Degradation of Secreted Proteins in Escherichia COW FRANCOIS BANEYX AND GEORGE GEORGIOUb Department of Chemical Engineering University of Texas at Austin Austin, Texas 78712 INTRODUCTION Protein degradation in the host cell is a major impediment to the production of recombinant proteins in microorganisms. In some cases, proteolysis can be substantially reduced by using site-specific mutagenesis to remove protease-sensitive sequences in the protein of interest.' Alternatively, proteins can be stabilized by inducing the formation of inclusion bodies that are usually resistant to degradation.' Although these approaches for alleviating the degradation of several proteolytically sensitive proteins have proved useful in several cases, they can present a number of problems. For example, the rational modification of the polypeptide sequence by site-directed mutagenesis requires detailed structural information that is not readily available. Usually, a number of different mutants have to be constructed and tested before obtaining a stabilized protein. Similarly, the refolding of aggregated proteins in an active conformation may result in very low yields, particularly in the case of eukaryotic proteins containing multiple disulfide bonds. In addition, there is some evidence that proteases can coaggregate with the polypeptide of interest and cause degradation upon resolubilization.* Gilbert and Talmadge suggested that secretion in the periplasmic space of gram-negative bacteria can increase the half-life of certain protein^.^ However, recent evidence indicates that proteolysis can be as severe in the periplasmic space as in the ~ytoplasm."~Despite the importance of secretion for biotechnology applications, very little information is available on protein catabolism in the periplasmic space of E. coli. This article describes both genetic and biochemical engineering approaches for minimizing the degradation of useful secreted proteins. As a model polypeptide for the study of proteolysis in the periplasmic space of E. coli, we have used a hybrid protein constructed by the in-frame fusion of the genes encoding the Staphylococcus aureus protein A and the E. coli enzyme TEM p-lactamase. Protein A-p-lactamase is a fully bifunctional hybrid protein that is efficiently secreted in the periplasmic space where it is subjected to severe d e g r a d a t i ~ nWe . ~ have previously determined that the enzymatic activity of the fusion protein accurately reflects the amount of intact protein A-p-lactamase present in the cells because hydrolysis takes place mainly in the p-lactamase d ~ m a i nThis . ~ property makes the fusion protein a useful model for studying the degradation of secreted proteins. aThis work was supported in part by Grant No. CBT-8657471 from the National Science Foundation. bTowhom all correspondence should be addressed. 301
ANNALS NEW YORK ACADEMY OF SCIENCES
302
MATERIALS AND METHODS Strains and Plasmids The protease-deficient strains used in this study are described in TABLE1. Plasmid pFB3, a pBR322 derivative encoding the protein A-P-lactamase fusion p r ~ t e i nwas , ~ used for all experiments. Cell Fractionation and Penicillinase Assays Cells were grown for 24 h in shake flasks containing either 25 mL of LB medium (Difco) supplemented with 0.2% glucose and 50 pg/mL ampicillin or M9 medium containing 0.2% glucose, 0.2% casein amino acid hydrolysate, 50 pg/mL ampicillin, TABLE1. Genotypes of Different E. coli Mutants Deficient in Cell Envelope Proteases Strain BW9051
Inactivated Protease(s) Wild Type
TA301 KS272
Protease IV Wild Type
KS474 SFlOO SF103 SFllO SF115
DegP OmpT Protease 111 DegP & OmpT DegP& Protease 111 DegP, OmpT & Protease 111
SF120
Genotype' F- thrl leuB6proA2pps2pheSll xthA his4 atgE3 thil aral4 lacy1 galK2xyl5 mtll rpsL31tsx33 supE44 BW9051 &ppA-Knr F- AlacX74galE galK rhi rspL (strA) AphoA KS272 degP42 (APstl::RKnr) KS272 AompT KS272ptr32: :RCmr KS272degP42 (APstl::RKnr) AompT KS272 degP42 (APsknKn')ptr32::RCm' KS272 degP42 (APsrl::nKnr) AompT ptr32::RCmr
Reference 8 9 10
10 11 12 11 12 12
a A indicates a deletion; R indicates an insertion. Abbreviations: Kn', kanamycin resistance; Cmr, chloramphenicol resistance.
