- Email: [email protected]
[14] Expression as trpE fusion
[ 14]
EXPRESSION AS trpE FUSION
161
thermore, the authors would like to thank Stephen Anderson, Cara Berman Marks, Paul Carter, and Mark Hude for critical comments on the manuscript. Paul Carter is also acknowledged for permitting reference to results prior to their publication. Carol Morito is greatly acknowledged for the construction of Figs. ! and 3. Finally, we thank KabiGen AB, the Swedish National Board of Technical Development, and the Swedish Natural Science Research Council for financial support in several projects which have made the development of the SPA fusion concept possible.
[ 14] E x p r e s s i o n
as trpE Fusion
By DANIEL G. YANSURA Although expression of a fusion protein is not as elegant as a more direct approach, often it is the easiest and quickest means of achieving expression of a particular protein. For m a n y small proteins or peptides, fusions appear to be the only practical way to accumulate useful levels of protein within the cell against losses due to proteolysis. This idea was first established for expression of somatostatin, a small 14-amino acid peptide; ~ however, much larger proteins may also benefit from this principle. 2 A second advantage of a fusion over direct expression is that problems due to poor translation initiation can be essentially avoided, a Overcoming such problems can be tedious and, for m a n y situations, a fusion protein may satisfy all the particular needs of the experimenter. Over the last several years, trpE or trpLE fusions have been used quite successfully, with more than 30 proteins or peptides reportedly expressed. The high level of expression of most of these fusions can be attributed to two factors: the relatively strong4 but controllable trp promoter, and the insoluble nature of the trpE or trpLE fusions, which usually leads to proteolytic protection within refractile bodies. In terms of expression, trpE and trpLE fusions appear to be quite comparable, the main difference being in the level of transcriptional control. While the trpE fusions are under both repressor and attenuation control, the trpLE fusions retain only repressor control. The trpLE fusions have deletions between the end of the leader and distal parts of the trpE polypeptide and have been derived l K. Itakura, T. Hirose, R. Crea, A. D. Riggs,H. L. Heyneker,F. Bolivar,and H. W. Boyer, Science 198, 1056 (1977). 2A. R. Davis, D. P. Nayak, M. Ueda, A. U Hiti, D. Dowbenko,and D. G. Kleid,Proc.Natl. Acad. Sci. U.S.A. 78, 5376 (1981). 3 B. E. Schoner, H. M. Hsiung,R. M. Belagaje,N. G. Mayne,and R. G. Schoner,Proc.Natl. Acad. Sci. U.S.A.81, 5403 (1984). 4 H. A. de Boer, L. J. Comstock, D. G. Yansura, and H. L. Heyneker, in "Promoter Structure and Function" (R. L. Rodriguezand M. J. Chamberlain,eds.), p. 462. Praeger, New York, 1982. METHODS IN ENZYMOLOGY, VOL. 185
Copyright© 1990by AcademicPress,Inc. All rights&reproductionin any formreserved.
162
EXPRESSION IN g . c . o f f
pWT111
pWT121
[14]
trpE 7 Hindlll AAA CCG ACT] CCA AGC TT Lys Pro Thr
trpE 7 Hindlll AAA CCG ACTICCA AGC TCC AAG C-FI E
Lys
pWT131
Pro
Thr
trpE 7 Hindlll AAA CCG ACTJCCA AGC TCC AAG CTC CAA GCT T Lys
Pro
Thr
FIG. l. Partial coding sequence of the trpE protein followed by HindlII restriction sites in three different reading frames for expression vectors pWT111, pWT121, and p W T I 3 1 ) 6
Numbers represent the amino acid positions of the trpE protein.
