214

EXPRESSIONIN B. subtilis

[ 19]

In the systems presented, foreign genes are expressed during logarithmic growth, taking advantage of the observation that the E. coli bacteriophage T5 promoter Pro5 is recognized by the vegetative (tr55) RNA polymerase of B. subtilis. Since there is rapid interchange of a factors in B. subtilis at later growth stages, 3t it is unlikely that PN2Sand P,~aI are recognized by the alternative forms of RNA polymerase. If it were necessary to produce foreign proteins at later growth stages, the system described here could be modified by replacing the vegII and N25 promoters with counterparts utilized by other forms of B. subtilis RNA polymerase. Alternatively, these growth-stage-specific promoters could be placed in the immediate 5' vicinity of Pv~i and Pms/o. Such a "twin" promoter would ensure constant synthesis of lac repressor. Furthermore, Deutchle et al. have recently demonstrated that the lac operator sequence can be separated from the promoter and still prevent RNA polymerase from entering into productive transcription. 32 Thus, transcription from a growth-stage-specific promoter placed 5' to the Pros/0 element would still be controlled by the lac operator. The plasmids and strains described in this article, together with complete nucleotide sequence and restriction maps, can be made available upon request. Acknowledgments I wish to thank Ursula Peschke and Verena Beuck for their assistance in early expression vector studies. The gifts of various expression cassettes from D. Stuber, R. G. Gentz, and J. Knowles are also acknowledged, as well as helpful comments on the manuscript from Jan Mous and Oktavian Shatz. Finally, I wish to thank Professor H. Bujard, whose excellent work on T5 promoters and belief in B. subtilis were instrumental in generation of the early expression systems. 3mR. Losick and J. Pero, Cell 25, 585 (198 I). 32 U. Deutchle, R. Gentz, and H. Bujard, Proc. Natl. Acad. Sci. U.S.A. 83, 4134 (1986).

[19] S y s t e m f o r S e c r e t i o n o f H e t e r o l o g o u s Bacillus subtilis

P r o t e i n s in

B y VASANTHA N A G A R A J A N

Introduction Secretion of heterologous proteins from Bacillus subtilis has several attractive properties. The secreted protein is usually soluble and active. Most importantly, secreted proteins from B. subtilis are located in the growth medium, in contrast to Escherichia coli, where the majority of METHODS IN ENZYMOLOGY, VOL. 185

Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.

[19]

SYSTEM FOR SECRETION OF HETEROLOGOUS PROTEINS

215

secreted proteins are localized in the periplasmic space. The protein can usually be recovered from the medium with relatively few contaminating proteins, simplifying further steps in the purification. Several vectors designed for the secretion of heterologous proteins from B. subtilis, based on the genes for several secreted proteins (a-amylase, alkaline protease, neutral protease, and levansucrase), have been described. 1-5 This article describes a secretion vector derived from the alkaline protease (subtilisin) gene (apr[BamP]) of Bacillus amyloliquefaciens. The promoter, translation initiation, and signal sequences are all provided by the apr[BamP] gene. The vector also provides origins of replication and selectable markers for both E. coli and B. subtilis, allowing constructions to be made in either organism. The E. coli origin and ampicillin resistance gene are derived from pBR322, while the pC194 chloramphenicol resistance gene and origin of replication provide replication functions and selection for B. subtilis. Description of apr[BamP]-Based Secretion Vector Although alkaline protease is synthesized as a preproprotease, 6,7 the first 30 amino acids are sufficient to function as a signal sequence. In order to simplify construction of heterologous gene fusions, a BamHI recognition sequence was created by site-directed mutagenesis two codons from the end of the signal (pre) peptide coding region (Fig. 1). Introduction of this BamHI site resulted in the addition of two amino acids (Asp and Pro) in the pro peptide sequence. The addition of these two amino acids did not interfere with the expression or activity of the alkaline protease. 3 The insertion of a heterologous gene at this BamHI site results in the inactivation of the apr[BamP] gene. The ability of the alkaline protease signal peptide to translocate heterologous proteins was demonstrated with staphylococcal protein A and bovine pancreatic ribonuclease A (RNase) as described below. A diagram of pGX2134, an E. coli-B, subtilis shuttle vector containing the apr gene with the introduced BamHI site is shown in Fig. 2.