and other additives as indicated. Samples (3 mL) were centrifuged at 8000 rpm for 5 min. The supernatant was saved and the pellet was resuspended in 3 mL of 50 mM potassium phosphate buffer, pH 6.5, and disrupted at 20,000 psi in a French Press. The extract was recovered by centrifugation (10,000 rpm for 10 min) and the insoluble fraction was discarded. Enzymatic activities towards penicillin G (penicillinase activities) were determined by spectrophotometry as d e ~ c r i b e d Because .~ protein A-P-lactamase is secreted efficiently in E. coli, the enzymatic activity in whole lysates corresponds to the amount of (intact) fusion protein in the periplasmic space. Fermentations Fermentations were performed in Bioflo I11 fermentors with a 2-L working volume in LB medium supplemented with 0.2% glucose and 50 pg/mL ampicillin.
BANEYX & GEORGIOU: DEGRADATION
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For constant pH fermentations, the culture medium was adjusted to the desired pH value and controlled by automatically adding 1 M NaOH or 1 M HCl. Fermentors were inoculated with 50-mL overnight shake-flask cultures of SF120(pFB3) cells. Samples (3 mL) were taken for the first 14 h of growth and after 24 h. General Methods
Western blotting was performed as previously described.' Samples (10 kg) were separated in 15% SDS polyacrylamide gels, transferred to nitrocellulose, and probed with p-lactamase antiserum. Protein concentrations were determined by using the BioRad protein assay with bovine serum albumin as a standard.
RESULTS AND DISCUSSION Protease-deficientStrains
At present, seven proteolytic activities associated with the cell envelope of E. coli have been isolated. Four of these, DegP, OmpT, Protease 111, and Protease IV (encoded by genes degP, ompT, ptr, and sppA, respectively), have been cloned and characterized.IJ3 In an effort to minimize the degradation of secreted recombinant proteins and to study the function and possible overlap in the action of secreted proteases, we have constructed a set of isogenic strains deficient in up to three different cell envelope-associated proteolytic enzymes. All the mutants were derived from the wild-type strain KS272 (TABLE1). The DegP-deficient strain KS474 contains a kanamycin-resistance gene inserted in the degP gene.'" A strain lacking OmpT activity was constructed by P1 transduction of an ompT deletion into the KS272 genome." Finally, a single mutant lacking Protease 111, the ptr gene product, was obtained by insertional mutagenesis.12The expression of the fusion protein was enhanced twofold to threefold in strains singly deficient in DegP, OmpT, or Protease I11 (FIGURE1). Double mutants lacking DegP and OmpT or DegP and Protease I11 exhibited a cumulative increase in protein A-P-lactamase expression, indicating that the three proteases hydrolyze the fusion protein independently. However, a further increase in production in a mutant lacking DegP, OmpT, and Protease I11 was only observed in minimal medium, suggesting that the growth conditions exert a strong influence on protein turnover (FIGURE1B).IZImmunological detection of protein A-(3-lactamase expressed from the degP ompTptr mutant revealed the presence of several low molecular weight bands, suggesting some residual degradation in the periplasmic space (data not shown). This result indicates that additional secreted proteases cleave the fusion protein. To determine whether a fourth cloned cell envelope protease, Protease IV (the sppA gene product), is involved in the hydrolysis of the fusion protein, we measured the level of expression in the Protease IV-deficient strain TA301 and in the parental strain BW9051 transformed with plasmid pFB3. TABLE2 shows that the expression of protein A-P-lactamase from these strains was very low in LB medium (compare with FIGURE 1A). A relatively small (40%) increase in penicillinase specific activity was observed in the Protease IV mutant TA301 relative to the parental strain
304
ANNALS NEW YORK ACADEMY OF SCIENCES
BW9051 when cells were grown in LB medium. This increase was lower than the typical twofold to threefold increase previously observed with mutants lacking OmpT, DegP, or Protease II111s12and may be related to fluctuations in f3-lactamase activity observed in cultures grown in LB medium and expressing the fusion protein at a low level. Because the stabilization effect observed in single protease-deficient mutants is observed both in rich and in minimal media (see FIGURE l), the effect of Protease IV deactivation was also examined in M9 medium supplemented with 10 kg/mL thiamine. TABLE2 shows that the protein A-f3-lactamase specific activities
A: LB Medium
.-0
1
.-
8
c 0 Q)
P,
w
zz Q
3
.-=c -* .- .= .o .L
i2 -
6-
4 -
2 -
2
3
4
5
6
7
BANEYX & GEORGIOU: DEGRADATION
305
TABLE2. Effect of Protease I V Inactivation on the Expression of Protein A-p-Lactamase Total Penicillinase Specific Activity" (U/mg)
?