from the deletions trpALE 1413 and trpALE 1417. 5 A more detailed review on the trp promoter and its use in expressing cloned genes was published by Nichols and Yanofsky in 1983. 6 Designing Fusion There are two major considerations when designing a trpE or trpLE fusion: the length of trpE polypeptide and the junction between the trpE polypeptide and the protein or peptide of interest. The length of trpE can be a significant factor in determining the levels of expression. In general, the longer trpE fusions give higher levels of expression. 7,s This may not be just a matter of increasing the mass of the total fusion, but probably involves better precipitation of the longer fusions within the cell, thus avoiding proteolysis. Comparison of relatively short versus long trpE lengths demonstrates this tendency whereby the shorter fusions are more soluble than the larger o n e s . 9,1° The length of trpE or trpLE polypepfide necessary to achieve maximum expression is probably in the range of 100-300 amino acids. Besides expression levels, however, producing a more soluble fusion may be a more important factor, depending on the ultimate use of the fusion. If one is interested in some enzymatic activity associated with the protein of interest, a shorter, more soluble fusion will almost certainly be more useful. For maximal enzymatic activity in gens G. F. Miozzari and C. Yanofsky, J. Bacteriol. 133, 1457 (1978). 6 B. P. Nichols and C. Yanofsky, this series, Vol. 101, p. 155. 7 R. Derynck, A. B. Roberts, M. E. Winlder, E. Y. Chen, and D. V. Goeddel, Cell 38, 287 (1984). s p. R. Szoka, A. B. Schreiber, H. Chart, and J. Murthy, DNA 5, 11 (1986). 9 N. Tanese, M. Roth, and S. P. Golf, Proc. Natl. Acad. Sci. U.S.A. 82, 4944 (1985). ~°C. A. Chlan, C. Coulter, and L. T. Feldman, J. Virol. 61, 1855 (1987).
[ 14]
EXPRESSION AS trpE FUSION
163
trpE 517 EcoRI Pstl EcoRI CAT GCA CAG[GAA TTC TGC AGA A'IF C His Ala Gin FIo. 2. Partial coding sequence for the trpLE protein of pmasmid pNCV with the EcoRI and PstI restriction sites available for cloning. The number represents the amino acid position in the trpE protein and not that of the LE. Note that this LE consists of the first nine amino acids of the trp leader fused to amino acids 339-517 of the trpE polypeptide.
eral, the lengths of trpE polypeptide in the fusions have been in the range of 17-42 amino acids, 9- I I although much larger fusions also retained some activity. The junction between the trpE polypeptide and the protein of interest can be designed to allow cleavage of the fusion at this point. There are a number of cleavable fusions reported using both chemical 7,s,~2 and enzymatic ~3,14means. Because many of the trpE fusion proteins are soluble only in the presence of chaotropic agents, cleavage by chemical means, if feasible, is usually preferred. Enzymatic cleavage, however, can be used in some cases if solubility problems can be overcome. Usually, the shorter trpE fusions are more amenable to this type of cleavage. Plasmids and D N A Construction The trp promoter and the gene encoding the fusion protein are usually carried on a multicopy plasmid for maximum expression. TrpR÷strains of Escherichia coli should be used in plasmid construction and expression experiments to help ensure plasmid stability; 6 strains HB101 and MM294 have been commonly used. There are a number of plasmids that can be used as a source of the trp promoter and trpE or trpLE coding sequences. One simply ligates the gene for the protein of interest, in frame, to some point in the trpE gene, using a convenient restriction site. Synthetic DNA is now readily available and can be used to link nonoverlapping restriction sites in the correct reading frame and also provide the desired junction. The complete nucleotide sequence of the trp operon has been published, ~5 and can be used to generate restriction sites within the trpE gene. " I. Sadowski and T. Pawson, Oncogene 1, 181 (1987). ~2T. C. Furman, J. Epp, H. M. Hsiung, J. Hoskins, G. L. Long, L. G. Mendelsohn, B. Schoner, D. P. Smith, and M. C. Smith, Bio/Technology 5, 1047 (1987). 13G. Allen, M. D. Winther, C. A. Henwood, J. Beesley, L. F. Sharry, J. O'Keefe, J. W. Bennett, R. E. Chapman, D. E. Hollis, B. A. Panaretto, P. Van Dooren, R. W. Edols, A. S. lnglis, P. C. Wynn, and G. P. M. Moore, J. Biotechnol. 5, 93 (1987). t4 T. Imai, T. Cho, H. Takamatsu, H. Hori, M. Saito, T. Masuda, S. Hirose, and K. Murakami, J. Biochem. 100, 425 (1986). ~5C. Yanofsky, T. Platt, I. P. Crawford, B. P. Nichols, G. E. Christie, H. Horowitz, M. Van Cleemput, and A. M. Wu, Nucleic Acids Res. 9, 6647 (1981).
164
EXPRESSION IN E. coli
[ 14]
trpE 323 Smal BamHI Sail Pstl Hindlll ATI" GAG ATC]CCC GGG GAT CCT CTA GAG TCG ACC TGC AGC CCA AGC 1-I-A FIG. 3. Sequence of the multiple cloning region of plasmid pATH2? s The number represents the amino acid position of the trpE protein.
