an immobilized monoclonal antibody raised against a portion of the protein or to use a unique peptide epitope fused to the protein in question [ 15]. The function of the protein can then be assessed by detecting a modification in the lysate or assay, or a modification within the immune complex (i.e. by photoatt3nity label binding of radiolabelled substrate, etc). Because of their large size, use of antibodies as described above can cause problems, especially ff the antigenic site is close to the active site (or effector site) of the protein under study. An alternative to using antibodies is to attach a specific 'handle' for purification to the coding region. An example is the attachment of six histidine residues to the amino- or carboxy-terminal ends. The presence of this histidine cluster allows for the specific purification of the expressed protein by metal-chelate-affinity chromatography using a nickel nitrilotriacetate column [16..]. The protein would then be assayed for function as described above. It should be noted that in the 'standard' synthetic reaction, one would expect, at best, the synthesis of about 100 pmol of globin per 100 gl of reaction mixture. Thus, any assay for a specific protein function must be very sensitive. Given the apparent advantages of cell-free translation systems, it would be nice ff they could produce more protein. A single report indicates that this is possible [17], although the work has yet to be confirmed by others. It was based on the fact that during protein synthesis products accumulate (e.g. ADP, GDP, AMP, PO4) that inhibit further synthesis. To avoid this build up, a continuousflow system was developed that allowed for the infusion of fresh reagents and the wash out of inhibitory materials. The results for one test case were spectacular. The quantity of protein obtained from a 20-40 h incubation was essentially 20-40 times as great as from a 1 h reac-
Gene expressionusingcell-free systemsMerrick 81 tion, i.e. the system displayed none of the usual slowing down. If reproducible, this methodology would allow for large-scale production, perhaps even to the 100-1000 ~tg range, and thus become a reasonable alternative to in vivo expression for preparative amounts of proteins. It should be noted that E. coli and wheat-germ extracts were used in this study and that these are significantly less efficient than reticulocyte lysates. If it is possible to use rabbit-reticulocyte lysate in the continuous flow system one would anticipate at least a lO-fold increase in product formation.
Conclusion Overall, the reticulocyte lysate is an ideal extract for expression because the reticulocyte is highly differentiated, which in mammalian species means it has no nucleus and very few organeUes. The extract is nearly always nuclease- and protease free, which allows for efficient use of mRNAs and the accumulation of protein product. While reticulocyte lysate is generally preferable because of its high synthetic rate and commercial availability, wheat-germ extracts have also been used extensively in the past. The advantages of the wheat-germ system are low price, low background levels of translation in the absence of added mRNA and a generally greater 7-methylguanosine cap-dependence than is seen in the reticulocyte system. In this report, cell-free expression systems that produce some biologically active proteins have been identified. In several cases, assay techniques have also been developed to test the products. These systems could now be used in conjunction with site-directed mutagenesis. It is envisaged that similar isolation and assay systems can be generated for a large number of proteins and thus obviate the difficulties often encountered in the expression of proteins in vivo, such as proteolysis, inclusion bodies, toxicity, etc.
Acknowledgements The author wishes to thank Ms Toni Bodnar for her expert editorial assistance. This work was supported in part by a grant from the National Institutes of Health, GM26796.
Annotated references and recommended reading • ••
Of interest Of outstanding interest
1.
MERRICK,In Ribosomes edited by Hill. American Society for Microbiology, 1990, pp292-299. .• HERSHEYJWB: ProteIn phosphorlyation controls translation rates. J Biol Chem 1989, 264:20823-20826. This is a nice, short overview of protein synthesis in eukaryotes. While the emphasis is on the control of protein synthesis by phosphorylation, it is a generally readable review, i.e. not just for the translation specialist. 3.
PAIN VM: Initiation of protein synthesis in mammalian cells. Biochem J 1986, 235:625-637.
4. 5.