Palva et al., Proc. Natl. Acad. Sci. U.S.A. 79, 5582 (1982). 2 Palva et aL, Gene 22, 229 (1983). 3 N. Vasantha and L. D. Thompson, J. Bacteriol. 165, 837 (1986). 4 Honjo et aL, J. BiotechnoL 4, 63 (1986). 5 Joyet et aL, in "Bacillus Molecular Genetics and Biotechnology Applications" (A. T. Ganesan and J. A. Hoch, eds.), p. 479. Academic Press, New York, 1986. 6 N. Vasantha et al., J. BacterioL 159, 811 (1984). v S. D. Power, R. M. Adams, a n d J . A. Wells, Proc. Natl. Acad. Sci. U.S.A. 83, 3096 (1986).

216

EXPRESSION IN B. subtilis

[ 19]

-107 -100 GTG AGA GGC AAA AAA GTA TGG ATC AGT TTG CTG TT'[ fmet Acg Gly Lys Lys Val Trp lie Ser Leu Leu Phe

AATCTGTCTATTGGTTATTCTGCAAATGAAAAAAAGGAGAGGATAAAGA

I PRO GCT TTA GCG TTA ATC Ala Leu Ala Leo fie

TTT ACG ATG GCG Phe Thr Met Ala

TTC GGC AGC ACA TCC TCT GCC CAG GCG GCA GGG AAA TCA AAC GGG (3AA Phe Gty Set Thr Set Set Ala G*n Ala Ala ~ Lys Ser Ash Gly Glu GGATCC BamHl

Signal p e p t i d e cleavage site

spa B apr-spa

C.~G

GCG

Gin

Ala

GCA Ala

GGG Gly

GAT Asp

CAA Gin

CGC Arg

CCG pro

AAA Lys

GAG Glu

GCA Ala

GCC Ala

GCA Ala

LINKER j, _ _ C apr-bprl

CAG

GCG

Gin

Ala

CAG

GCG

Gin

Ala

GCA Ala

GGG Gly

GAT Asp

AAT ASh

GGT Gly

bpr

bpr D apr-bpr2

AAA Lys

GAG Glu

ACA Thr

FIG. 1. The entire DNA sequence of apr[BamP]has been published. ~ (A) DNA sequence of the signal peptide coding region and location of the introduced BamHI site. DNA sequences across the fusion junction of (B) apr-spa(pGX2136), (C) apr-bpr1(pGX2211), and (D) apt- bpr2(pGX2214) are also shown. PvulI

B

a

m

~ Sal

amp r ~

Sma

Eoo.,

II

Cm r BamHI

Clal

I

I

Pvull

I

I~'//A

s.,,

I

I

apr [Barn P]

[ Mz..5-,9-2

4ol

Fro. 2. pGX2134 is an E. coli-B, subtilis shuttle vector containing the apr[BamP] sequence on an EcoRI-Sall fragment. A linear map of the EcoRI-Sall fragment with the relevant restriction sites is shown below. I , pre; ~, pro; r'l, mature coding sequence of

apr[BamP].

[ 19]