Some plasmids, the so-called trpE expression vectors, have been rather useful in constructing specific fusion expression plasmids. These vectors have convenient restriction sites engineered at various distances into the trpE gene, often with more than one coding phase available for cloning. For short trpE fusions, vectors pWT 11 l, pWT 12 l, and pWTl 31 have been constructed.~6 These contain the trp promoter, leader, and coding sequence for the first seven amino acids of the trpE, followed by a HindIII restriction site in all three reading frames (Fig. 1). For intermediate length fusions, pNCV ~7 which carries the deletion trpALE1413,5 can be used. This plasmid has EcoRI and PstI restriction sites, engineered at the end of the coding sequence for 188 amino acids of trpLE. This trpLE consists of the first nine amino acids of the leader fused to amino acids 339-517 of the trpE polypeptide (Fig. 2). For longer trpE fusions, the vector pATH2 has been extensively used. ~s This vector contains the trp promoter, leader, and coding sequence for the first 323 amino acids of trpE, followed by the multiple cloning region ofpUC12 (Fig. 3). trpE and trpLE fusion expression plasmids can be maintained in trpR+ strains provided sufficient tryptophan is present in the media. In minimal media, cultures containing these plasmids should normally be supplemented with tryptophan to approximately 20/zg/ml.
trp Induction Induction of the trpE or trpLE fusion generally involves tryptophan starvation of bacterial cultures containing the expression plasmids. Because some plasmids are unstable in their host, it is advisable to start with bacterial cells recently transformed with the appropriate expression plasmids. Such cells are usually grown overnight to saturation in a rich medium, such as LB, ~9 containing the appropriate antibiotic. The overnight
16W. Tacon, N. Carey, and S. Emtage, Mol. Gen, Genet. 177, 427 (1980). 17 T. Maniatis, E. F. Fritsch, and J. Sambrook, in "Molecular Cloning: A Laboratory Manual," p. 426. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. ts C. L. Dieckmann and A. Tzagoloff, J. Biol. Chem. 260, 1513 (1985).
[14]
EXPRESSION AS trpE FUSION
165
12 !!~i~i~iii~ii~!i~! !il ~¸~¸~' ¸ ~zi~ii~iiiiiiiiiii~iiii~!iiiii!i ~'
FIG. 4. SDS-polyacrylamide gel analysis of the expression o f a trpLE fusion. Escherichia coli, strain W3110, containing plasmid pBR322 and a plasmid encoding a trpLE-relaxin A chain fusion, was grown overnight in LB. The cultures were diluted 100-fold into minimal medium containing 3-indoleacrylic acid and grown for 7 hr. Samples of these cultures equivalent to 1 ml with an absorbance at 600 nm of l were then removed for analysis. The pelleted cells were resuspended in 250 gl of 0.1 M 2-mercaptoethanol, 2% (w/v) SDS, 60 m M Tris (pH 6.8), 10% (v/v) glycerol, heated to 95 ° for 2 min, and loaded onto an SDSpolyacrylamide gel. After electrophoresis, the gel was stained with Coomassie brilliant blue. Lane l shows the control culture containing pBR322. Lane 2 shows the culture containing the trpLE-relaxin A chain fusion with an arrow pointing to the fusion protein band.
culture is then diluted 25 to 50-fold into M919 with 0.5% (w/v) casamino acids and the appropriate antibiotic. After 1 - 2 hr growth, 3-indoleacrylic acid 2° is added to a final concentration of 25 /zg/ml. Growth is then continued for 2 - 5 hr, at which time samples may be removed to assay for the particular fusion protein. Samples removed from induced cultures are usually analyzed by S D S polyacrylamide gels (Fig. 4). Expression levels oftrpE or trpLE fusions are usually quite high, and the fusion can then be visualized after staining with Coomassie brilliant blue. For many fusions, the appearance of a new protein band on the gel after staining is the only way to verify and estimate the level of expression. One should therefore include on the gel samples of ~9j. H. Miller, in "Experiments in Molecular Genetics," p. 431. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972. 20 W. F. Doolittle and C. Yanofsky, J. Bacteriol. 95, 1283 0968).
