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Chromatog, Forum 1, 31 37 5 Dean, P. D. G., Johnson W. S. and Middle, F. A. (1985) Affinity chromatography: a practical approach, IRL Press 6 Burgess, R. (ed.) (1987) Protein purification: micro to macro, Alan R. Liss 7 Janson, J. C. (1984) Trends Biotechhal. 2, 31-38 8 Groman, E. V. and Wilchek, M. {1987) Trends Biotechnol. 5, 220-224 9 Cabrera, K. E. and Wilchek, M. {1987) in Protein purification: micro to macro (Burgess, R., ed.), pp. 163-175, Alan R. Liss 10 Cabrera, K. E. and Wilchek, M. (1988) Trends Analyt. Chem. 7, 2, 58-63 11 Cabrera, K. E. and Wilchek, M. (1988) Makromol. Chem. Macromol. Syrup. 19, 145-154 12 Ohlson, S., Gudmundsson, B. M., Wikstrom, P. and Larsson, P. O. (1988) Clin. Chem. 34, 2039-2043 13 Ito, N., Noguchi, K., Kazama, M. and
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14 15 16 17
18 19 20 21 22 23
Kasai, K. I. (1987) J. Chromatogr. 400, 163 167 Ostlund, C. (1986) Trends in Biotechhal. 4, 288-293 Ohlson, S., Hansson, L., Larsson, P. O. and Mosbach, K. (1978) FEBS Lett. 93, 5-9 Ohlson, S., Hansson, L., Glad, M. and Mosbach, K. (1989) Trends in Biotechno]. 7, 179-186 Wainer, I. W., Jadaud, P., Schonbaum, G. R., Kakodkar, S. V. and Henry, M. P. (1988) Chromatographia 25, 903-907 Waiters, R. R. (1982) J. Chromatogr. 249, 19 28 Horstmann, B. J., Kenny, C. N. and Chase, H. A. (1986) J. Chromatogr. 361, 179-190 Fowell, S. L. and Chase, H. A. (1986) I. Biotechnol. 4, 355-368 Taylor, R. F. (1985) Anal. Chim. Acta. 172, 241-248 Hochu]i, E. (1988) J. Chromutogr. 444, 293-302 Yang, C. and Tsao, G. T. (1982) Adv.
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Protein engineering for thermostability Yoshiaki Nosoh and Takeshi Sekiguchi Studies with small, monomeric proteins indicate that, to some extent, the effects of amino acid substitutions can be predicted. However, conformational and other changes may complicate the prediction. Site-directed mutagenesis is leading both to a better understanding of protein stability and to the production of more stable proteins. A variety of e n z y m e s is n o w u s e d in various fields of b i o t e c h n o l o g y i n d u s t r i e s and, w i t h further d e v e l o p m e n t s in b i o t e c h n o l o g y , the v o l u m e a n d range of e n z y m e s r e q u i r e d will increase. M a n y e n z y m e s isolated f r o m m e s o p h i l e s h a v e b e e n u s e d for b i o t e c h n o l o g y , but these are of limited use due to their d e n a t u r a t i o n on e x p o s u r e to c o n d i t i o n s w h i c h are often e n c o u n t e r e d in i n d u s t r i a l processes (e.g. heat, organic solvents a n d v a r i o u s chemicals). E n z y m e s stable
Y. Nosoh and T. Sekiguchi are at the Department of Fundamental Science, College of Science and Engineering, Iwaki Meisei University, Iwaki, Fukushima 970, Japan.
u n d e r these c o n d i t i o n s are, therefore, required. E n z y m e s f r o m t h e r m o philes are generally m o r e stable t h a n the c o r r e s p o n d i n g ones from m e s o philes, and t h e r m o p h i l e s are the traditional source of stable e n z y m e s . Protein e n g i n e e r i n g t h r o u g h sitedirected m u t a g e n e s i s has b e c o m e a p r o m i s i n g alternative strategy for protein stabilization 1. Stability in a folded p r o t e i n is a balance b e t w e e n the stabilizing (mostly h y d r o p h o b i c ) interactions, a n d the t e n d e n c y t o w a r d s destabilization c a u s e d by the loss of conform a t i o n a l e n t r o p y as the p r o t e i n a d o p t s the u n f o l d e d form. Consequently, the stability of a p r o t e i n m a y be altered b y c h a n g i n g a m i n o acids w h i c h affect either stabilizing
@ 1990, Elsevier Science Publishers Ltd (UK) 0167 9430/89/$2.00 -
Biochem. Bioeng. 25, 19-41 24 Nishikawa, A. H., Bailon, P. and Ramel, A. H. (1976) J. Macromol. Sci. Chem. A10, 149-190 25 Kennedy, J. F. and Barnes, J. A. (1983) f Chromatogr. 281, 83-93 26 Fowell, S. L. and Chase, H. A. (1986) J. Biotechnol. 4, 1-13 27 Wu, D. and Walters, R. R. (1988) J. Chromatogr. 458, 169-174 28 Narayanan, S. R., Knochs, S. J. and Crane, L. J. J. Chromatogr. (in press) 29 Chase, H. A. (1988) Makromo]. 17, Chem., Macromol. Syrup. 467-482 30 Secher, D. S. and Burke, D. C. (1980) Nature 285,446-450 31 Zimmerman, T. S. and Fulcher, S. (1982) US Patent No. 4361509 32 Dodd, I., Jalalpour, S., Southwick, W. et al. (1986) FEBS Lett, 209, 13-17 33 Bonnerjea, J., Oh, S., Hoare, M. and Duni]l, P. (1986) Biotechnology 4, 954-958 34 Katoh, S. (1987) TrendsBiotechnol. 5, 328-331
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interactions in the folded protein, or destabilizing interactions in the unfolded form, or b o t h (Table 1).
Mechanisms and control of protein stability This r e v i e w s h o w s h o w p r o t e i n e n g i n e e r i n g can be u s e d b o t h to explore the m o l e c u l a r basis of protein stability a n d to m o d i f y p r o t e i n stability (Fig. 1). En tropic stabi]ization The d e n a t u r a t i o n of p r o t e i n s can be w r i t t e n s i m p l y as N , ~ U--* X w h e r e the folded state (N) is reversibly c h a n g e d to the u n f o l d e d state (U), (reversible d e n a t u r a t i o n or unfolding), a n d U i r r e v e r s i b l y to X (irreversible inactivation). N has a l o w e r free energy t h a n U. A n y t h i n g that stabilizes N and/or decreases the destabilizing forces of U will increase the free energy change for unfolding, a n d t h e r e b y stabilize the protein. T h e irreversible i n a c t i v a t i o n d e p e n d s on the c o n f o r m a t i o n of the u n f o l d e d f o r m (U): m o r e c o m p a c t e d f o r m s of U will be i n a c t i v a t e d m o r e s l o w l y a n d will thus stabilize the protein.