SD
Strain Genotype LB M9 Wild Type 1.7 ? 0.3 5.8 ? 0.8 BW905 1(pFB3) 2.4 ? 0.4 5.1 ? 0.7 SPPA TMOl(pFB3) aTotal penicillinase specific activities correspond to the ratio of the sum of activities in the extract and supernatant to the protein content in these fractions. SD stands for standard deviation expressed in U/mg and was calculated from at least six experiments. were increased relative to LB broth. However, there was no difference between the Protease IV mutant and the parental strain. It was concluded that Protease IV either is not involved or, at most, plays a very minor role in the degradation of the model substrate. This result is consistent with the observation that Protease IV is located exclusively on the inner membrane of E. coli, where it hydrolyzes the cleaved signal sequence of secreted proteins.14 Influence of the Growth Conditions In contrast to the inner membrane, the outer membrane of E. coli is freely permeable to small solutes.1s Consequently, the composition of the growth medium exerts a direct effect o n the periplasmic space microenvironment, which in turn may affect proteolysis. The fermentation p H was shown to exert a significant effect on the expression of protein A-p-lactarnase.l6 FIGURE 2 shows the maximum penicillinase activity obtained during the first 14 hours of fermentation as a function of the culture pH in the ompT degPptr mutant SF120 harboring plasmid pFB3. For this and other protease-deficient strains,I7 optimum activities were observed under mildly acidic growth conditions (5.5 IpH I6.0) and they rapidly decreased when the pH was maintained outside this range. To determine whether the observed increase in expression was related to a decrease in proteolysis, the proteins from the triple protease-deficient strain SF120(pFB3) grown at different p H values were analyzed by Western blotting. FIGURE 3 shows that the intensity of the band corresponding to the fusion protein increases at lower pH. In addition, the band corresponding to one of the major degradation products of protein A-p-lactamase7 (arrow 2 in FIGURE 3) becomes more pronounced as the pH rises. This result, along with direct measurement of the rate of degradation of the fusion protein in fermentors maintained at pH 6.0 or 7.0,17 suggests that acidic p H reduces the degradation of the model substrate. The growth temperature also influenced the degradation rate of the fusion protein with approximately a fourfold decrease in turnover rates at 30°C compared to 42 'C.I7 In contrast, the dissolved oxygen concentration in the range between 80% and 30% did not have any effect on the expression of the fusion protein (data not shown). Hydroxylated compounds increase the thermodynamic stability of proteins in vitro by a mechanism known as preferential hydration.18JY In the case of TEM p-lactamase, the presence of 0.3 M sucrose increases the free energy of folding by
ANNALS NEW YORK ACADEMY OF SCIENCES
306
approximately 2.0 kJ/mol (P. Valax, unpublished results). In vivo, the addition of sucrose into the growth medium was found to inhibit the formation of p-lactamase inclusion bodies in the periplasmic space, thus favoring the formation of the correctly folded protein.20 Sucrose and other nonmetabolizable carbohydrates diffuse freely into the periplasmic space, but cannot be transported through the inner membrane and thus are excluded from the cytoplasm. Detailed studies have shown that the inhibition of inclusion body formation in the periplasmic space is related specifically