EXPRESSION IN E. ¢oli
166
[ 1 5]
induced cultures containing the parent trpE or trpLE expression plasmid as a control. If the fusion protein is to be used to raise antibodies against the protein of interest, one can purify the fusion by preparative SDS gel electrophoresis once the prospective protein band has been identified.2~-23 Comments Occasionally, one cannot successfully express a protein of interest as a
trpE or trpLE fusion. Usually, the bacterial cultures for these fusions stop growing completely when induced, and no new protein band can be seen on an SDS-polyacrylamide gel analysis of a total cell lysate. The hepatitis B surface antigen, for example, when fused to 190 amino acids of trpLE, displayed this type of expression problem. Removal of the last 24 amino acids of the hepatitis surface antigen, however, restored expression to a high level.24 It is not clear what the nature of this problem is, but for many fusions such trimming of the protein of interest may be an acceptable alternative. 21 L. K. Pape, T. J. Koerner, and A. Tzagoioff, J. Biol. Chem. 260, 15362 (1985). 22 D. G. Kleid, D. Yansura, B. Small, D. Dowbenko, D. M. Moore, M. J. Grubman, P. D. McKercher, D. O. Morgan, B. H. Robertson, and H. L. Baehrach, Science 214, 1125 (1981). 23 I. Sadowski, J. C. Stone, and T. Pawson, Mol. Cell. Biol. 6, 4396 (1986). 24 D. Yansura and D. G. Kleid, unpublished observation (1980).
[ 15] E n g i n e e r i n g
E s c h e r c h i a coli t o S e c r e t e H e t e r o l o g o u s Gene Products
B y J O A N A . S T A D E R a n d T H O M A S J. SILHAVY
The development of recombinant DNA technology has revolutionized the way we approach research problems in almost all biological systems, but its use in applied technology still favors only a few organisms and cell types. Certainly in terms of prokaryotes, Escherichia coli is one of the most favored organisms for many applications because of the ease with which the genome can be manipulated. Controlling factors such as promoter efficiency and regulation, transformation efficiency, protease levels, and the secretion of protein into the growth medium, is within our current capability. Still, despite all the progress, in many instances success is achieved empirically and the practice can best be described as an art. In this article, we attempt to bring to light some of the problems encountered in secreting heterologous proteins from E. coli and discuss some of the successes that have been achieved to date. METHODS IN ENZYMOLOGY, VOL. 185
Copyright© 1990by AcademicPress,Inc. All riots of reproductionin any formreserved.
EXPRESSION AS trpE FUSION
161
thermore, the authors would like to thank Stephen Anderson, Cara Berman Marks, Paul Carter, and Mark Hude for critical comments on the manuscript. Paul Carter is also acknowledged for permitting reference to results prior to their publication. Carol Morito is greatly acknowledged for the construction of Figs. ! and 3. Finally, we thank KabiGen AB, the Swedish National Board of Technical Development, and the Swedish Natural Science Research Council for financial support in several projects which have made the development of the SPA fusion concept possible.
[ 14] E x p r e s s i o n
as trpE Fusion
By DANIEL G. YANSURA Although expression of a fusion protein is not as elegant as a more direct approach, often it is the easiest and quickest means of achieving expression of a particular protein. For m a n y small proteins or peptides, fusions appear to be the only practical way to accumulate useful levels of protein within the cell against losses due to proteolysis. This idea was first established for expression of somatostatin, a small 14-amino acid peptide; ~ however, much larger proteins may also benefit from this principle. 2 A second advantage of a fusion over direct expression is that problems due to poor translation initiation can be essentially avoided, a Overcoming such problems can be tedious and, for m a n y situations, a fusion protein may satisfy all the particular needs of the experimenter. Over the last several years, trpE or trpLE fusions have been used quite successfully, with more than 30 proteins or peptides reportedly expressed. The high level of expression of most of these fusions can be attributed to two factors: the relatively strong4 but controllable trp promoter, and the insoluble nature of the trpE or trpLE fusions, which usually leads to proteolytic protection within refractile bodies. In terms of expression, trpE and trpLE fusions appear to be quite comparable, the main difference being in the level of transcriptional control. While the trpE fusions are under both repressor and attenuation control, the trpLE fusions retain only repressor control. The trpLE fusions have deletions between the end of the leader and distal parts of the trpE polypeptide and have been derived l K. Itakura, T. Hirose, R. Crea, A. D. Riggs,H. L. Heyneker,F. Bolivar,and H. W. Boyer, Science 198, 1056 (1977). 2A. R. Davis, D. P. Nayak, M. Ueda, A. U Hiti, D. Dowbenko,and D. G. Kleid,Proc.Natl. Acad. Sci. U.S.A. 78, 5376 (1981). 3 B. E. Schoner, H. M. Hsiung,R. M. Belagaje,N. G. Mayne,and R. G. Schoner,Proc.Natl. Acad. Sci. U.S.A.81, 5403 (1984). 4 H. A. de Boer, L. J. Comstock, D. G. Yansura, and H. L. Heyneker, in "Promoter Structure and Function" (R. L. Rodriguezand M. J. Chamberlain,eds.), p. 462. Praeger, New York, 1982. METHODS IN ENZYMOLOGY, VOL. 185
Copyright© 1990by AcademicPress,Inc. All rights&reproductionin any formreserved.