TIBTECH- JANUARY 1990 [Vol, 8]
17
~Table I Factors to consider in stabilizing proteins a
inactivation than the wild-type protein even though, in most cases, the disulfide bond strengths were high enough to stabilize the mutant protein against thermal inactivation. This suggests that either the cysteine substitution and/or the crosslinkformation disrupted other stabilizing interactions, increasing the rate of unfolding and, therefore, the rate of irreversible inactivation.
Interaction or bond Disulfide bond Hydrogen bond Electrostatic interaction Hydrophobic interaction Conformational factors Stability of secondary structure Compact packing Conformational flexibility Internal hydrophobicity Entropic stability Protection Deamination of carboxyamide Oxidation of sulfhydryl groups Intramolecular S-H/S-S exchange Oxidation of tryptophan or methionine
• Dihydrofolate reductase
aThese factors were deduced by comparing the structures of proteins of different stabilities (proteins isolated from mesophiles and thermophiles, wild-type and aminoacid-substituted proteins, wild-type and chemically modified proteins).
Disulfide bonds Disulfide bonds in proteins restrict the degree of freedom for the unfolded state U and thereby stabilize the folded state N (Ref. 2). The first type of protein stabilization performed by genetic manipulation was the introduction of disulfide bonds. One or two amino acids in a protein were replaced with cysteines; a disulfide bond forms in vivo or in vitro. If the introduced disulfide bond caused no, or little tertiary structural change, the crosslink stabilizes the protein. Disulfide bonds have been engineered into T4 lysozyme 3,4, subtilisin 5'6, dihydrofolate reductase 7 and the )~repressor 8, with stabilization occurring in some cases but not others.
• Subtilisin
Subtilisin naturally contains neither disulfide bonds nor free cysteine residues. Disulfide bonds which formed between the introduced Cys 22 and Cys 87 or between the introduced Cys 24 and Cys 87 did not stabilize the protein 5. A protein with the former of these types of bond was less thermostable than the wildtype protein 5. Recently, five singledisulfide-bonded forms of subtilisin were produced 6. None was more stable against irreversible thermal
A disulfide bond was introduced into E. coli dihydrofolate reductase by replacing Pro 39 with Cys and crosslinking this with the natural Cys 85 (Ref. 7). The crosslinked enzyme was about 7.56 kJmo1-1 more stable than the non-crosslinked or the wild-type enzyme, with respect to reversible denaturation by guanidine hydrochloride. This may indicate that the increased free energy change for the unfolding of the crosslinked enzyme is due to the entropic stabilization of the unfolded form. However, the crosslinked enzyme was not more resistant to thermal inactivation than the wild-type protein. The X-ray crystal structure analyses showed that the flexibility of the region containing residues 80-90 increases on crosslink formation. The disulfide bond appears to be too flexible to restrict the flexible motion of the region. This
~Fig. 1 Protein
']
• Lysozyme
Isoleucine (Ile) at position 3 in T4 phage lysozyme was replaced by Cys, followed by in vitro oxidation 3 (Fig. 2). The crosslink between the introduced Cys 3 and native Cys 97 imparted stability against both reversible thermal unfolding and irreversible thermal inactivation 4. The stability against reversible unfolding of the crosslinked enzyme was possibly due to the entropic effect on the unfolded state of the protein (thermodynamic stabilization). Its stabilization against irreversible inactivation was considered to be due to the unfolded state of the crosslinked enzyme being more compact in structure than the native enzyme (kinetic stabilization).
Protein stabilization
I I
Mechanism of protein stability
Relationship of amino acid substitution to controlling protein stability and understanding its mechanisms. Proteins are stabilized through site-directed mutagenesis based on the mechanism of protein unfolding. Site-directed mutagenesis is also employed to reveal the mechanism of stabilization (e.g. comparison of X-ray crystal structures and the thermodynamic and kinetic evaluations in the wild-type and engineered proteins).
18
TIBTECH - JANUARY 1990 [Vol. 8]
--Fig. 2
A82P
i23
may be w h y the cross-linked protein is not more stable against thermal inactivation.
Other amino acid substitutions Glycine has no ~-carbon and allows more conformational freedom in a polypeptide chain than alanine. Thus, changing Gly to Ala might increase protein stability by reducing the entropy loss on unfolding. Similarly, substituting proline for other amino acids might decrease the entropy of polypeptide backbone. T4 lysozyme 9 and X repressor 1° have been stabilized in this way by sitedirected mutagenesis. In T4 lysozyme 9, two kinds of amino acid substitution (Gly 77 to Ala and Ala 82 to Pro) were performed (Fig. 2). The Gly 77 --~ Ala substitution produced a 1.68 kJ mo1-1 increase in stability (for the reversible thermal denaturation): Gly 77 is in an o~-helix, so the increased sta bil ity might be attributed to better t i c l ix formation ~a. However, entropic stabilization could also be responsible for the increase in protein stability 9. The observed increase in thermodynamic stabilization is about half the value expected from theory. The substitution of Ala for Gly produces localized changes in the protein structure which may affect the entropic contribution to the free energy change for unfolding. The Ala82 --+ Pro substitution also increased the stability of T4 lysozyme by 3.6 kJ mo1-1 (Ref. 9). In the X repressor, the replacement of two Gly residues in e~-helices increased the free energy change for reversible unfolding by 1.68-3.78 kJ tool -~ (Ref. 10). Since the two substitution sites are in o~-helices, as in the case of T4 lysozyme 4, the increased stability could also be ascribed to the entropic effect. Entropic stabilization might be a general strategy for enhancing protein stability. Altering stabilizing factors We have discussed stabilization by reduction of destabilizing interactions in the unfolded form of the protein. The other way to increase stabilization is to increase t h e number of, or strengthen, the stabilizing interactions in the folded protein. In tern al hydroph obicity
Protein folding is driven by the hydrophobic properties of nonpolar
I
G77A
1251 -r__L,1~~ " ~, 1 ~ (313 .~ '~ /
90
\+,. T157AA
'"'
Gin IO5
C975
I
Glu 22 Thr ~ k Gly 21 ~ TyrO25 Lys
i
Ser
20 4
GIn6~
I3AA
/
T
3o
C54V The X-ray crystal structure of T4 lysozyme. Some of the amino acid substitutions that have been made (including those described in the text) are indicated by arrows: 13AA, lie 3 to various amino acids; C54V, Cys 54 to Val; C97T, Cys 97 to Thr; G77A, Gly 77 to Ala; A82P, Ala 82 to Pro; T157AA, Thr 157 to various amino acids. The structure is adapted from Ref. 28.
amino acid residues 2. Strengthening the internal hydrophobicity in a protein by amino acid substitution may increase protein stability. Glutamine (Gin), at position 49 in the interior of the o:-subunit of E. c01i tryptophan synthetase, was replaced with other amino acids 12. The stability of each resulting protein was calculated from the free energy changes between the native and unfolded proteins (thermodynamics) and between the native and transition states of unfolding (kinetics). Both measurements of stability correlated well with the hydrophobicity of the substituted amino acids, except where the proteins contained introduced aromatic amino acid residues. The authors concluded that increasing the internal hydrophobicity per se stabilized the protein thermodynamically and kinetically. Furthermore, they concluded that extra energy which resulted from increased hydrophobicity might also contribute to increased stability ~2.