to the stabilizing effect of these additives and not to metabolic changes in the Because it has been suggested that increased thermodynamic stability correlates with greater stability against p r o t e o l y ~ i s , ~we ~-~ ~ investigated the hypothesis that the addition of nonmetabolizable sugars may suppress the degradation of protein A-P-lactamase. Shake-flask cultures of KS474(pFB3) were grown in LB medium for 24 hours in the presence of 0.2% glucose, 50 kg/mL ampicillin, and 0.1 to 0.3 M NaCI, sucrose, or raffinose. NaCl does not affect the thermodynamic stability of proteins and was added to control cultures to account for the effect of changes in osmotic pressure. No difference could be observed in the presence or absence of raffinose. In both cases, the total penicillinase specific activity was approximately 48 U/mg of total soluble protein. Cultures grown in the presence of sucrose consistently gave about 20% lower activities, but this difference was not statistically significant. These results indicate that the addition of protein-stabilizing agents does not affect the degradation of protein A-P-lactamase in vivo. Nevertheless, the failure to inhibit degradation may be dependent on the specific recombinant protein. Stabilizing cosolvents such as sucrose increase the free energy of unfolding, which is a global measure of thermodynamic stability. It is possible that the local unfolding of certain flexible, protease-accessible regions is not affected even though the protein is more
70 60 50 40
30 20 10 0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
FIGURE 2. Effect of the culture pH on the expression of protein A-P-lactamase from the degP ompTptr strain SF120 harboring plasmid pFB3. The maximum penicillinase activities in the extract and supernatant fractions obtained during the first 14 hours of fermentation are plotted versus the pH. Adapted from reference 17.
307
BANEYX 8~GEORGIOU: DEGRADATION
234M SPA-bla -+ 1-
50
2-
39 27
FIGURE 3. Western blot of the total cell extract proteins from samples corresponding to the maximum penicillinase activity obtained during constant-pH fermentations with SF120(pFB3) cells. Lanes: 1, pH 5.5; 2, pH 6.0; 3, pH 6.5; 4, pH 7.0; M, molecular weight markers. The positions of protein A-p-lactamase (SpA-bla) and a major carboy-terminal degradation product of the fusion protein (arrow 2) are shown. Adapted from reference 17.
stable against unfolding. This may be the case for our model polypeptide. Hence, we believe that the usefulness of sugar addition to the growth medium of cells synthesizing unstable secreted polypeptides must be examined on a case-by-case basis.
CONCLUSIONS Overall, our results show that strains deficient in multiple secreted proteases are useful for reducing proteolysis in vivo. Specifically, we have demonstrated that (i) both OmpT and Protease I11 play a significant role in the degradation of secreted proteins, (ii) at least three proteases independently degrade the fusion protein, (iii) mutations in secreted protease genes have an additive effect on the stabilization of protease-sensitive polypeptides, and (iv) Protease IV does not participate in the catabolism of protein A-P-lactamase. The protease-deficient strains described in this study can be used in combination with optimal growth conditions (e.g., low pH) in order to reduce the degradation of secreted recombinant proteins in E. cofi.
ACKNOWLEDGMENTS We are grateful to Todd Roth and Amanda Ayling for their help with part of this work.