162
EXPRESSION IN g . c . o f f
pWT111
pWT121
[14]
trpE 7 Hindlll AAA CCG ACT] CCA AGC TT Lys Pro Thr
trpE 7 Hindlll AAA CCG ACTICCA AGC TCC AAG C-FI E
Lys
pWT131
Pro
Thr
trpE 7 Hindlll AAA CCG ACTJCCA AGC TCC AAG CTC CAA GCT T Lys
Pro
Thr
FIG. l. Partial coding sequence of the trpE protein followed by HindlII restriction sites in three different reading frames for expression vectors pWT111, pWT121, and p W T I 3 1 ) 6
Numbers represent the amino acid positions of the trpE protein.
from the deletions trpALE 1413 and trpALE 1417. 5 A more detailed review on the trp promoter and its use in expressing cloned genes was published by Nichols and Yanofsky in 1983. 6 Designing Fusion There are two major considerations when designing a trpE or trpLE fusion: the length of trpE polypeptide and the junction between the trpE polypeptide and the protein or peptide of interest. The length of trpE can be a significant factor in determining the levels of expression. In general, the longer trpE fusions give higher levels of expression. 7,s This may not be just a matter of increasing the mass of the total fusion, but probably involves better precipitation of the longer fusions within the cell, thus avoiding proteolysis. Comparison of relatively short versus long trpE lengths demonstrates this tendency whereby the shorter fusions are more soluble than the larger o n e s . 9,1° The length of trpE or trpLE polypepfide necessary to achieve maximum expression is probably in the range of 100-300 amino acids. Besides expression levels, however, producing a more soluble fusion may be a more important factor, depending on the ultimate use of the fusion. If one is interested in some enzymatic activity associated with the protein of interest, a shorter, more soluble fusion will almost certainly be more useful. For maximal enzymatic activity in gens G. F. Miozzari and C. Yanofsky, J. Bacteriol. 133, 1457 (1978). 6 B. P. Nichols and C. Yanofsky, this series, Vol. 101, p. 155. 7 R. Derynck, A. B. Roberts, M. E. Winlder, E. Y. Chen, and D. V. Goeddel, Cell 38, 287 (1984). s p. R. Szoka, A. B. Schreiber, H. Chart, and J. Murthy, DNA 5, 11 (1986). 9 N. Tanese, M. Roth, and S. P. Golf, Proc. Natl. Acad. Sci. U.S.A. 82, 4944 (1985). ~°C. A. Chlan, C. Coulter, and L. T. Feldman, J. Virol. 61, 1855 (1987).
[ 14]
EXPRESSION AS trpE FUSION
163
trpE 517 EcoRI Pstl EcoRI CAT GCA CAG[GAA TTC TGC AGA A'IF C His Ala Gin FIo. 2. Partial coding sequence for the trpLE protein of pmasmid pNCV with the EcoRI and PstI restriction sites available for cloning. The number represents the amino acid position in the trpE protein and not that of the LE. Note that this LE consists of the first nine amino acids of the trp leader fused to amino acids 339-517 of the trpE polypeptide.