The stabilities of the proteins with introduced aromatic amino acid residues were lower than w o u l d be predicted from consideration of the hydrophobicity of the residues 12. This may be due to the conformational distortion around the substitution site. A similar result was obtained with kanamycin nucleotidyltransferase 13. The reverse experiment was performed with barnase14: the hydrophobicity of the core was reduced. Ile at position 96 of barnase was replaced by Val or Ala (Ref. 14), and the free energy change for reversible urea denaturation of the protein was reduced by 5.0 or 16.8 kJ mo1-1, respectively. These stability changes were larger in magnitude than those expected simply on the basis of reduced hydrophobicity. The authors suggested that the substitution of Ile with Val or Ala created a cavity with the size of one or three methylene carbons, respectively, which destabilized the protein.
TIBTECH - J A N U A R Y 1990 [Vol. 8]
In contrast to the results obtained with the three previous examples TM 14, there was good agreement between the free energy change for reversible thermal inactivation of T4 lysozyme and its derivative proteins (substitutions of Ile 3; Fig. 2), and hydrophobicity of the side chains substituted ~5. Results with all four proteins, however, indicate that increasing internal hydrophobicity could be a useful strategy in stabilizing proteins.
Hydrogen bonding Addition or strengthening of noncovalent interactions other than hydrophobic interactions has been considered as a way of stabilizing proteins 1. However, high resolution protein structures usually do not reveal obvious sites where amino acid substitutions could strengthen a hydrogen bond or electrostatic interaction through closer contact between side chains 16. On the other hand, it is known that destruction of a hydrogen bond by a single amino acid substitution often reduces protein stability. When Thr 15 7 in T4 lysozyme was replaced by other amino acids, there was a strong correlation between a major reduction in stability and a replacement residue's inability to form a hydrogen bond with the amide of Asp 159 (which interacts with the hydroxyl group of Thr 157 in the wild-type protein) 17 (Fig. 2). A similar decrease in protein stability was reported with dihydrofolate reductase when Val was substituted for Thr 113 (Ref. 18). These results indicate that, if it were possible rationally to design additional hydrogen bonding, this could stabilize proteins.
Protection against chemical destabilization Irreversible inactivation of some proteins is triggered by deamination of aspmagine residues followed by hydrolysis of the peptide bonds at the aspartic acid residues, or by cleavage at the ~-position of cysteine, or by intramolecular thio/disulfide exchange, or by destruction of methionine residuesL Substituting for asparagine, cysteine and methionine might prevent such inactivation. Some evidence to support this was obtained in studies on yeast triosephosphate isomerase, a dimeric protein containing twelve asparagine residues per monomer, three of them
19
at the interfacial surface. When two of the three surface Asn residues (14 and 78) were altered to Thr and Ile, respectively 19, the half-life of the enzyme, relative to that of the wildtype enzyme at 100°C, was doubled. Similarly, in T4 lysozyme, when the natural Cys 54 and Cys 97 residues were mutated to Val and Thr, respectively, the enzyme was stabilized 2° (Fig. 2). In subtilisin, H202 inactivation was prevented by replacing Met 222 with Ala or Ser (Ref. 21).
Stabilization without knowledge of tertiary structure In almost all the targets of protein engineering, X-ray crystal structures are known. Even when the tertiary structure is not at hand, however, stabilization through site-directed mutagenesis may be possible, provided that a homologous protein of known structure is available. Using this strategy, a neutral protease from Bacillus stearothermophilus was stabilized using thermolysin as a model protein 22. Three kinds of substitution, Gly 61 to Ala, Gly 144 to Ala and Thr 66 to Ser, were examined. The first two stabilized the protein (possibly due to an increase in internal hydrophobicity and stabilization of o:-helix), and the last destabilized it al. Double and triple substitutions suggested that the stabilization effects were not additive, but cooperative. If neither the tertiary structures of a particular protein nor of any homologous model protein are available, another strategy - biological thermal adaptation - could be considered. A plasmid (pUB110)-borne kanamycin nucleotidyltransferase gene, originally isolated from a mesophile, was exposed to elevated temperatures in a host thermophilic bacterium 23. This treatment induced two kinds of mutation, Asp 80 to Tyr and Thr 130 to Lys. The mutation occurred naturally in a thermophile. The two substituted proteins are more thermostable than the original mesophilic protein. The role of Tyr 80 in thermostabilization was examined thermodynamically and kinetically13. Concluding remarks The work discussed demonstrates the contribution to protein stability of both stabilizing 12-15 and destabilizing 3-8 interactions. A few, or even
just a single, amino acid replacement may modify a protein's stability. It is too early yet for any general mechanisms of protein stabilization to have been proposed. Site-directed mutagenesis has been used to provide information on the kinds of interactions (or bonds) which should be considered. With small monomer proteins such as T4 lysozyme4,15, subti]isin 6, ~.repressor 24 and nuclease 25, it is relatively easy to compare the X-ray crystal structures and the energetics of denaturation of the wild-type and substituted proteins. Both structural and energetic analyses are important in correlating substitutions with stability since, in most cases, the changes in stability are greater than or less than those expected from simple consideration of the changes made; conformational and other factors must contribute to these deviations from prediction. The role of water in the energetics of denaturation should also be considered 26. If the X-ray crystal structure of the protein to be engineered is not available, appropriate amino acid substitutions can be guided in using the known structure of a homologous protein 22 and known differences between proteins in mesophiles and thermophiles 11. When no model protein is available, the strategies of thermal mutation 23 and random chemical mutation 27 could be attempted. In oligomeric proteins, subunit dissociation should also be considered as part of the inactivation process. Appropriate amino acid substitutions on the interfacial surface can stabilize the protein TM. Protein engineering, then, is a promising tool, not only for stabilizing proteins, but also for dissecting the mechanism of protein unfolding.
