activity, and increased the purity of extracellular I-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30°C was difficult due to poor cell ...
Vol. 56, No. 1
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1990, p. 104-111
0099-2240/90/010104-08$02.00/0 Copyright © 1990, American Society for Microbiology
Effects of Temperature on Escherichia coli Overproducing 3-Lactamase or Human Epidermal Growth Factor JEFFREY J.
CHALMERS,lt EUNKI KIM,' JOHN N. TELFORD,2
EDITH Y. WONG,3 WILLIAM C. TACON,3 MICHAEL L. SHULER,' AND DAVID B. WILSON2* School of Chemical Engineering' and Section of Biochemistry, Molecular and Cell Biology,2 Cornell University, Ithaca, New York 14853, and Department of Biological Sciences, Monsanto Company, Chesterfield, Missouri 631983 Received 14 April 1989/Accepted 4 October 1989
The effects of temperature on strains of Escherichia coli which overproduce and excrete either P-lactamase human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for P-lactamase were grown and induced with isopropyl-"D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20°C. The lower temperature greatly reduced the formation of periplasmic j8-lactamase inclusion bodies, increased significantly the total amount of I8-lactamase activity, and increased the purity of extracellular I-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30°C was difficult due to poor cell reproduction and P-lactamase production. However, at 20°C, continuous production and excretion of f8-lactamase were obtained for >450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37°C, whereas little cell lysis was observed at 21 and 25°C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble. or
Advances in molecular biology allow the production of high levels of a desired protein, using plasmid vectors in Escherichia coli. In some cases, 50% of the cell protein is the target protein. However, major problems encountered with E. coli as a host include segregational and structural instability of the plasmid, especially in continuous culture (10); precipitation of the target protein within the cell; and lysis of cells after induction (especially when a strong promoter is used). When large amounts of a protein are produced, it almost always precipitates inside the cell cytoplasm in what have been called inclusion bodies (17, 23, 24). The protein found in these inclusion bodies is inactive. To obtain active protein, the inclusion bodies usually must be solubilized with denaturing agents, and then the denaturing agents must be removed slowly under the appropriate conditions of pH, ionic strength, protein concentration, and redox potential for optimal protein refolding and disulfide bond formation (18, 20, 22). The process of solubilizing, denaturing, and renaturing proteins into an active form can be difficult, time-consuming, and expensive on a commercial scale and often produces low yields of active protein. We have previously reported that E. coli RB791 containing plasmid pTacll will produce and excrete large amounts of normally periplasmic 1-lactamase into the extracellular solution (6, 7). The release of >95% of the P-lactamase activity to the extracellular compartment is not due to lysis, as indicated by low levels (90%). Quantification of the number of inclusion bodies per cell at various temperatures is summarized in Table 3. Electron micrographs of negatively stained sectioned cells shows that cells grown at 37'C contained many periplasmic inclusion bodies (average of 3.2 per cell) as had been reported previously (8). However, cells grown at 30'C contained fewer inclusion bodies (average, 0.5 per cell), while cells grown at 25 or 20'C contained about 0.35 inclusion body per cell. Thus, the total amount of P-lactamase in inclusion bodies at 20'C is about 10% of that at 37°C. Further evidence for a decrease in inclusion bodies in cells grown at 20'C came from phase-contrast light microscopy as >95% of the cells grown at 37'C showed bulges and distortions which were not present at 20 or 25°C, where 80% of the transformants with plasmid recovered from cells subjected to extended exposure to IPTG do produce and excrete normal levels of P-lactamase activity (unpublished data). These results suggest that the plasmid remained largely unchanged during chemostat operation. That most of the plasmid has apparently remained unaltered suggests that the host cell has changed. Colonies recovered directly from the chemostat at 252 h showed reduced productivity (ca. 85% of normal) and low excretion (ca. 30%) when cultured at 37'C in shake flasks. Also, cells
108
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CHALMERS ET AL.
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150
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FIG. 3. Response of a chemostat at 200C with RB791(pKN) to induction with 10-4 M IPTG. The OD6. (0) and the total (A) and supernatant (-) concentrations of P-lactamase are plotted as a function of time. At point A, IPTG was added to the culture and feed reservoir to obtain a final concentration of 10-4 M. At point B, the dilution rate was reduced from 0.113 to 0.033 h-1. At point C, samples of cells were removed for further analysis (see text). In this experiment, glucose was the limiting nutrient in a medium which also contained casein amino
acids, M9 salt solution, and 50 ,ug of neomycin per ml, which provided selective pressure.