SYSTEM FOR SECRETION OF HETEROLOGOUS PROTEINS

217

Construction of Hybrid Gene Fusions Plasmid pGX2912 is a previously described plasmid with the protein A gene that has a Bcl site at the twenty-second and twenty-third codons of mature protein A gene (spa) and a PvuII sites following the gene.8 pGX2134 was digested with BamHI and PvuII and ligated to the BclIPvuII fragment containing the spa gene from pGX2912. The ligation mixture was transformed into E. coli and the desired plasmid (pGX2136) was isolated. Bacillus subtilis strain GX4935 (trpC2 metBlO lys-3 AaprE Anpr) was transformed with pGX2136, and colonies containing the proper plasmid expressing the protein A gene were screened by colony immunoassay (see below). A BamHI site was created preceding the mature sequence of bovine pancreatic ribonuclease (bpr) by site-directed mutagenesis of the bpr gene which had been subcloned into M 13mpl 9. 9 The replicative form of this M 13 phage was isolated, and a BamHI-PvuII fragment containing the bpr gene was isolated and ligated into pGX2134 which had also been digested with BamHI and PvuII. The ligation was done at a high DNA concentration (100/~g/ml) to generate long concatemers. Both E. coli and B. subtilis were transformed with the ligated DNA. Two out of the 12 E. coli transformants screened had the correct plasmid based on restriction analysis and were designated as pGX2211 and pGX2212. Site-directed mutagenesis was performed on pGX2211 (an E. coli-B, subtilis-Ml3 shuttle vector containing apr-bprl) to delete the 12 nucleotides (Fig. l) between the end of the signal peptide coding region and mature bpr. The resulting plasmid carrying apr-bpr2 was designated pGX2214. Bacillus subtilis GX4935 was transformed with pGX2211 and pGX2214 by the competent cell transformation protocol of Gryczan et al.l° Selection was for chloramphenicol resistance on TBAB plates supplemented with 5 pg/ml of chloramphenicol. Transformants were screened for RNase activity by colony screening as described below. Once positive colonies expressing protein A or RNase were identified, plasmid DNA was isolated and its structure verified by restriction analysis. The bacteria carrying the correct plasmid were stored by resuspension in Spizzen minimal medium [0.2% (w/v) ammonium sulfate, 0.14% (w/v) dibasic potassium phosphate, 0.6% (w/v) monobasic potassium phosphate, 0.1% (w/v) sodium citrate] plus 20% (v/v) glycerol, frozen in a dry iceethanol bath, and maintained at --70*.

8 S. R. Fahnestock and K. E. Fisher, J. BacterioL 165, 796 (1986). 9 N. Vasantha and D. Filpula, Gene 76, 53 (1989). ~oT. S. Gryczan, S. Contente, and D. Dubnau, MoL Gen. Genet. 177, 459

218

EXPRESSION IN B. subtilis

[ 19]

Colony Screening Transformants were patched onto two filters overlaid on TBAB [tryprose blood agar base (Difco, Detroit, MI), 33 g/liter] + Cm (5 /~g/ml) plates. ~ The top filter was a cellulose acetate membrane filter [Schleicher & Schuell (Keene, NH) OE67] and the bottom filter was a nitrocellulose filter (Schleicher & Schuell BA85). The plates were incubated for 17 hr at 37% The bacteria were retained on the cellulose acetate filter and secreted proteins passed through the cellulose acetate filter and bound to the nitrocellulose filter below. The nitrocellulose filter was removed, and processed as follows: I. Incubate the filter for l hr in l 0 ml of TS buffer [ 10 m M Tris-hydrochloride, pH 8.0, 0.9% (w/v) sodium chloride] containing 3% (w/v) bovine serum albumin (BSA) at room temperature. 2. Add l0/~l of rabbit anti-RNase serum and incubate the filter for 1 hr at 25 ° . 3. Wash three times with TS buffer to remove the antibody. 4. Add 5/11 of horseradish peroxidase conjugate of goat anti-rabbit IgG (Bio-Rad, Richmond, CA) to 10 ml of TS buffer and incubate the filter for 1 hr at room temperature. 5. Wash with TS buffer three times and develop using 4-chloro-lnaphthol and hydrogen peroxide. (Dissolve 36 mg of 4-chloro-l-naphthol in 12 ml of methanol and add 48 ml of TS buffer. Mix thoroughly and add 120/zl of 30% hydrogen per oxide. Use immediately and do not store.) RNase Activity Screen The nitrocellulose filter can also be screened for RNase activity by overlaying the filter on an RNA-agarose plate [1% (w/v) yeast RNA in 50 m M Tris-HC1, pH 7.0, l0 m M EDTA, and 1% (w/v) agarose] for 4 to 8 hr and incubated at 37 °. The filter is removed and the plate is treated with 0.1 M sulfuric acid; zones of clearing indicate RNase activity. Analysis of Expression of Heterologous Protein There are several methods to determine how efficiently a heterologous protein is secreted from B. subtilis. Immunoblot analysis of a culture supernatant cannot determine whether a protein is present in the growth medium due to secretion or cell lysis. Proteolysis of the accumulated protein in the culture supernatant can complicate the interpretation of the results of an immunoblot. Therefore, pulse-chase experiments can be used to determine the efficiency of secretion of a protein and its stability. H C. W. Saunders, et al., J. Bacteriol. 169, 2917 (1987).