eral, the lengths of trpE polypeptide in the fusions have been in the range of 17-42 amino acids, 9- I I although much larger fusions also retained some activity. The junction between the trpE polypeptide and the protein of interest can be designed to allow cleavage of the fusion at this point. There are a number of cleavable fusions reported using both chemical 7,s,~2 and enzymatic ~3,14means. Because many of the trpE fusion proteins are soluble only in the presence of chaotropic agents, cleavage by chemical means, if feasible, is usually preferred. Enzymatic cleavage, however, can be used in some cases if solubility problems can be overcome. Usually, the shorter trpE fusions are more amenable to this type of cleavage. Plasmids and D N A Construction The trp promoter and the gene encoding the fusion protein are usually carried on a multicopy plasmid for maximum expression. TrpR÷strains of Escherichia coli should be used in plasmid construction and expression experiments to help ensure plasmid stability; 6 strains HB101 and MM294 have been commonly used. There are a number of plasmids that can be used as a source of the trp promoter and trpE or trpLE coding sequences. One simply ligates the gene for the protein of interest, in frame, to some point in the trpE gene, using a convenient restriction site. Synthetic DNA is now readily available and can be used to link nonoverlapping restriction sites in the correct reading frame and also provide the desired junction. The complete nucleotide sequence of the trp operon has been published, ~5 and can be used to generate restriction sites within the trpE gene. " I. Sadowski and T. Pawson, Oncogene 1, 181 (1987). ~2T. C. Furman, J. Epp, H. M. Hsiung, J. Hoskins, G. L. Long, L. G. Mendelsohn, B. Schoner, D. P. Smith, and M. C. Smith, Bio/Technology 5, 1047 (1987). 13G. Allen, M. D. Winther, C. A. Henwood, J. Beesley, L. F. Sharry, J. O'Keefe, J. W. Bennett, R. E. Chapman, D. E. Hollis, B. A. Panaretto, P. Van Dooren, R. W. Edols, A. S. lnglis, P. C. Wynn, and G. P. M. Moore, J. Biotechnol. 5, 93 (1987). t4 T. Imai, T. Cho, H. Takamatsu, H. Hori, M. Saito, T. Masuda, S. Hirose, and K. Murakami, J. Biochem. 100, 425 (1986). ~5C. Yanofsky, T. Platt, I. P. Crawford, B. P. Nichols, G. E. Christie, H. Horowitz, M. Van Cleemput, and A. M. Wu, Nucleic Acids Res. 9, 6647 (1981).
164
EXPRESSION IN E. coli
[ 14]
trpE 323 Smal BamHI Sail Pstl Hindlll ATI" GAG ATC]CCC GGG GAT CCT CTA GAG TCG ACC TGC AGC CCA AGC 1-I-A FIG. 3. Sequence of the multiple cloning region of plasmid pATH2? s The number represents the amino acid position of the trpE protein.
Some plasmids, the so-called trpE expression vectors, have been rather useful in constructing specific fusion expression plasmids. These vectors have convenient restriction sites engineered at various distances into the trpE gene, often with more than one coding phase available for cloning. For short trpE fusions, vectors pWT 11 l, pWT 12 l, and pWTl 31 have been constructed.~6 These contain the trp promoter, leader, and coding sequence for the first seven amino acids of the trpE, followed by a HindIII restriction site in all three reading frames (Fig. 1). For intermediate length fusions, pNCV ~7 which carries the deletion trpALE1413,5 can be used. This plasmid has EcoRI and PstI restriction sites, engineered at the end of the coding sequence for 188 amino acids of trpLE. This trpLE consists of the first nine amino acids of the leader fused to amino acids 339-517 of the trpE polypeptide (Fig. 2). For longer trpE fusions, the vector pATH2 has been extensively used. ~s This vector contains the trp promoter, leader, and coding sequence for the first 323 amino acids of trpE, followed by the multiple cloning region ofpUC12 (Fig. 3). trpE and trpLE fusion expression plasmids can be maintained in trpR+ strains provided sufficient tryptophan is present in the media. In minimal media, cultures containing these plasmids should normally be supplemented with tryptophan to approximately 20/zg/ml.
trp Induction Induction of the trpE or trpLE fusion generally involves tryptophan starvation of bacterial cultures containing the expression plasmids. Because some plasmids are unstable in their host, it is advisable to start with bacterial cells recently transformed with the appropriate expression plasmids. Such cells are usually grown overnight to saturation in a rich medium, such as LB, ~9 containing the appropriate antibiotic. The overnight
16W. Tacon, N. Carey, and S. Emtage, Mol. Gen, Genet. 177, 427 (1980). 17 T. Maniatis, E. F. Fritsch, and J. Sambrook, in "Molecular Cloning: A Laboratory Manual," p. 426. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. ts C. L. Dieckmann and A. Tzagoloff, J. Biol. Chem. 260, 1513 (1985).