308
ANNALS NEW YORK ACADEMY OF SCIENCES REFERENCES
1992.In Stability of Protein Pharmaceuticals: Chemical and 1. BANEYX, F. & G. GEORGIOU. Physical Pathways of Protein Degradation. Plenum. New York. 2. BABBITT, P. C., B. L. WEST,D. D. BUETCHER, I. D. KUNTZ& G. L. KENYON. 1990. Bio/Technology8: 945-949. 3. TALMADGE, K. & W. GILBERT. 1982. Proc. Natl. Acad. Sci. U.S.A. 7 9 1830-1833. 4. ANBA,J., A. BERNADAC, C. LAZDUNSKI & J. M. PAGES.1988. Biochimie 7 0 727-733. 5. GREENWOOD, J. M., N. R. GILKES,D. G. KILBURN, R. C. MILLER, JR.& R. A. J. WARREN. FEBS Lett. 244: 127-131. H. TAATJES, W. BANNWARTH, D. STUEBER & 6. GENTZ,R.,Y. KUYS,C. ZWIEB,D. TAATJES, I. IBRAHIMI. 1988.J. Bacteriol. 170 2212-2220. 7. BANEYX, F. & G. GEORGIOU. 1989. Enzyme Microb. Technol. 11: 559-567. 8. MILCAREK, C. & B. WEISS.1972. J. Mol. Biol. 6 8 303-318. 9. SUZUKI, T., A. ITOH,S. ICHIHARA & S. MIZUSHIMA. 1987.J. Bacteriol. 169 2523-2528. 10. STRAUCH, K. L., K. JOHNSON & J. BECKWITH. 1989. J. Bacteriol. 171: 2689-2696. F. & G. GEORGIOU. 1990. J. Bacteriol. 172: 491-494. 11. BANEYX, F. & G. GEORGIOU. 1991. J. Bacteriol. 173: 2696-2703. 12. BANEYX, 13. LAZDUNSKI, A. M. 1989. FEMS Microbiol. Rev. 63: 265-276. 14. ICHIHARA, S., N. BEPPU& S. MIZUSHIMA. 1984. J. Biol. Chem. 259 9853-9857. 15. CHENG, Y-S.E. & D. ZIPSER.1979. J. Biol. Chem. 254 4698-4706. 16. DECAD,G. M. & H. NIKAIDO.1976. J. Bacteriol. 128: 325-336. T. PALUMBO, D. THOMAS & G. GEORGIOU. 1991.Appl. Microbiol. 17. BANEYX, F., A. AYLING, Biotechnol. 3 6 14-20. 1981. J. Biol. Chem. 256 7193-7201. 18. LEE,J. C. & S . N. TIMASHEFF. 1981. Biochemistry21: 6536-6544. 19. ARAKAWA, T. & S. N. TIMASHEFF. G. A. & G. GEORGIOU. 1988. Biotechnol. Prog. 4 97-101. 20. BOWDEN, 1990. J. Biol. Chem. 265: 16760-16766. 21. BOWDEN, G. A. & G. GEORGIOU. 22. MCLENDON, G. & E. RADANY. 1978. J. Biol. Chem. 253: 6335-6337. 1986. J. Biol. Chem. 261: 15430-15436. 23. ROTE,K. V. & M. RECHSTEINER. 1989. J. Biol. Chem. 264: 7590-7595. 24. PARSELL, D. A. & R. T. SAUER.
ANNALS NEW YORK ACADEMY OF SCIENCES
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MATERIALS AND METHODS Strains and Plasmids The protease-deficient strains used in this study are described in TABLE1. Plasmid pFB3, a pBR322 derivative encoding the protein A-P-lactamase fusion p r ~ t e i nwas , ~ used for all experiments. Cell Fractionation and Penicillinase Assays Cells were grown for 24 h in shake flasks containing either 25 mL of LB medium (Difco) supplemented with 0.2% glucose and 50 pg/mL ampicillin or M9 medium containing 0.2% glucose, 0.2% casein amino acid hydrolysate, 50 pg/mL ampicillin, TABLE1. Genotypes of Different E. coli Mutants Deficient in Cell Envelope Proteases Strain BW9051
Inactivated Protease(s) Wild Type
TA301 KS272
Protease IV Wild Type
KS474 SFlOO SF103 SFllO SF115
DegP OmpT Protease 111 DegP & OmpT DegP& Protease 111 DegP, OmpT & Protease 111
SF120
Genotype' F- thrl leuB6proA2pps2pheSll xthA his4 atgE3 thil aral4 lacy1 galK2xyl5 mtll rpsL31tsx33 supE44 BW9051 &ppA-Knr F- AlacX74galE galK rhi rspL (strA) AphoA KS272 degP42 (APstl::RKnr) KS272 AompT KS272ptr32: :RCmr KS272degP42 (APstl::RKnr) AompT KS272 degP42 (APsknKn')ptr32::RCm' KS272 degP42 (APsrl::nKnr) AompT ptr32::RCmr
Reference 8 9 10
10 11 12 11 12 12
a A indicates a deletion; R indicates an insertion. Abbreviations: Kn', kanamycin resistance; Cmr, chloramphenicol resistance.