[ 19]

SYSTEM FOR SECRETION OF HETEROLOGOUS PROTEINS

219

Protocol for P u l s e - C h a s e Analysis 1. Start overnight cultures of the desired strain and a negative control strain on a TBAB + Cm plate from the frozen stock and incubate at 30 °. 2. Inoculate the strains into 20 ml of medium S7 [10 m M ammonium sulfate, 50 m M potassium phosphate, pH 7.0, 2 m M MgC12, 0.7 m M CaC12, 50 g M MnC12, 5 p M FeC13, 1 # M ZnCh, 2 # M thiamin, 1% glucose, 20 m M L-glutamate (adjusted to pH 7.0 with KOH), tryptophan (50 gg/ml), methionine (50 gg/ml), and lysine (50 #g/ml)] in a 125-ml flask such that the absorbance of the initial inoculum is OD~00 = 0.05. No antibiotic is added because the shift down and addition of antibiotic results in a long growth lag. 3. Grow at 37 ° with aeration at 120 rpm; doubling time of the cultures is normally 60 to 85 min; if longer, add 0.001% yeast extract. 4. At an absorbance of OD~o -- 0.5, spin 2.0 ml of the culture for 5 min at 6000 rpm at room temperature. 5. Wash the cells with 2 ml of prewarmed synthetic medium lacking methionine. 6. Resupend the cells in 2 ml of prewarmed synthetic medium lacking methionine. 7. After a 10-min incubation at 37* with aeration, add 50-100/zCi of L-[35S]methionine. After 3 min, add 30/zl of chase solution (1 mg of puromycin per milliliter, 5 mg of methionine per milliliter). Withdraw samples (500 pl) after 2, 5, and 10 min, and add them to a microfuge tube containing 25 #1 of BSA (5 #g/ml). Spin for 20 sec at room temperature in a microfuge to separate the cell and supernatant fractions. Precipitate proteins in both the cell pellet and supernatant with trichloroacetic acid [5% (v/v) final concentration]. 9. After 1 hr at 40, spin down the precipitate at 4" and wash with 80% (v/v) acetone. 10. Air dry the acetone pellet and add 50/A of lysozyme (1 mg/ml) in buffer A [50 m M Tris- 10 m M E D T A + 1 m M phenylmethylsulfonyl fluoride (PMSF)] to the cell pellet and buffer A alone to the supernatant fraction. 11. Vortex thoroughly and incubate at 37 ° for 10 min; add 15 #1 of 4X sample preparation buffer (Laemmli) 12 to all samples, vortex, and boil for 5 min. 12. Spin at room temperature for 15 min in a microfuge and transfer the supernatant to a microfuge tube. Process the supernatant for immunoprecipitation using any standard protocol, a Resuspend the immunoprecipitate in 30 gl of Laemmli sample buffer 12 and run 10 gl per lane on a polyacrylamide gel. J2 U. K. Laemmli, Nature (London) 227, 680 (1970).

220

EXPRESSION IN B. subtilis

[19]

Demonstration of Synthesis of Precursor Proteins Pulse-chase experiments do not always reveal the presence of a precursor protein due to the rapid processing of the precursor. Precursors for secreted proteins can be observed by inhibiting protein translocation. In E. co~i, precursors of secreted proteins have been observed by labeling cells in the presence of 2-phenylcthanol (PEA), which disrupts the proton-motive force.~3 Precursor species for protein A, human serum albumin, ribonucleasc, and fl-lactamase have been observed by labeling B. #ubtilis in the presence of PEA. 3,9,t~,~4Figure 3 shows results from a culture orB. subtilis cells containing pGX2214 labeled with L-[35S]methionine in the presence of varying amounts of PEA. Cultures treated with 0 to 0.6% PEA showed a protein with an apparent mobility of 14 kDa, identical to that of mature RNase, in both the cell and supernatant fractions. The cultures treated with 0.6% PEA showed an additional protein with an apparent mobility of 17 kDa in the cellular fraction. Prolonged exposure of the fluorogram revealed a 17-kDa protein in the cellular fraction of cells treated with 0.8% PEA (not shown). However, the 17-kDa protein was never observed in the supcrnatant fraction. The expected size of the precursor protein would be 3 kDa larger than the mature protein due to the signal sequence, and thus the 17-kDa cell-associated protein is likely to be the unprocessed precursor protein.