References 1 Nosoh, Y. and Sekiguchi, T. (1988) Biocatalysis 1, 257-273 2 Schulz, G. E. and Schirmer, R. H. (1979) Principles of protein structure, Springer-Verlag 3 Perry, L. ]. and Wetzel, R. (1984) Science 226,555-557 4 Wetzel, R., Perry, L. J., Baase, W. A. and Becktel, W. J. (1988) Proc. Notl Acad. Sci. USA 85,401-405 5 Wells, J. A. and Powers, D. B. (1986) J. Biol. Chem. 261, 6564-6570 6 Mitchinson, C. and Wells, J. A. (1989)
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Biochemistry 28, 4807-4815 7 Villafranca, J. E., Howell, E. E., Oatley, S. J., Xuong, N. and Kraut, J. (1987) Biochemistry 26, 2182-2189 8 Sauer, R. T., Hehir, K., Stearman, R. S. et al. (1986) Biochemistry 25, 5992-5998 9 Matthews, B. W., Nicholson, H. and Becktel, W. J. (1987) Proc. Natl Acad. Sci. USA 84, 6663-6667 10 Hecht, M. H., Sturtevant, J. M. and Sauer, R. T. (1986) Proteins Struct. Funct. Genet. 1, 43-46 11 Men6ndez-Arias, L. and Argos, P. (1989) J. Mol. Biol. 206, 397-406 12 Yutani, K., Ogasawara, K., Tsujita, T. and Sugino, Y. (1987) Proc. Nat] Acad. Sci. USA 84, 4441-4444 13 Matsumura, M., Yahanda, S., Yasumura, S., Yutani, K. and Aiba, S.
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(1988) Ear. J. Biochem. 171,715-720 14 Kellis, J. T. Jr, Nyberg, K., Sali, D. and Fersht, A. R. (1988) Nature 333, 784-786 15 Matsumura, M., Becktel, W. J. and Matthews, B. W. (1988) Nature 334, 406-410 16 Shortle, D. (1989) J. Biol. Chem. 264, 5315-5318 17 Alber, T., Dao-pin, S., Wilson, K. et al. (1987) Nature 330, 41-46 18 Perry, K. M., Onuffer, J. J., Touchette, N. A. et al. (1987) Biochemistry 26, 2674-2682 19 Ahern, T. J., Casal, J. I., Petsko, G. A. and Klibanov, A. M. (1987) Proc. Natl Acad. Sci. USA 84, 675-679 20 Perry, L. J. and Wetzel, R. (1987) Protein Eng. 1, 101-105 21 Estel, D. A., Graycer, T. P. and Wells, []
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Vaccinia virus: a versatile tool for molecular biologists
22 23 24 25 26 27 28
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J. A. (1985) ]. Biol. Chem. 260, 6518-6521 Imanaka, T., Shibazaki, M. and Takagi, M. (1986) Nature 324, 695-697 Liao, H., McKenzie, T. and Hageman, R. (1986) Proc. Nat] Acad. Sci. USA 83,576-580 Reidharr-Olson, J. F. and Sauer, R. T. (1988) Science 241, 53-57 Shortle, D. and Meeker, A. K. (1989) Biochemistry 28, 936-944 Shortle, D., Meeker, A. K. and Freire, E. (1988) Biochemistry 27, 4761-4768 Hecht, M. H., Sturtevant, J. M. and Sauer, R. T. (1984) Proc. Natl Acad. Sci. USA 81, 5685-5689 Matthews, B. W. and Remington, S. J. (1974) Proc. Natl Acad. Sci. USA 71, 4178-4182 []
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vectors, recent reports have i n d i c a t e d that the vaccinia virus s y s t e m is a promising w a y of p r o d u c i n g significant a m o u n t s of correctly p r o c e s s e d and m o d i f i e d eukaryotic proteins in
mammalian cells 2'3.
Jeffrey N. Miner and Dennis E. Hruby
Vaccinia biology
Continued advances in genetic engineering have made possible the high-level expression of correctly processed cellular, viral and bacterial polypeptides. This article focuses on viral expression vectors and, more specifically, the vaccinia virus expression system. Vaccinia virus has been used to express a variety of proteins with useful immunogenic, catalytic or pharmaceutical properties. We discuss briefly the biology o f v a c c i n i a and its significance in the use of vaccinia as an expression vector, the variety of vaccinia systems currently in use and, finally, we summarize some recent developments which bode well for future applications of vaccinia virus technology. The synthesis of protein p r o d u c t s is, perhaps, the p r i m a r y use of recombinant DNA t e c h n o l o g y in both a p p l i e d and basic biological science. T h e p r o d u c t i o n of enzymes, m e m brane proteins, receptors and neuropeptides in order to make use of their catalytic or p h a r m a c e u t i c a l properties, and the synthesis of viral structural proteins to be u s e d as immunogens for vaccines, all d e p e n d u p o n the availability of efficient e x p r e s s i o n vectors 1. It is clearly i m p o r t a n t to have the o p t i o n
]. N. Miner and D. E. Hruby are at the Center for Gene Research, Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA.
of choosing from a n u m b e r of different e x p r e s s i o n systems, each of w h i c h has a particular i n h e r e n t set of advantages (and disadvantages). The choice of an e x p r e s s i o n system d e p e n d s primarily on the n a t u r e of the protein p r o d u c t being e x p r e s s e d and its anticipated uses. What a m o u n t of protein is required? Does it n e e d to be biologically active? Are eukaryotic or prokaryotic post-translational modifications n e c e s s a r y for p r o p e r activity or solubility? Must the protein be e x p o r t e d from the cell? The answers to these types of questions will d e t e r m i n e largely w h i c h e x p r e s s i o n system is appropriate. Alt h o u g h a large n u m b e r of excellent alternatives exist, i n c l u d i n g phage, bacterial, yeast, viral and cellular
Vaccinia, a large d o u b l e - s t r a n d e d DNA poxvirus, replicates in the c y t o p l a s m of infected ceils in relative isolation from host n u c l e a r functions. U p o n infection, after penetration of the virus into the cytoplasm, the first of two m e m b r a n o u s e n v e l o p e s is r e m o v e d , leaving the core of the virus particle from w h i c h early-gene e x p r e s s i o n c o m m e n c e s . The early-gene class is activated b y a functional t r a n s c r i p t i o n c o m p l e x e n c o d e d by vaccinia and packaged w i t h i n the vaccinia virion. P r o d u c t s from these early genes trigger the release of the viral DNA into the cytoplasm, followed by DNA replication, d o w n - r e g u l a t i o n of earlygene transcription and the initiation of late-gene expression. The late genes e n c o d e structural p r o d u c t s w h i c h f u n c t i o n d u r i n g the formation of m a t u r e virions, as well as e n z y m e s to be packaged for use d u r i n g the initiation of infection. A c o m p l e x series of m o r p h o g e n i c steps leads to the assembly of p r o g e n y viral particles 2. Certain aspects of vaccinia biology make this virus particularly amenable to genetic m a n i p u l a t i o n : • vaccinia is p r o b a b l y a safe viral system to w o r k w i t h as it has b e e n