[14]
EXPRESSION AS trpE FUSION
165
12 !!~i~i~iii~ii~!i~! !il ~¸~¸~' ¸ ~zi~ii~iiiiiiiiiii~iiii~!iiiii!i ~'
FIG. 4. SDS-polyacrylamide gel analysis of the expression o f a trpLE fusion. Escherichia coli, strain W3110, containing plasmid pBR322 and a plasmid encoding a trpLE-relaxin A chain fusion, was grown overnight in LB. The cultures were diluted 100-fold into minimal medium containing 3-indoleacrylic acid and grown for 7 hr. Samples of these cultures equivalent to 1 ml with an absorbance at 600 nm of l were then removed for analysis. The pelleted cells were resuspended in 250 gl of 0.1 M 2-mercaptoethanol, 2% (w/v) SDS, 60 m M Tris (pH 6.8), 10% (v/v) glycerol, heated to 95 ° for 2 min, and loaded onto an SDSpolyacrylamide gel. After electrophoresis, the gel was stained with Coomassie brilliant blue. Lane l shows the control culture containing pBR322. Lane 2 shows the culture containing the trpLE-relaxin A chain fusion with an arrow pointing to the fusion protein band.
culture is then diluted 25 to 50-fold into M919 with 0.5% (w/v) casamino acids and the appropriate antibiotic. After 1 - 2 hr growth, 3-indoleacrylic acid 2° is added to a final concentration of 25 /zg/ml. Growth is then continued for 2 - 5 hr, at which time samples may be removed to assay for the particular fusion protein. Samples removed from induced cultures are usually analyzed by S D S polyacrylamide gels (Fig. 4). Expression levels oftrpE or trpLE fusions are usually quite high, and the fusion can then be visualized after staining with Coomassie brilliant blue. For many fusions, the appearance of a new protein band on the gel after staining is the only way to verify and estimate the level of expression. One should therefore include on the gel samples of ~9j. H. Miller, in "Experiments in Molecular Genetics," p. 431. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972. 20 W. F. Doolittle and C. Yanofsky, J. Bacteriol. 95, 1283 0968).
EXPRESSION IN E. ¢oli
166
[ 1 5]
induced cultures containing the parent trpE or trpLE expression plasmid as a control. If the fusion protein is to be used to raise antibodies against the protein of interest, one can purify the fusion by preparative SDS gel electrophoresis once the prospective protein band has been identified.2~-23 Comments Occasionally, one cannot successfully express a protein of interest as a
trpE or trpLE fusion. Usually, the bacterial cultures for these fusions stop growing completely when induced, and no new protein band can be seen on an SDS-polyacrylamide gel analysis of a total cell lysate. The hepatitis B surface antigen, for example, when fused to 190 amino acids of trpLE, displayed this type of expression problem. Removal of the last 24 amino acids of the hepatitis surface antigen, however, restored expression to a high level.24 It is not clear what the nature of this problem is, but for many fusions such trimming of the protein of interest may be an acceptable alternative. 21 L. K. Pape, T. J. Koerner, and A. Tzagoioff, J. Biol. Chem. 260, 15362 (1985). 22 D. G. Kleid, D. Yansura, B. Small, D. Dowbenko, D. M. Moore, M. J. Grubman, P. D. McKercher, D. O. Morgan, B. H. Robertson, and H. L. Baehrach, Science 214, 1125 (1981). 23 I. Sadowski, J. C. Stone, and T. Pawson, Mol. Cell. Biol. 6, 4396 (1986). 24 D. Yansura and D. G. Kleid, unpublished observation (1980).
[ 15] E n g i n e e r i n g
E s c h e r c h i a coli t o S e c r e t e H e t e r o l o g o u s Gene Products
B y J O A N A . S T A D E R a n d T H O M A S J. SILHAVY
The development of recombinant DNA technology has revolutionized the way we approach research problems in almost all biological systems, but its use in applied technology still favors only a few organisms and cell types. Certainly in terms of prokaryotes, Escherichia coli is one of the most favored organisms for many applications because of the ease with which the genome can be manipulated. Controlling factors such as promoter efficiency and regulation, transformation efficiency, protease levels, and the secretion of protein into the growth medium, is within our current capability. Still, despite all the progress, in many instances success is achieved empirically and the practice can best be described as an art. In this article, we attempt to bring to light some of the problems encountered in secreting heterologous proteins from E. coli and discuss some of the successes that have been achieved to date. METHODS IN ENZYMOLOGY, VOL. 185
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