[17]
E X P R E S S I O N OF H E T E R O L O G O U S GENES
[17]
Expression
of Heterologous subtilis
By D E N N I S
199
Genes in Bacillus
J. H E N N E R
There are several reasons why Bacillus subtilis might be considered useful as a host for expression of a protein of interest. It is a well-studied prokaryote, with extensive genetic systems, and well-understood physiology; there is a wealth of studies on transcription and translation in it. Additionally, a great deal of experience has been gained by growing various Bacillus species for industrial production. Over the years, a number of expression systems have been constructed which are reasonably simple to use. Perhaps the greatest advantage of B. subtilis is its ability to secrete proteins directly into the culture medium; however, as discussed below, this may have both positive and negative aspects. Secretion A major rationale for using B. subtilis as a host for expression of proteins is its ability to secrete proteins directly into the culture medium, accumulating them to high levels, and in a relatively pure state. A further advantage of secretion is that the proteins made are often correctly folded, disulfide-bonded, and soluble. However, the ability of an expression system to secrete proteins efficiently is not well understood. Even attempts to secrete heterologous proteins in the better characterized host Escherichia coli are not uniformly successful. Certain proteins seem to be efficiently secreted, while other, even closely related, proteins are secreted poorly. In our experience, most prokaryotic proteins are secreted efficiently from both organisms, while the behavior of eukaryotic proteins in a secretion system cannot be predicted. The underlying mechanism(s) for this failure of some proteins to be efficiently secreted has not been well documented in either B. subtilis or E. coli. One report demonstrated the accumulation of large amounts of the precursor of human interferon a2 in a detergent-insoluble fraction in B. subtilis when an attempt was made to express it with the a-amylase signal sequence) Our attempts to find similar material accumulating using human growth hormone failed to detect any significant accumulation of precursor material. The "competence" of different prokaryotic organisms to secrete heterologous proteins remains open to investigation. C. H. Schein, K. Kashiwagi, A. Fujisawa, and C. Weissmann,
METHODS IN ENZYMOLOGY, VOL. 185
Bio/Technology4, 719 (1986).
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
200
EXPRESSION IN B. subtilis
[ 17]
Proteases and Protein Stability Bacillus subtilis sccrctcs several protcases into the culturc medium which have the potential to degrade other proteins that arc also secreted to the medium. This problem has bccn well documented. 2,a Thc genes for both of the major protcascs have bccn isolated and used to create deletions in thc chromosome of B. subtilis.4.5 Strains carrying such deletions arc viable; however, such strains exhibited only modcst improvcmcnts in thc stabilityof hctcrologous protcins. There arc several other minor protcases, at least two of which have been cloned and chromosomal deletions creatcd.6,7The elimination of all the proteases will probably lead to improvemcnts in the stabilityof heterologous proteins in the culture medium. Anothcr stabilityproblem which should bc distinguishcd from protcolysis is the stabilityof the protein to the physical conditions in the culture medium. W c found that several proteins of interest to us were very unstable in sterileculture media during agitation. This instabilitywas exhibited by a rapid aggregation and/or loss of rcactivityin an immunological assay and may bc caused by surface denaturation resulting from the agitation and acration found during fermentation. Denaturation of the protein could also lead to increased susceptibilityto protcolysis.
Plasmid Instability There have also bccn a large number of anecdotal reports of plasmid instability in B. subtilis. Howcvcr, in our hands, plasmid instability has never led to practical problems. The only plasmids that exhibited unacccptablc levels of deletions or rearrangements were those which had a region repeated on the plasmid, or those which constitutively expressed a protein at high levels.Both problems can bc avoided by taking care in construction of plasmids so as to avoid duplicating a region of D N A and by using promoters that arc regulated,s Another simple way to avoid plasmid instability in B. subtilis is to integrate thc expression system into the chromosome. 9 It is vcry simple to 2 I. Ulmancn, K. Lundstrom, P. Lchtovaara, M. Sarvas, M. Ruohoncn, and I. Palva, ]. Bacteriol. 162, 176 (1985). a K. Nakamura, T. Furusato, T. Shiroza, and K. Yamane, Biochem. Biophys. Res. Commun. 128, 601 (1985). 4 M. L. Stahl and E. Ferrari, J. Bacterio1158, 411 (1984). s M.Y. Yang, E. Ferrari, and D. J. Henner, J. Bacterio1160, 15 (1984). 6 A. Sloma, A. Ally, D. Ally, and J. Pero, J. Bacteriol. 170, 5557 (1988). 7 M. Ruppen and E. Ferrari, personal communication (1988). s S. F. J. Le Grice, this volume [18]. 9 C. W. Saunders, B. J. Schmidt, M. S. Mirot, L. D. Thompson, and M. S. Guyer, J. Bacteriol. 157, 718 (1984).