(~ 1990, Elsevier Science Publishers Ltd (UK)
0167 - 9430/89/$2.00
TIBTECH - J A N U A R Y 1990 [Vol. 8]
Chromatog, Forum 1, 31 37 5 Dean, P. D. G., Johnson W. S. and Middle, F. A. (1985) Affinity chromatography: a practical approach, IRL Press 6 Burgess, R. (ed.) (1987) Protein purification: micro to macro, Alan R. Liss 7 Janson, J. C. (1984) Trends Biotechhal. 2, 31-38 8 Groman, E. V. and Wilchek, M. {1987) Trends Biotechnol. 5, 220-224 9 Cabrera, K. E. and Wilchek, M. {1987) in Protein purification: micro to macro (Burgess, R., ed.), pp. 163-175, Alan R. Liss 10 Cabrera, K. E. and Wilchek, M. (1988) Trends Analyt. Chem. 7, 2, 58-63 11 Cabrera, K. E. and Wilchek, M. (1988) Makromol. Chem. Macromol. Syrup. 19, 145-154 12 Ohlson, S., Gudmundsson, B. M., Wikstrom, P. and Larsson, P. O. (1988) Clin. Chem. 34, 2039-2043 13 Ito, N., Noguchi, K., Kazama, M. and
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Kasai, K. I. (1987) J. Chromatogr. 400, 163 167 Ostlund, C. (1986) Trends in Biotechhal. 4, 288-293 Ohlson, S., Hansson, L., Larsson, P. O. and Mosbach, K. (1978) FEBS Lett. 93, 5-9 Ohlson, S., Hansson, L., Glad, M. and Mosbach, K. (1989) Trends in Biotechno]. 7, 179-186 Wainer, I. W., Jadaud, P., Schonbaum, G. R., Kakodkar, S. V. and Henry, M. P. (1988) Chromatographia 25, 903-907 Waiters, R. R. (1982) J. Chromatogr. 249, 19 28 Horstmann, B. J., Kenny, C. N. and Chase, H. A. (1986) J. Chromatogr. 361, 179-190 Fowell, S. L. and Chase, H. A. (1986) I. Biotechnol. 4, 355-368 Taylor, R. F. (1985) Anal. Chim. Acta. 172, 241-248 Hochu]i, E. (1988) J. Chromutogr. 444, 293-302 Yang, C. and Tsao, G. T. (1982) Adv.
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Protein engineering for thermostability Yoshiaki Nosoh and Takeshi Sekiguchi Studies with small, monomeric proteins indicate that, to some extent, the effects of amino acid substitutions can be predicted. However, conformational and other changes may complicate the prediction. Site-directed mutagenesis is leading both to a better understanding of protein stability and to the production of more stable proteins. A variety of e n z y m e s is n o w u s e d in various fields of b i o t e c h n o l o g y i n d u s t r i e s and, w i t h further d e v e l o p m e n t s in b i o t e c h n o l o g y , the v o l u m e a n d range of e n z y m e s r e q u i r e d will increase. M a n y e n z y m e s isolated f r o m m e s o p h i l e s h a v e b e e n u s e d for b i o t e c h n o l o g y , but these are of limited use due to their d e n a t u r a t i o n on e x p o s u r e to c o n d i t i o n s w h i c h are often e n c o u n t e r e d in i n d u s t r i a l processes (e.g. heat, organic solvents a n d v a r i o u s chemicals). E n z y m e s stable
Y. Nosoh and T. Sekiguchi are at the Department of Fundamental Science, College of Science and Engineering, Iwaki Meisei University, Iwaki, Fukushima 970, Japan.
u n d e r these c o n d i t i o n s are, therefore, required. E n z y m e s f r o m t h e r m o philes are generally m o r e stable t h a n the c o r r e s p o n d i n g ones from m e s o philes, and t h e r m o p h i l e s are the traditional source of stable e n z y m e s . Protein e n g i n e e r i n g t h r o u g h sitedirected m u t a g e n e s i s has b e c o m e a p r o m i s i n g alternative strategy for protein stabilization 1. Stability in a folded p r o t e i n is a balance b e t w e e n the stabilizing (mostly h y d r o p h o b i c ) interactions, a n d the t e n d e n c y t o w a r d s destabilization c a u s e d by the loss of conform a t i o n a l e n t r o p y as the p r o t e i n a d o p t s the u n f o l d e d form. Consequently, the stability of a p r o t e i n m a y be altered b y c h a n g i n g a m i n o acids w h i c h affect either stabilizing
@ 1990, Elsevier Science Publishers Ltd (UK) 0167 9430/89/$2.00 -
Biochem. Bioeng. 25, 19-41 24 Nishikawa, A. H., Bailon, P. and Ramel, A. H. (1976) J. Macromol. Sci. Chem. A10, 149-190 25 Kennedy, J. F. and Barnes, J. A. (1983) f Chromatogr. 281, 83-93 26 Fowell, S. L. and Chase, H. A. (1986) J. Biotechnol. 4, 1-13 27 Wu, D. and Walters, R. R. (1988) J. Chromatogr. 458, 169-174 28 Narayanan, S. R., Knochs, S. J. and Crane, L. J. J. Chromatogr. (in press) 29 Chase, H. A. (1988) Makromo]. 17, Chem., Macromol. Syrup. 467-482 30 Secher, D. S. and Burke, D. C. (1980) Nature 285,446-450 31 Zimmerman, T. S. and Fulcher, S. (1982) US Patent No. 4361509 32 Dodd, I., Jalalpour, S., Southwick, W. et al. (1986) FEBS Lett, 209, 13-17 33 Bonnerjea, J., Oh, S., Hoare, M. and Duni]l, P. (1986) Biotechnology 4, 954-958 34 Katoh, S. (1987) TrendsBiotechnol. 5, 328-331
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interactions in the folded protein, or destabilizing interactions in the unfolded form, or b o t h (Table 1).
Mechanisms and control of protein stability This r e v i e w s h o w s h o w p r o t e i n e n g i n e e r i n g can be u s e d b o t h to explore the m o l e c u l a r basis of protein stability a n d to m o d i f y p r o t e i n stability (Fig. 1). En tropic stabi]ization The d e n a t u r a t i o n of p r o t e i n s can be w r i t t e n s i m p l y as N , ~ U--* X w h e r e the folded state (N) is reversibly c h a n g e d to the u n f o l d e d state (U), (reversible d e n a t u r a t i o n or unfolding), a n d U i r r e v e r s i b l y to X (irreversible inactivation). N has a l o w e r free energy t h a n U. A n y t h i n g that stabilizes N and/or decreases the destabilizing forces of U will increase the free energy change for unfolding, a n d t h e r e b y stabilize the protein. T h e irreversible i n a c t i v a t i o n d e p e n d s on the c o n f o r m a t i o n of the u n f o l d e d f o r m (U): m o r e c o m p a c t e d f o r m s of U will be i n a c t i v a t e d m o r e s l o w l y a n d will thus stabilize the protein.