and other additives as indicated. Samples (3 mL) were centrifuged at 8000 rpm for 5 min. The supernatant was saved and the pellet was resuspended in 3 mL of 50 mM potassium phosphate buffer, pH 6.5, and disrupted at 20,000 psi in a French Press. The extract was recovered by centrifugation (10,000 rpm for 10 min) and the insoluble fraction was discarded. Enzymatic activities towards penicillin G (penicillinase activities) were determined by spectrophotometry as d e ~ c r i b e d Because .~ protein A-P-lactamase is secreted efficiently in E. coli, the enzymatic activity in whole lysates corresponds to the amount of (intact) fusion protein in the periplasmic space. Fermentations Fermentations were performed in Bioflo I11 fermentors with a 2-L working volume in LB medium supplemented with 0.2% glucose and 50 pg/mL ampicillin.
BANEYX & GEORGIOU: DEGRADATION
303
For constant pH fermentations, the culture medium was adjusted to the desired pH value and controlled by automatically adding 1 M NaOH or 1 M HCl. Fermentors were inoculated with 50-mL overnight shake-flask cultures of SF120(pFB3) cells. Samples (3 mL) were taken for the first 14 h of growth and after 24 h. General Methods
Western blotting was performed as previously described.' Samples (10 kg) were separated in 15% SDS polyacrylamide gels, transferred to nitrocellulose, and probed with p-lactamase antiserum. Protein concentrations were determined by using the BioRad protein assay with bovine serum albumin as a standard.
RESULTS AND DISCUSSION Protease-deficientStrains
At present, seven proteolytic activities associated with the cell envelope of E. coli have been isolated. Four of these, DegP, OmpT, Protease 111, and Protease IV (encoded by genes degP, ompT, ptr, and sppA, respectively), have been cloned and characterized.IJ3 In an effort to minimize the degradation of secreted recombinant proteins and to study the function and possible overlap in the action of secreted proteases, we have constructed a set of isogenic strains deficient in up to three different cell envelope-associated proteolytic enzymes. All the mutants were derived from the wild-type strain KS272 (TABLE1). The DegP-deficient strain KS474 contains a kanamycin-resistance gene inserted in the degP gene.'" A strain lacking OmpT activity was constructed by P1 transduction of an ompT deletion into the KS272 genome." Finally, a single mutant lacking Protease 111, the ptr gene product, was obtained by insertional mutagenesis.12The expression of the fusion protein was enhanced twofold to threefold in strains singly deficient in DegP, OmpT, or Protease I11 (FIGURE1). Double mutants lacking DegP and OmpT or DegP and Protease I11 exhibited a cumulative increase in protein A-P-lactamase expression, indicating that the three proteases hydrolyze the fusion protein independently. However, a further increase in production in a mutant lacking DegP, OmpT, and Protease I11 was only observed in minimal medium, suggesting that the growth conditions exert a strong influence on protein turnover (FIGURE1B).IZImmunological detection of protein A-(3-lactamase expressed from the degP ompTptr mutant revealed the presence of several low molecular weight bands, suggesting some residual degradation in the periplasmic space (data not shown). This result indicates that additional secreted proteases cleave the fusion protein. To determine whether a fourth cloned cell envelope protease, Protease IV (the sppA gene product), is involved in the hydrolysis of the fusion protein, we measured the level of expression in the Protease IV-deficient strain TA301 and in the parental strain BW9051 transformed with plasmid pFB3. TABLE2 shows that the expression of protein A-P-lactamase from these strains was very low in LB medium (compare with FIGURE 1A). A relatively small (40%) increase in penicillinase specific activity was observed in the Protease IV mutant TA301 relative to the parental strain
304
ANNALS NEW YORK ACADEMY OF SCIENCES