[ 1 8]
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201
integrate nonreplicative plasmids into the chromosome by homologous recombination, and a number of vectors have been designed for this purpose. These integrated plasmids are replicated by the chromosomal replication machinery, and are very stable. Expression vectors for secretion of proteins that is based on the B. amyloliquefaciens alkaline protease (subtilisin) promoter and signal sequence are discussed in [18]. These vectors have been successfully used to express both prokaryotic and eukaryotic proteins. The vectors are reasonably simple to use, and constructions can be made in either E. cull or B. subtilis. This expression system would be appropriate for the expression of most prokaryotic secreted proteins, and is also worth considering for eukaryotic proteins which can be demonstrated to be reasonably stable in the culture media of the B. subtilis host strain. A series of vectors which allow the regulated expression of proteins in B. subtilis is described in [19]. Although primarily designed for intracellular expression, this system might be also utilized for secreted proteins. I am not aware of any proteins which have been successfully expressed in B. subtilis but not in E. coli, and I suspect that E. coli will be the first choice of most investigators. However, there has not been a detailed comparison of the two organisms as hosts, and the vectors described here can be used in either organism, making such comparisons easy to do. This system should also be useful to researchers working with B. subtilis who wish to overproduce proteins in this organism. A brief description of two plasmids which can be used in one step to insertionally inactivate a gene in B. subtilis and place that same gene under the control of a tightly regulated promoter is presented in [20]. These plasmids would be of use for studies of gene regulation and expression in B. subtilis. [ 18] R e g u l a t e d P r o m o t e r f o r H i g h - L e v e l E x p r e s s i o n o f Heterologous Genes in Bacillus subtilis B y S T U A R T F . J. L E G R I C E
Introduction A desirable feature of prokaryotic vector design is the ability to control tightly expression of a gene whose product might be detrimental to the host. In Escherichia coli, the most commonly employed systems have incorporated transcriptional control mediated through either the lac, i trpfl H. A. de Boer, L. J. Comstock, and M. Vasser, Proc. NatL Acad. Sci. U.S.A. 80, 21 (1983). 2 B. P. Nichols and C. Yanofsky, this series, Vol. 101, p. 155.
METHODS IN ENZYMOLOGY, VOL. 185
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
E X P R E S S I O N OF H E T E R O L O G O U S GENES
[17]
Expression
of Heterologous subtilis
By D E N N I S
199
Genes in Bacillus
J. H E N N E R
There are several reasons why Bacillus subtilis might be considered useful as a host for expression of a protein of interest. It is a well-studied prokaryote, with extensive genetic systems, and well-understood physiology; there is a wealth of studies on transcription and translation in it. Additionally, a great deal of experience has been gained by growing various Bacillus species for industrial production. Over the years, a number of expression systems have been constructed which are reasonably simple to use. Perhaps the greatest advantage of B. subtilis is its ability to secrete proteins directly into the culture medium; however, as discussed below, this may have both positive and negative aspects. Secretion A major rationale for using B. subtilis as a host for expression of proteins is its ability to secrete proteins directly into the culture medium, accumulating them to high levels, and in a relatively pure state. A further advantage of secretion is that the proteins made are often correctly folded, disulfide-bonded, and soluble. However, the ability of an expression system to secrete proteins efficiently is not well understood. Even attempts to secrete heterologous proteins in the better characterized host Escherichia coli are not uniformly successful. Certain proteins seem to be efficiently secreted, while other, even closely related, proteins are secreted poorly. In our experience, most prokaryotic proteins are secreted efficiently from both organisms, while the behavior of eukaryotic proteins in a secretion system cannot be predicted. The underlying mechanism(s) for this failure of some proteins to be efficiently secreted has not been well documented in either B. subtilis or E. coli. One report demonstrated the accumulation of large amounts of the precursor of human interferon a2 in a detergent-insoluble fraction in B. subtilis when an attempt was made to express it with the a-amylase signal sequence) Our attempts to find similar material accumulating using human growth hormone failed to detect any significant accumulation of precursor material. The "competence" of different prokaryotic organisms to secrete heterologous proteins remains open to investigation. C. H. Schein, K. Kashiwagi, A. Fujisawa, and C. Weissmann,
METHODS IN ENZYMOLOGY, VOL. 185
Bio/Technology4, 719 (1986).