recovered at 252 h were used to inoculate a new chemostat which operated for more than 200 h in the presence of 1O' M IPTG. This satellite chemostat behaved similarly to the last 200 h of operation of the chemostat in Fig. 3 with respect to total cell concentration (OD) and total P-lactamase production. From the data in Fig. 3 it can be calculated that the specific productivity was 25 U of ,-lactamase activity per OD per h shortly after induction, and the productivity fell to nearly zero before recovering to ca. 3.0 U/OD per h (the 150- to 400-h period). Reduced specific productivity upon extended exposure to IPTG has been observed in all chemostat experiments. Figure 4 shows the results of an experiment in which P-lactamase production was induced with a lower concentration of IPTG (10-' M) at point A. When P-lactamase production was induced with 10-5 M IPTG, the rate of production rapidly increased and equally rapidly decreased without the concurrent decrease in OD. This rapid drop in productivity suggests that a specific inhibition of P-lactamase production or rapid degradation is the cause of the drop and not simply a selection for cells which produce less P-lactamase (the rapid drop took place in less than one generation time). TABLE 4. Specific activity of ,B-lactamase in the supernatant and percentage of extracellular protein which is f-lactamase in the supernatant at various times during the operation of the continuous culture presented in Fig. 2 Time (h)
3-Lactamase
(U/ml)
Sp act (U/mg)
% Protein ,-lactamase'
43.5 109 145
27.8 54.5 92 160 143 180 123 160
728 1,500 1,600 2,090 2,820 2,710 2,250 2,770
21 43 46 60 81 77 64 79
179 210 299 322 346
a One milligram of purified
P-lactamase corresponds to 3,500 U of activity.
In batch culture, we have shown that 10-5 M IPTG results in only partial induction of the tac promoter with little excretion (7); the sustained exposure to 10-5 M IPTG in chemostat culture results in a higher level of P-lactamase production than in batch-grown cells. At point B, IPTG was added to give a final concentration in the chemostat and feed reservoir of i0-' M. After both changes in IPTG concentration, the rate of P-lactamase productivity rapidly increased and decreased while the cell concentration stayed relatively constant. The specific productivities shortly after induction with 10-5 M IPTG were 11 and 10 U/OD per h after point B (10-' M IPTG). The sustained level of specific productivity was 2 U/OD per h (225 to 350 h). The decreased responsiveness of cells that had undergone prior IPTG induction to induction with l0o' M IPTG has been observed under a variety of culture conditions and is indicative of complex metabolic and regulatory interactions. The same general observations (temperature effect and rapid drop in productivity) have been seen with a different strain, JM109, containing the pKN plasmid. These similar observations indicate that these results are not specific to this particular strain of E. coli. With the JM109(pKN) combination, continuous production and excretion of Ilactamase has been observed for >880 h or 47 generations (J. J. Chalmers, Ph.D. thesis). Effects of temperature on hEGF production. When RB791(pCU) cells were grown and induced with IPTG at 31 and 37°C, >50% of the total cellular protein was found in the media. Also, the proteins in the media were almost identical to those in cell lysates, indicating cell lysis at these temperatures (data not shown). However, very little cell lysis, based on the excretion of total protein and 3-galactosidase, was observed at 21 and 25°C. Transmission electron microscopy of cells grown at different temperatures showed a large number of disintegrated cells at 31 and 37°C. However, almost all cells were intact at 21 and 25°C. Excessive synthesis of plasmid-encoded proteins in induced cultures at higher temperature probably decreased the production or transport of outer membrane proteins destroying the integrity of the cell envelope, causing cell lysis (6).
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350 OD6.
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supernatant (-) concentrations of ,B-lactamase are plotted as a function of time. At point A, IPTG was added to the culture and feed reservoir to obtain a final concentration of l0-5 M IPTG. At point B, IPTG was added to the culture and feed reservoir to obtain a final concentration of 1o-4 M. Note the lack of cell washout in this experiment as compared with the experiment presented in Fig. 2. The dilution rate was constant throughout the experiment at 0.06 h-1, and glucose was the limiting nutrient in a medium which also contained casein amino acids, M9 salt solution, and 50 ,ug of neomycin per ml, which provided selective pressure.