TIBTECH- JANUARY 1990 [Vol, 8]
17
~Table I Factors to consider in stabilizing proteins a
inactivation than the wild-type protein even though, in most cases, the disulfide bond strengths were high enough to stabilize the mutant protein against thermal inactivation. This suggests that either the cysteine substitution and/or the crosslinkformation disrupted other stabilizing interactions, increasing the rate of unfolding and, therefore, the rate of irreversible inactivation.
Interaction or bond Disulfide bond Hydrogen bond Electrostatic interaction Hydrophobic interaction Conformational factors Stability of secondary structure Compact packing Conformational flexibility Internal hydrophobicity Entropic stability Protection Deamination of carboxyamide Oxidation of sulfhydryl groups Intramolecular S-H/S-S exchange Oxidation of tryptophan or methionine
• Dihydrofolate reductase
aThese factors were deduced by comparing the structures of proteins of different stabilities (proteins isolated from mesophiles and thermophiles, wild-type and aminoacid-substituted proteins, wild-type and chemically modified proteins).
Disulfide bonds Disulfide bonds in proteins restrict the degree of freedom for the unfolded state U and thereby stabilize the folded state N (Ref. 2). The first type of protein stabilization performed by genetic manipulation was the introduction of disulfide bonds. One or two amino acids in a protein were replaced with cysteines; a disulfide bond forms in vivo or in vitro. If the introduced disulfide bond caused no, or little tertiary structural change, the crosslink stabilizes the protein. Disulfide bonds have been engineered into T4 lysozyme 3,4, subtilisin 5'6, dihydrofolate reductase 7 and the )~repressor 8, with stabilization occurring in some cases but not others.
• Subtilisin
Subtilisin naturally contains neither disulfide bonds nor free cysteine residues. Disulfide bonds which formed between the introduced Cys 22 and Cys 87 or between the introduced Cys 24 and Cys 87 did not stabilize the protein 5. A protein with the former of these types of bond was less thermostable than the wildtype protein 5. Recently, five singledisulfide-bonded forms of subtilisin were produced 6. None was more stable against irreversible thermal
A disulfide bond was introduced into E. coli dihydrofolate reductase by replacing Pro 39 with Cys and crosslinking this with the natural Cys 85 (Ref. 7). The crosslinked enzyme was about 7.56 kJmo1-1 more stable than the non-crosslinked or the wild-type enzyme, with respect to reversible denaturation by guanidine hydrochloride. This may indicate that the increased free energy change for the unfolding of the crosslinked enzyme is due to the entropic stabilization of the unfolded form. However, the crosslinked enzyme was not more resistant to thermal inactivation than the wild-type protein. The X-ray crystal structure analyses showed that the flexibility of the region containing residues 80-90 increases on crosslink formation. The disulfide bond appears to be too flexible to restrict the flexible motion of the region. This
~Fig. 1 Protein
']
• Lysozyme
Isoleucine (Ile) at position 3 in T4 phage lysozyme was replaced by Cys, followed by in vitro oxidation 3 (Fig. 2). The crosslink between the introduced Cys 3 and native Cys 97 imparted stability against both reversible thermal unfolding and irreversible thermal inactivation 4. The stability against reversible unfolding of the crosslinked enzyme was possibly due to the entropic effect on the unfolded state of the protein (thermodynamic stabilization). Its stabilization against irreversible inactivation was considered to be due to the unfolded state of the crosslinked enzyme being more compact in structure than the native enzyme (kinetic stabilization).
Protein stabilization
I I
Mechanism of protein stability
Relationship of amino acid substitution to controlling protein stability and understanding its mechanisms. Proteins are stabilized through site-directed mutagenesis based on the mechanism of protein unfolding. Site-directed mutagenesis is also employed to reveal the mechanism of stabilization (e.g. comparison of X-ray crystal structures and the thermodynamic and kinetic evaluations in the wild-type and engineered proteins).
18
TIBTECH - JANUARY 1990 [Vol. 8]
--Fig. 2
A82P
i23
may be w h y the cross-linked protein is not more stable against thermal inactivation.
Other amino acid substitutions Glycine has no ~-carbon and allows more conformational freedom in a polypeptide chain than alanine. Thus, changing Gly to Ala might increase protein stability by reducing the entropy loss on unfolding. Similarly, substituting proline for other amino acids might decrease the entropy of polypeptide backbone. T4 lysozyme 9 and X repressor 1° have been stabilized in this way by sitedirected mutagenesis. In T4 lysozyme 9, two kinds of amino acid substitution (Gly 77 to Ala and Ala 82 to Pro) were performed (Fig. 2). The Gly 77 --~ Ala substitution produced a 1.68 kJ mo1-1 increase in stability (for the reversible thermal denaturation): Gly 77 is in an o~-helix, so the increased sta bil ity might be attributed to better t i c l ix formation ~a. However, entropic stabilization could also be responsible for the increase in protein stability 9. The observed increase in thermodynamic stabilization is about half the value expected from theory. The substitution of Ala for Gly produces localized changes in the protein structure which may affect the entropic contribution to the free energy change for unfolding. The Ala82 --+ Pro substitution also increased the stability of T4 lysozyme by 3.6 kJ mo1-1 (Ref. 9). In the X repressor, the replacement of two Gly residues in e~-helices increased the free energy change for reversible unfolding by 1.68-3.78 kJ tool -~ (Ref. 10). Since the two substitution sites are in o~-helices, as in the case of T4 lysozyme 4, the increased stability could also be ascribed to the entropic effect. Entropic stabilization might be a general strategy for enhancing protein stability. Altering stabilizing factors We have discussed stabilization by reduction of destabilizing interactions in the unfolded form of the protein. The other way to increase stabilization is to increase t h e number of, or strengthen, the stabilizing interactions in the folded protein. In tern al hydroph obicity
Protein folding is driven by the hydrophobic properties of nonpolar
I
G77A
1251 -r__L,1~~ " ~, 1 ~ (313 .~ '~ /
90
\+,. T157AA
'"'
Gin IO5
C975
I
Glu 22 Thr ~ k Gly 21 ~ TyrO25 Lys
i
Ser
20 4
GIn6~
I3AA
/
T
3o
C54V The X-ray crystal structure of T4 lysozyme. Some of the amino acid substitutions that have been made (including those described in the text) are indicated by arrows: 13AA, lie 3 to various amino acids; C54V, Cys 54 to Val; C97T, Cys 97 to Thr; G77A, Gly 77 to Ala; A82P, Ala 82 to Pro; T157AA, Thr 157 to various amino acids. The structure is adapted from Ref. 28.
amino acid residues 2. Strengthening the internal hydrophobicity in a protein by amino acid substitution may increase protein stability. Glutamine (Gin), at position 49 in the interior of the o:-subunit of E. c01i tryptophan synthetase, was replaced with other amino acids 12. The stability of each resulting protein was calculated from the free energy changes between the native and unfolded proteins (thermodynamics) and between the native and transition states of unfolding (kinetics). Both measurements of stability correlated well with the hydrophobicity of the substituted amino acids, except where the proteins contained introduced aromatic amino acid residues. The authors concluded that increasing the internal hydrophobicity per se stabilized the protein thermodynamically and kinetically. Furthermore, they concluded that extra energy which resulted from increased hydrophobicity might also contribute to increased stability ~2.