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
200
EXPRESSION IN B. subtilis
[ 17]
Proteases and Protein Stability Bacillus subtilis sccrctcs several protcases into the culturc medium which have the potential to degrade other proteins that arc also secreted to the medium. This problem has bccn well documented. 2,a Thc genes for both of the major protcascs have bccn isolated and used to create deletions in thc chromosome of B. subtilis.4.5 Strains carrying such deletions arc viable; however, such strains exhibited only modcst improvcmcnts in thc stabilityof hctcrologous protcins. There arc several other minor protcases, at least two of which have been cloned and chromosomal deletions creatcd.6,7The elimination of all the proteases will probably lead to improvemcnts in the stabilityof heterologous proteins in the culture medium. Anothcr stabilityproblem which should bc distinguishcd from protcolysis is the stabilityof the protein to the physical conditions in the culture medium. W c found that several proteins of interest to us were very unstable in sterileculture media during agitation. This instabilitywas exhibited by a rapid aggregation and/or loss of rcactivityin an immunological assay and may bc caused by surface denaturation resulting from the agitation and acration found during fermentation. Denaturation of the protein could also lead to increased susceptibilityto protcolysis.
Plasmid Instability There have also bccn a large number of anecdotal reports of plasmid instability in B. subtilis. Howcvcr, in our hands, plasmid instability has never led to practical problems. The only plasmids that exhibited unacccptablc levels of deletions or rearrangements were those which had a region repeated on the plasmid, or those which constitutively expressed a protein at high levels.Both problems can bc avoided by taking care in construction of plasmids so as to avoid duplicating a region of D N A and by using promoters that arc regulated,s Another simple way to avoid plasmid instability in B. subtilis is to integrate thc expression system into the chromosome. 9 It is vcry simple to 2 I. Ulmancn, K. Lundstrom, P. Lchtovaara, M. Sarvas, M. Ruohoncn, and I. Palva, ]. Bacteriol. 162, 176 (1985). a K. Nakamura, T. Furusato, T. Shiroza, and K. Yamane, Biochem. Biophys. Res. Commun. 128, 601 (1985). 4 M. L. Stahl and E. Ferrari, J. Bacterio1158, 411 (1984). s M.Y. Yang, E. Ferrari, and D. J. Henner, J. Bacterio1160, 15 (1984). 6 A. Sloma, A. Ally, D. Ally, and J. Pero, J. Bacteriol. 170, 5557 (1988). 7 M. Ruppen and E. Ferrari, personal communication (1988). s S. F. J. Le Grice, this volume [18]. 9 C. W. Saunders, B. J. Schmidt, M. S. Mirot, L. D. Thompson, and M. S. Guyer, J. Bacteriol. 157, 718 (1984).
[ 1 8]
R E G U L A T E D P R O M O T E R FOR H I G H - L E V E L EXPRESSION
201
integrate nonreplicative plasmids into the chromosome by homologous recombination, and a number of vectors have been designed for this purpose. These integrated plasmids are replicated by the chromosomal replication machinery, and are very stable. Expression vectors for secretion of proteins that is based on the B. amyloliquefaciens alkaline protease (subtilisin) promoter and signal sequence are discussed in [18]. These vectors have been successfully used to express both prokaryotic and eukaryotic proteins. The vectors are reasonably simple to use, and constructions can be made in either E. cull or B. subtilis. This expression system would be appropriate for the expression of most prokaryotic secreted proteins, and is also worth considering for eukaryotic proteins which can be demonstrated to be reasonably stable in the culture media of the B. subtilis host strain. A series of vectors which allow the regulated expression of proteins in B. subtilis is described in [19]. Although primarily designed for intracellular expression, this system might be also utilized for secreted proteins. I am not aware of any proteins which have been successfully expressed in B. subtilis but not in E. coli, and I suspect that E. coli will be the first choice of most investigators. However, there has not been a detailed comparison of the two organisms as hosts, and the vectors described here can be used in either organism, making such comparisons easy to do. This system should also be useful to researchers working with B. subtilis who wish to overproduce proteins in this organism. A brief description of two plasmids which can be used in one step to insertionally inactivate a gene in B. subtilis and place that same gene under the control of a tightly regulated promoter is presented in [20]. These plasmids would be of use for studies of gene regulation and expression in B. subtilis. [ 18] R e g u l a t e d P r o m o t e r f o r H i g h - L e v e l E x p r e s s i o n o f Heterologous Genes in Bacillus subtilis B y S T U A R T F . J. L E G R I C E
Introduction A desirable feature of prokaryotic vector design is the ability to control tightly expression of a gene whose product might be detrimental to the host. In Escherichia coli, the most commonly employed systems have incorporated transcriptional control mediated through either the lac, i trpfl H. A. de Boer, L. J. Comstock, and M. Vasser, Proc. NatL Acad. Sci. U.S.A. 80, 21 (1983). 2 B. P. Nichols and C. Yanofsky, this series, Vol. 101, p. 155.
METHODS IN ENZYMOLOGY, VOL. 185
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