BW9051 when cells were grown in LB medium. This increase was lower than the typical twofold to threefold increase previously observed with mutants lacking OmpT, DegP, or Protease II111s12and may be related to fluctuations in f3-lactamase activity observed in cultures grown in LB medium and expressing the fusion protein at a low level. Because the stabilization effect observed in single protease-deficient mutants is observed both in rich and in minimal media (see FIGURE l), the effect of Protease IV deactivation was also examined in M9 medium supplemented with 10 kg/mL thiamine. TABLE2 shows that the protein A-f3-lactamase specific activities
A: LB Medium
.-0
1
.-
8
c 0 Q)
P,
w
zz Q
3
.-=c -* .- .= .o .L
i2 -
6-
4 -
2 -
2
3
4
5
6
7
BANEYX & GEORGIOU: DEGRADATION
305
TABLE2. Effect of Protease I V Inactivation on the Expression of Protein A-p-Lactamase Total Penicillinase Specific Activity" (U/mg)
?
SD
Strain Genotype LB M9 Wild Type 1.7 ? 0.3 5.8 ? 0.8 BW905 1(pFB3) 2.4 ? 0.4 5.1 ? 0.7 SPPA TMOl(pFB3) aTotal penicillinase specific activities correspond to the ratio of the sum of activities in the extract and supernatant to the protein content in these fractions. SD stands for standard deviation expressed in U/mg and was calculated from at least six experiments. were increased relative to LB broth. However, there was no difference between the Protease IV mutant and the parental strain. It was concluded that Protease IV either is not involved or, at most, plays a very minor role in the degradation of the model substrate. This result is consistent with the observation that Protease IV is located exclusively on the inner membrane of E. coli, where it hydrolyzes the cleaved signal sequence of secreted proteins.14 Influence of the Growth Conditions In contrast to the inner membrane, the outer membrane of E. coli is freely permeable to small solutes.1s Consequently, the composition of the growth medium exerts a direct effect o n the periplasmic space microenvironment, which in turn may affect proteolysis. The fermentation p H was shown to exert a significant effect on the expression of protein A-p-lactarnase.l6 FIGURE 2 shows the maximum penicillinase activity obtained during the first 14 hours of fermentation as a function of the culture pH in the ompT degPptr mutant SF120 harboring plasmid pFB3. For this and other protease-deficient strains,I7 optimum activities were observed under mildly acidic growth conditions (5.5 IpH I6.0) and they rapidly decreased when the pH was maintained outside this range. To determine whether the observed increase in expression was related to a decrease in proteolysis, the proteins from the triple protease-deficient strain SF120(pFB3) grown at different p H values were analyzed by Western blotting. FIGURE 3 shows that the intensity of the band corresponding to the fusion protein increases at lower pH. In addition, the band corresponding to one of the major degradation products of protein A-p-lactamase7 (arrow 2 in FIGURE 3) becomes more pronounced as the pH rises. This result, along with direct measurement of the rate of degradation of the fusion protein in fermentors maintained at pH 6.0 or 7.0,17 suggests that acidic p H reduces the degradation of the model substrate. The growth temperature also influenced the degradation rate of the fusion protein with approximately a fourfold decrease in turnover rates at 30°C compared to 42 'C.I7 In contrast, the dissolved oxygen concentration in the range between 80% and 30% did not have any effect on the expression of the fusion protein (data not shown). Hydroxylated compounds increase the thermodynamic stability of proteins in vitro by a mechanism known as preferential hydration.18JY In the case of TEM p-lactamase, the presence of 0.3 M sucrose increases the free energy of folding by
ANNALS NEW YORK ACADEMY OF SCIENCES
306