Determination of Signal Peptide-Processing Site It is important to confirm that a secreted protein has the correct N-terminal amino acid sequence. One method is first to purify the protein and then determine its N-terminal amino acid sequence. Another simple method to determine the N-terminal sequence is to radiolabcl the protein with one amino acid and then determine the position of labeled residue in the sequence. For example, lysine is the third and fourth residue in the alkaline protcase signal peptide and the first and eighth wsidue in bovine pancreatic ribonuclease (Fig. 1A, D). A culture of B. subtilis carrying pGX2214 was labeled with [3H]lysine and processed as described below. The majority of the counts released by the Edman degradation were in cycles one and eight. These results are consistent with correct processing of the signal pcptide, indicating that B. subtilis can secrete ribonucleasc with the same N terminus as the native enzyme.

t3 C. J. Daniels, et al. Proc. Natl. Acad. Sci. U.S.A. 78, 5396 (1981). ,4 N. Vasantha and L. D. Thompson, Gene 49, 23 (1986).

[ 19]

SYSTEMFOR SECRETION OF HETEROLOGOUSPROTEINS

I

II

A C

221

C

S

B

S C S C

C

D

S

C S P M

~i ~i~ i~ i i~ ~i~ ii ~i !i~ ~i ~ ~

~

Fro. 3. Bacillus subtilis cellscarryingpGX2214 (apr- bpr2) werelabeledwith [3SS]methionine for 3 rain. (I) Control culture with no PEA; (II) PEA-treatedculture. PEA at 0.2% (A), 0.4% (B), 0.6% (C), and 0.8% (D) was present during methionine starvation. The cellular (C) and supernatant (S) fractions were separated and immunoprecipitated. P, precursor, M, mature. N-Terminal Analysis of Protein b y Radiolabeling Steps 1 through 4 are as described previously for pulse-labeling experiments. 5. Wash the cells with 2 ml of prewarmed $7 medium lacking lysine. 6. Resuspend the cells in 1 ml of $7 medium lacking lysine and containing 2 m M PMSF (a protease inhibitor is necessary to prevent proteolysis of the secreted protein). Incubate for 10 min with aeration (at 37*). 7. Add [3H]lysine (200 aCi) and label for 5 min. 8. Add 30/~1 of chase solution (1 mg/ml puromycin, 5 mg/ml lysine). 9. After 10 min, separate the cell and supematant fractions and immunoprecipitate as described above. 10. The immunoprecipitate is dissolved in 40/tl of 2% SDS, boiied for 5 min, and centrifuged in a microfuge for 15 min. The supernatant is removed and 5 al is analyzed by polyacrylamide gel electrophoresis to ensure that only a single species is present. 1 I. The rest of the solubilized protein (35/d) is diluted to 100 #1 with water, 800/tl of 80% cold acetone is added, and the protein is precipitated for 17 hr at - 2 0 °. 12. The precipitated protein is centrifuged for 15 min in a microfuge at

222

s.XPR~SSIOt~ XN B. subtilis

[19]

TABLE I MEDIUM OPTIMIZATIONa Medium

Cell density

Protein A (mg/liter)

Pen assay + MnCI2 2% yeast extract GY medium Medium A

4.8 4.3 9.7 10.2

3 12 32 300

~Cell density (absorbance at 600 nm) and amount of protein A [S. Lotdahl, B. Guss, M. Uhlen, L. Philipson, and M. Lindberg, Proc. Natl. Acad. Sci. U.S.A. 80, 697 (1983)] were determined in a 12-hrculture. 4 ° and the supernatant is gently aspirated. The pellet is washed again with 80% acetone to remove all traces of SDS. 13. Dissolve the precipitate in 30% acetic acid and subject it to automated Edman degradation.