The stabilities of the proteins with introduced aromatic amino acid residues were lower than w o u l d be predicted from consideration of the hydrophobicity of the residues 12. This may be due to the conformational distortion around the substitution site. A similar result was obtained with kanamycin nucleotidyltransferase 13. The reverse experiment was performed with barnase14: the hydrophobicity of the core was reduced. Ile at position 96 of barnase was replaced by Val or Ala (Ref. 14), and the free energy change for reversible urea denaturation of the protein was reduced by 5.0 or 16.8 kJ mo1-1, respectively. These stability changes were larger in magnitude than those expected simply on the basis of reduced hydrophobicity. The authors suggested that the substitution of Ile with Val or Ala created a cavity with the size of one or three methylene carbons, respectively, which destabilized the protein.
TIBTECH - J A N U A R Y 1990 [Vol. 8]
In contrast to the results obtained with the three previous examples TM 14, there was good agreement between the free energy change for reversible thermal inactivation of T4 lysozyme and its derivative proteins (substitutions of Ile 3; Fig. 2), and hydrophobicity of the side chains substituted ~5. Results with all four proteins, however, indicate that increasing internal hydrophobicity could be a useful strategy in stabilizing proteins.
Hydrogen bonding Addition or strengthening of noncovalent interactions other than hydrophobic interactions has been considered as a way of stabilizing proteins 1. However, high resolution protein structures usually do not reveal obvious sites where amino acid substitutions could strengthen a hydrogen bond or electrostatic interaction through closer contact between side chains 16. On the other hand, it is known that destruction of a hydrogen bond by a single amino acid substitution often reduces protein stability. When Thr 15 7 in T4 lysozyme was replaced by other amino acids, there was a strong correlation between a major reduction in stability and a replacement residue's inability to form a hydrogen bond with the amide of Asp 159 (which interacts with the hydroxyl group of Thr 157 in the wild-type protein) 17 (Fig. 2). A similar decrease in protein stability was reported with dihydrofolate reductase when Val was substituted for Thr 113 (Ref. 18). These results indicate that, if it were possible rationally to design additional hydrogen bonding, this could stabilize proteins.
Protection against chemical destabilization Irreversible inactivation of some proteins is triggered by deamination of aspmagine residues followed by hydrolysis of the peptide bonds at the aspartic acid residues, or by cleavage at the ~-position of cysteine, or by intramolecular thio/disulfide exchange, or by destruction of methionine residuesL Substituting for asparagine, cysteine and methionine might prevent such inactivation. Some evidence to support this was obtained in studies on yeast triosephosphate isomerase, a dimeric protein containing twelve asparagine residues per monomer, three of them
19
at the interfacial surface. When two of the three surface Asn residues (14 and 78) were altered to Thr and Ile, respectively 19, the half-life of the enzyme, relative to that of the wildtype enzyme at 100°C, was doubled. Similarly, in T4 lysozyme, when the natural Cys 54 and Cys 97 residues were mutated to Val and Thr, respectively, the enzyme was stabilized 2° (Fig. 2). In subtilisin, H202 inactivation was prevented by replacing Met 222 with Ala or Ser (Ref. 21).
Stabilization without knowledge of tertiary structure In almost all the targets of protein engineering, X-ray crystal structures are known. Even when the tertiary structure is not at hand, however, stabilization through site-directed mutagenesis may be possible, provided that a homologous protein of known structure is available. Using this strategy, a neutral protease from Bacillus stearothermophilus was stabilized using thermolysin as a model protein 22. Three kinds of substitution, Gly 61 to Ala, Gly 144 to Ala and Thr 66 to Ser, were examined. The first two stabilized the protein (possibly due to an increase in internal hydrophobicity and stabilization of o:-helix), and the last destabilized it al. Double and triple substitutions suggested that the stabilization effects were not additive, but cooperative. If neither the tertiary structures of a particular protein nor of any homologous model protein are available, another strategy - biological thermal adaptation - could be considered. A plasmid (pUB110)-borne kanamycin nucleotidyltransferase gene, originally isolated from a mesophile, was exposed to elevated temperatures in a host thermophilic bacterium 23. This treatment induced two kinds of mutation, Asp 80 to Tyr and Thr 130 to Lys. The mutation occurred naturally in a thermophile. The two substituted proteins are more thermostable than the original mesophilic protein. The role of Tyr 80 in thermostabilization was examined thermodynamically and kinetically13. Concluding remarks The work discussed demonstrates the contribution to protein stability of both stabilizing 12-15 and destabilizing 3-8 interactions. A few, or even
just a single, amino acid replacement may modify a protein's stability. It is too early yet for any general mechanisms of protein stabilization to have been proposed. Site-directed mutagenesis has been used to provide information on the kinds of interactions (or bonds) which should be considered. With small monomer proteins such as T4 lysozyme4,15, subti]isin 6, ~.repressor 24 and nuclease 25, it is relatively easy to compare the X-ray crystal structures and the energetics of denaturation of the wild-type and substituted proteins. Both structural and energetic analyses are important in correlating substitutions with stability since, in most cases, the changes in stability are greater than or less than those expected from simple consideration of the changes made; conformational and other factors must contribute to these deviations from prediction. The role of water in the energetics of denaturation should also be considered 26. If the X-ray crystal structure of the protein to be engineered is not available, appropriate amino acid substitutions can be guided in using the known structure of a homologous protein 22 and known differences between proteins in mesophiles and thermophiles 11. When no model protein is available, the strategies of thermal mutation 23 and random chemical mutation 27 could be attempted. In oligomeric proteins, subunit dissociation should also be considered as part of the inactivation process. Appropriate amino acid substitutions on the interfacial surface can stabilize the protein TM. Protein engineering, then, is a promising tool, not only for stabilizing proteins, but also for dissecting the mechanism of protein unfolding.