approximately 2.0 kJ/mol (P. Valax, unpublished results). In vivo, the addition of sucrose into the growth medium was found to inhibit the formation of p-lactamase inclusion bodies in the periplasmic space, thus favoring the formation of the correctly folded protein.20 Sucrose and other nonmetabolizable carbohydrates diffuse freely into the periplasmic space, but cannot be transported through the inner membrane and thus are excluded from the cytoplasm. Detailed studies have shown that the inhibition of inclusion body formation in the periplasmic space is related specifically to the stabilizing effect of these additives and not to metabolic changes in the Because it has been suggested that increased thermodynamic stability correlates with greater stability against p r o t e o l y ~ i s , ~we ~-~ ~ investigated the hypothesis that the addition of nonmetabolizable sugars may suppress the degradation of protein A-P-lactamase. Shake-flask cultures of KS474(pFB3) were grown in LB medium for 24 hours in the presence of 0.2% glucose, 50 kg/mL ampicillin, and 0.1 to 0.3 M NaCI, sucrose, or raffinose. NaCl does not affect the thermodynamic stability of proteins and was added to control cultures to account for the effect of changes in osmotic pressure. No difference could be observed in the presence or absence of raffinose. In both cases, the total penicillinase specific activity was approximately 48 U/mg of total soluble protein. Cultures grown in the presence of sucrose consistently gave about 20% lower activities, but this difference was not statistically significant. These results indicate that the addition of protein-stabilizing agents does not affect the degradation of protein A-P-lactamase in vivo. Nevertheless, the failure to inhibit degradation may be dependent on the specific recombinant protein. Stabilizing cosolvents such as sucrose increase the free energy of unfolding, which is a global measure of thermodynamic stability. It is possible that the local unfolding of certain flexible, protease-accessible regions is not affected even though the protein is more
70 60 50 40
30 20 10 0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
FIGURE 2. Effect of the culture pH on the expression of protein A-P-lactamase from the degP ompTptr strain SF120 harboring plasmid pFB3. The maximum penicillinase activities in the extract and supernatant fractions obtained during the first 14 hours of fermentation are plotted versus the pH. Adapted from reference 17.
307
BANEYX 8~GEORGIOU: DEGRADATION
234M SPA-bla -+ 1-
50
2-
39 27
FIGURE 3. Western blot of the total cell extract proteins from samples corresponding to the maximum penicillinase activity obtained during constant-pH fermentations with SF120(pFB3) cells. Lanes: 1, pH 5.5; 2, pH 6.0; 3, pH 6.5; 4, pH 7.0; M, molecular weight markers. The positions of protein A-p-lactamase (SpA-bla) and a major carboy-terminal degradation product of the fusion protein (arrow 2) are shown. Adapted from reference 17.
stable against unfolding. This may be the case for our model polypeptide. Hence, we believe that the usefulness of sugar addition to the growth medium of cells synthesizing unstable secreted polypeptides must be examined on a case-by-case basis.
CONCLUSIONS Overall, our results show that strains deficient in multiple secreted proteases are useful for reducing proteolysis in vivo. Specifically, we have demonstrated that (i) both OmpT and Protease I11 play a significant role in the degradation of secreted proteins, (ii) at least three proteases independently degrade the fusion protein, (iii) mutations in secreted protease genes have an additive effect on the stabilization of protease-sensitive polypeptides, and (iv) Protease IV does not participate in the catabolism of protein A-P-lactamase. The protease-deficient strains described in this study can be used in combination with optimal growth conditions (e.g., low pH) in order to reduce the degradation of secreted recombinant proteins in E. cofi.
ACKNOWLEDGMENTS We are grateful to Todd Roth and Amanda Ayling for their help with part of this work.
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ANNALS NEW YORK ACADEMY OF SCIENCES REFERENCES
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