M e d i u m Optimization The accumulation of the proper product willbe a balance between the final cell density, the specificproductivity,and the degradation of the product. Bacillus subtilis strain GX4935 containing pGX2143 ( a p r - s p a in pPL703) was grown in the following media: Medium A (tryptone, 33 g; yeast extract, 20 g; NaC1, 7.4 g; Na2HPO4, 8 g; KH2PO 4, 4 g; casamino acids, 20 g; glucose, 10 g; MnCI2, 0.06 mM; and NaOH to pH 7.5) Pen assay broth + 50/zM MnC12 2% yeast extract Glycerol-yeast (GY) extract medium (50 m M potassium phosphate, 2 m M MgC12, 0.7 m M CaC12, 50 # M MnC12, 5 # M FeC13, 1/zM ZnC12, 2 g M thiamin, 1% glycerol, 1% yeast extract, and 20 m M sodium glutamate) The highest accumulation of protein A was in medium A (Table I). Some proteolysis of the protein A was still observed, even though the host strain has deletions of the two major extracellular proteases. ~5,1~Although the yield of product was lower, proteolysis of the protein A was less in a glycerol-yeast extract medium. Optimization of the medium for each heterologous protein would probably be worthwhile. 15M. Y. Yang, E. Ferrari, and D. J. Henner, J. Bacteriol. 160, 15 (1984). 16S. R. Fahnestockand K. E. Fisher, Appl. Environ. Microbiol. 379 (1987).

[20]

INDUCIBLE EXPRESSION OF REGULATORY GENES

223

Acknowledgments I thank Mark Guyer, Stephen Fahnestock, Charles Saunders, and Ethel N. Jackson for critical discussions during the course of this work and Leo Thompson for the construction of aDr-spa. This work was performed at Gene× Corporation, Gaithersburg MD 20877.

[20] Inducible Expression of Regulatory Bacillus subtilis

G e n e s in

B y DENNIS J. H E N N E R

Introduction The ability to manipulate the expression of a gene of interest easily can be a valuable means to regulate gene expression. In Bacillus subtilis, the homologous recombination of nonreplicative plasmids into the chromosome makes it particularly simple to inactivate genes, to produce gene duplications, and to put any gene under the control of a regulated promoter. By this means, one can study the consequences of the loss of expression of an essential gene, or the inappropriate expression of a gene product. There are many examples of such studies in other organisms. For example, a system devised to put the Escherichia coli signal peptidase under control of the araC promoter was used to show that the signal peptidase was essential for cell viability.~ In yeast, the plasma membrane ATPase was put under the control of a galactose-inducible promoter, and it was shown that the cells were not viable unless inducer was present3 This article describes a very simple system for placing genes under control of an inducible promoter in B. subtilis. Design of Regulatory System As previously described in this volume by Le Grice) the simplest approach to an inducible promoter system in B. subtilis was to import one from E. coll. The hybrid promoter that we constructed, designated spac-1, contains the RNA polymerase recognition site from an early promoter of the B. subtilis phage SPO- 1 and the lac operator.' The lacI gene, encoding the lac repressor, was placed under the control of the Bacillus licheniformis penicillinase transcriptional and translational control signals to ensure expression in B. subtilis. 4 ' R. E. Dalbey and W. Wickner, J. Biol. Chem. 260, 15925 (1985). 2 A. Cid, R. Perona, and R. Serrano, Curr. Genet. 12, 105 (1987). 3 S. F. J. Le Grice, this volume, [18]. 4 D. Yansura and D. J. Henner, Proc. Natl. Acad Sci. U.S.A. 81, 439 (1984).

METHODS IN ENZYMOLOGY, VOL. 185

Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.

System for secretion of heterologous proteins in Bacillus subtilis.

214 EXPRESSIONIN B. subtilis [ 19] In the systems presented, foreign genes are expressed during logarithmic growth, taking advantage of the observa...
804KB Sizes 0 Downloads 0 Views