References 1 Nosoh, Y. and Sekiguchi, T. (1988) Biocatalysis 1, 257-273 2 Schulz, G. E. and Schirmer, R. H. (1979) Principles of protein structure, Springer-Verlag 3 Perry, L. ]. and Wetzel, R. (1984) Science 226,555-557 4 Wetzel, R., Perry, L. J., Baase, W. A. and Becktel, W. J. (1988) Proc. Notl Acad. Sci. USA 85,401-405 5 Wells, J. A. and Powers, D. B. (1986) J. Biol. Chem. 261, 6564-6570 6 Mitchinson, C. and Wells, J. A. (1989)
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Biochemistry 28, 4807-4815 7 Villafranca, J. E., Howell, E. E., Oatley, S. J., Xuong, N. and Kraut, J. (1987) Biochemistry 26, 2182-2189 8 Sauer, R. T., Hehir, K., Stearman, R. S. et al. (1986) Biochemistry 25, 5992-5998 9 Matthews, B. W., Nicholson, H. and Becktel, W. J. (1987) Proc. Natl Acad. Sci. USA 84, 6663-6667 10 Hecht, M. H., Sturtevant, J. M. and Sauer, R. T. (1986) Proteins Struct. Funct. Genet. 1, 43-46 11 Men6ndez-Arias, L. and Argos, P. (1989) J. Mol. Biol. 206, 397-406 12 Yutani, K., Ogasawara, K., Tsujita, T. and Sugino, Y. (1987) Proc. Nat] Acad. Sci. USA 84, 4441-4444 13 Matsumura, M., Yahanda, S., Yasumura, S., Yutani, K. and Aiba, S.
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(1988) Ear. J. Biochem. 171,715-720 14 Kellis, J. T. Jr, Nyberg, K., Sali, D. and Fersht, A. R. (1988) Nature 333, 784-786 15 Matsumura, M., Becktel, W. J. and Matthews, B. W. (1988) Nature 334, 406-410 16 Shortle, D. (1989) J. Biol. Chem. 264, 5315-5318 17 Alber, T., Dao-pin, S., Wilson, K. et al. (1987) Nature 330, 41-46 18 Perry, K. M., Onuffer, J. J., Touchette, N. A. et al. (1987) Biochemistry 26, 2674-2682 19 Ahern, T. J., Casal, J. I., Petsko, G. A. and Klibanov, A. M. (1987) Proc. Natl Acad. Sci. USA 84, 675-679 20 Perry, L. J. and Wetzel, R. (1987) Protein Eng. 1, 101-105 21 Estel, D. A., Graycer, T. P. and Wells, []
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Vaccinia virus: a versatile tool for molecular biologists
22 23 24 25 26 27 28
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J. A. (1985) ]. Biol. Chem. 260, 6518-6521 Imanaka, T., Shibazaki, M. and Takagi, M. (1986) Nature 324, 695-697 Liao, H., McKenzie, T. and Hageman, R. (1986) Proc. Nat] Acad. Sci. USA 83,576-580 Reidharr-Olson, J. F. and Sauer, R. T. (1988) Science 241, 53-57 Shortle, D. and Meeker, A. K. (1989) Biochemistry 28, 936-944 Shortle, D., Meeker, A. K. and Freire, E. (1988) Biochemistry 27, 4761-4768 Hecht, M. H., Sturtevant, J. M. and Sauer, R. T. (1984) Proc. Natl Acad. Sci. USA 81, 5685-5689 Matthews, B. W. and Remington, S. J. (1974) Proc. Natl Acad. Sci. USA 71, 4178-4182 []
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vectors, recent reports have i n d i c a t e d that the vaccinia virus s y s t e m is a promising w a y of p r o d u c i n g significant a m o u n t s of correctly p r o c e s s e d and m o d i f i e d eukaryotic proteins in
mammalian cells 2'3.
Jeffrey N. Miner and Dennis E. Hruby
Vaccinia biology
Continued advances in genetic engineering have made possible the high-level expression of correctly processed cellular, viral and bacterial polypeptides. This article focuses on viral expression vectors and, more specifically, the vaccinia virus expression system. Vaccinia virus has been used to express a variety of proteins with useful immunogenic, catalytic or pharmaceutical properties. We discuss briefly the biology o f v a c c i n i a and its significance in the use of vaccinia as an expression vector, the variety of vaccinia systems currently in use and, finally, we summarize some recent developments which bode well for future applications of vaccinia virus technology. The synthesis of protein p r o d u c t s is, perhaps, the p r i m a r y use of recombinant DNA t e c h n o l o g y in both a p p l i e d and basic biological science. T h e p r o d u c t i o n of enzymes, m e m brane proteins, receptors and neuropeptides in order to make use of their catalytic or p h a r m a c e u t i c a l properties, and the synthesis of viral structural proteins to be u s e d as immunogens for vaccines, all d e p e n d u p o n the availability of efficient e x p r e s s i o n vectors 1. It is clearly i m p o r t a n t to have the o p t i o n
]. N. Miner and D. E. Hruby are at the Center for Gene Research, Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA.
of choosing from a n u m b e r of different e x p r e s s i o n systems, each of w h i c h has a particular i n h e r e n t set of advantages (and disadvantages). The choice of an e x p r e s s i o n system d e p e n d s primarily on the n a t u r e of the protein p r o d u c t being e x p r e s s e d and its anticipated uses. What a m o u n t of protein is required? Does it n e e d to be biologically active? Are eukaryotic or prokaryotic post-translational modifications n e c e s s a r y for p r o p e r activity or solubility? Must the protein be e x p o r t e d from the cell? The answers to these types of questions will d e t e r m i n e largely w h i c h e x p r e s s i o n system is appropriate. Alt h o u g h a large n u m b e r of excellent alternatives exist, i n c l u d i n g phage, bacterial, yeast, viral and cellular
Vaccinia, a large d o u b l e - s t r a n d e d DNA poxvirus, replicates in the c y t o p l a s m of infected ceils in relative isolation from host n u c l e a r functions. U p o n infection, after penetration of the virus into the cytoplasm, the first of two m e m b r a n o u s e n v e l o p e s is r e m o v e d , leaving the core of the virus particle from w h i c h early-gene e x p r e s s i o n c o m m e n c e s . The early-gene class is activated b y a functional t r a n s c r i p t i o n c o m p l e x e n c o d e d by vaccinia and packaged w i t h i n the vaccinia virion. P r o d u c t s from these early genes trigger the release of the viral DNA into the cytoplasm, followed by DNA replication, d o w n - r e g u l a t i o n of earlygene transcription and the initiation of late-gene expression. The late genes e n c o d e structural p r o d u c t s w h i c h f u n c t i o n d u r i n g the formation of m a t u r e virions, as well as e n z y m e s to be packaged for use d u r i n g the initiation of infection. A c o m p l e x series of m o r p h o g e n i c steps leads to the assembly of p r o g e n y viral particles 2. Certain aspects of vaccinia biology make this virus particularly amenable to genetic m a n i p u l a t i o n : • vaccinia is p r o b a b l y a safe viral system to w o r k w i t h as it has b e e n
(~ 1990, Elsevier Science Publishers Ltd (UK)
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