KoZ~. M: An analysis of 5'-non-coding sequences from 699 verteorate mRNAs. Nucl Acids Res 1987, 15:8125-8148. KOZAKM: Comparison of initiation o f protein synthesis in procaryotes, eucaryotes and organelles. Microbiol Rev 1983, 47:1-45.
PELLETIERJ, KAPLAN G, RACANIELLOVR, SONENBERG N: Capindependent translation of poliovirus mRNA is conferred by s e q u e n c e elements within the 5' non-coding region. Mol Cell Biol 1988, 8:1103-1112. 7. PELLETIERJ, SONENBERG N: Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNK Nature 1988, 334:320-325. 8. DASSOMC, JACKSON RJ: On the fidelity of mRNA transla•• tion in the nuclease-treated rabbit-reticulocyte lysate system. Nucl Acids Res 1989, 17:3129-3144. This is an excellent guide to some of the precautions that must be taken in the setting-up of the conditions for translation of specific mRNAs. As might be anticipated, no two mRNAs are the same. Fortunately, there appear to be only a few variables that must be determined for each mRN& 6.
9. . o
KOZAKM: Context effects and inefficient initiation at nonAUG codons in eukaryotic cell-free translation systems. Mol Cell Biol 1989, 9:5073-5080. In this paper the influence of the bases surrounding the initiating AUG are characterized in both wheat-germ and rabbit-reticulocyte lysates. For individuals who wish to tailor their mRNA to initiate only at the correct AUG codon, helpful hints for site-directed mutagenesis and lysate optimization are provided. YAFFEMB, FARRGW, STERNLICHTH: Translation of f~-tubulin mRNA in vitro generates multiple molecular forms. J Biol (70em 1988, 263:16023-16031. 11. YAFFEMB, FARR GW, STERNI/CHT H: Kinetics of J3-tubulin ~. exchange following translation. Evidence for a slow conformational change in [3-tubulin necessary for incorporation into heterodimers. J Biol Chem 1989, 264:19045-19051. s is a follow up to [ 8 * . ] and indicates the depth of analysis possible with proteins expressed in reticulocyte tysates. For those who feel that little more than sodium dodecyt sulfate polyacrylamide gel dectrophoresis molecular weights can be determined for translation products, this paper should be a real eye-opener. The system described is currently being studied in conjunction with site-directed mutagenesis. 12. HIREMATHLS, HIREMATHST, RYCHLIKW, HOSHI S, DOMIER LIo •, RHOADSRE: In vitro synthesis, phosphorylation and localization on 48S initiation complexes of human protein synthesis initiation factor 4E. J Biol Chem 1989, 264:1132-1138. A study of the participation of the in vitro synthesized 7-methylguanosine cap-binding subunit of eIF-4F (here termed elF-4E) in the process of translation. The translation product behaved normally and is now a candidate for investigating the alterations in function caused by site-directed mutagenesis. This also indicates that multiple types of analysis can be performed on the translation product. 13. DEVERAND MERRICKIn Guanine Nucleotide Binding Proteins edited by Bosch et at Plenum Publishing Corporation, 1989, pp35-48. 14. HWANGY, MILLERDL A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein. J Biol Chem 1987, 262:13081-13085. 15. FIELDJ, NIKAWAJ, BROEKD, MACDONALDB, RODGERSL, WmSON IA, LERNERRA, WIGLER M: Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 1988, 8:2159-2165. 16. LEGRICE SFJ, GRUNINGER-LE1TCH F: Rapid purification of •• homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate aflh~ty chromatography. Eur J Biochem 1990, 187:307-314. The addition of six histidine residues to the amino terminus of (HIV)-I reverse transcriptase facilitates (via site-directed mutagenesis) one-step purification using a 'nickel' column. In spite of the complex reactions catalyzed by the enzyme, the additional amino acids did not appear to alter its function. 10.
17.
SPIRINAS, BARANOVVI, RYABOVALA, OVODOV SY, ALAKHOV YB: A continuous cell-free translation system capable of producing, polypeptides in high yields. Science 1988, 242:1162-1164.