The relative amount of hEGF, expressed as the specific productivity (micrograms of hEGF per milligram of total protein), increased as the culturing temperature decreased. A specific productivity of 70 ,ug of hEGF per mg of total protein was obtained at 21°C (Fig. 4). The fraction of hEGF in the media, soluble cell lysate, and insoluble pellet varied at different temperatures. The increased fraction of hEGF in the medium at 31 and 37°C was due to cell lysis at these temperatures. Some 5 and 20% of hEGF was insoluble at 21 and 25°C, respectively (Fig. 5). Whether insoluble hEGF exists as inclusion bodies is not clear, since the presence of inclusion bodies was not detected by electron microscopy even at very high magnification (data not shown). Note that the total amount of hEGF made at 21 and 25°C was almost twice that at 370C. The optimum temperature for the excretion of hEGF into the media was 25°C. At this temperature, hEGF was the major protein in the medium based on the results of SDSpolyacrylamide gel electrophoresis of total excreted proteins in the media (data not shown). At 25°C, high productivity (11 jig of hEGF per OD per ml) was obtained with 40% excretion in the absence of significant cell lysis (Fig. 6). DISCUSSION
These results can be compared with the previous reports on the effects of temperature on the cytoplasmic production
of IFN-,B (12), IFN-L2, IFN-2, and IFN-induced murine protein MX (16). With two secreted proteins (3-lactamase and hEGF), we have demonstrated a decrease in the formation of insoluble protein at lower growth temperatures. Unlike the previous reports, we have shown a significant increase in total plasmid-encoded protein synthesis with hEGF and a smaller increase with 3-lactamase. The mechanism by which decreased growth temperature might increase the total amount of P-lactamase and hEGF formed is not known, nor is why such increases are observed for
secreted proteins tested and not for the cytoplasmic proteins studied in other reports (12, 16). However, it should be noted that ,-lactamase degradation is essentially nil extracellularly, but measurable in cell lysates. Although cell lysis at higher temperatures was observed with hEGF production, total protein production was higher at 30 and 37°C than at 20 and 25°C, suggesting that the reduced hEGF production at higher temperatures was not due to decreased protein synthesis capacity. Although decreased growth temperature was beneficial for both P-lactamase and hEGF production, differences in the responses were observed. Extensive cell lysis was observed at 30 and 37°C upon induction of hEGF synthesis from the tac promoter, while P-lactamase synthesis from the same promoter did not cause significant lysis. The two primary differences in these systems are the nature of the protein itself and the signal sequence used. P-Lactamase is a normal E. coli periplasmic protein and was produced with its own signal sequence. Obviously, hEGF is a non-E. coli protein and was fused to the OmpA signal sequence. Our previous work with ,B-lactamase has shown that overproduction from the tac promoter can reduce synthesis (but not processing) of two outer membrane proteins (OmpA and OmpC), and excreting cells have an altered outer membrane (7). This work also demonstrates that decreasing growth temperature allows successful chemostat operation for E. coli RB791(pKN). At 37°C, cellular reproduction is at an insufficient rate to maintain a chemostat at D = 0.10 h-1, and cell yield and P-lactamase production are low at D = 0.03 h-1. A rather stable culture can be obtained and maintained at 20°C for an extended period in the presence of neomycin (50 0.06 h-1. Although such cultures are mg/liter) at D dominated by cells capable of forming neomycin-resistant colonies, the population is not homogeneous and contains a significant number of apparently plasmid-free cells. Nonetheless, such chemostats provide a tool to begin to probe the physiology of protein-excreting E. coli at low temperatures.
110
CHALMERS ET AL.
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ACKNOWLEDGMENTS The technical assistance of Shirin Zartoshty is gratefully acknowledged. We also acknowledge the supportive advice and assistance of Bruce Bishop at Monsanto. This work was supported, in part, by a grant from the National Science Foundation, EET 8513612. The work on hEGF production was supported, in part, by a grant from Monsanto Co. LITERATURE CITED 1. Amann, E., J. Brosius, and M. Ptashne. 1983. Vectors bearing a hybrid trp-lac promoter for regulated gene expression of cloned genes in Escherichia coli. Gene 25:167-178. 2. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-527. 3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248-252. 4. Brent, R., and M. Ptashne. 1981. Mechanism of action of the lexA gene product. Proc. Natl. Acad. Sci. USA 78:4204-4208. 5. Fikes, J. D., and P. J. Bassford. 1987. Export of unprocessed precursor maltose-binding protein to the periplasm of Escherichia coli cells. J. Bacteriol. 169:2352-2359. 6. Georgiou, G, J. J. Chalmers, and M. L. Shuler. 1985. Continuous immobilized recombinant protein production from E. coli capable of selective protein excretion: a feasibility study. Biotechnol. Prog. 1:75-79. 7. Georgiou, G., M. L. Shuler, and D. B. Wilson. 1988. Release of periplasmic enzymes and other physiological effects of,B-lactamase overproduction in Escherichia coli. Biotechnol. Bioeng. 32:741-748. 8. Georgiou, G., J. N. Telford, M. L. Shuler, and D. B. Wilson. 1986. Localization of inclusion bodies in Escherichia coli overproducing P-lactamase or alkaline phosphatase. Appl. Environ. Microbiol. 52:1157-1161. 9. Hall, M. N., M. Schwartz, and T. J. Silhavy. 1982. Sequence information within the LamB gene is required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12. J. Mol. Biol. 156:93-112. 10. Imanaka, T., and S. Aiba. 1981. A perspective on the application of genetic engineering: stability of recombinant plasmid. Ann. N.Y. Acad. Sci. 369:1-14. 11. Kitano, K., S. Fryimoto, M. Nakoa, T. Watanabe, and Y. Nakao. 1987. Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells. J. Biotechnol. 5:77-86. 12. Mizukami, T., Y. Komatsu, N. Hosoi, and T. Oka. 1986. Production of active interferon-B in E. coli I. Preferential production by lower culture temperature. Biotechnol. Lett. 8:605-610. 13. Nugent, M. E., S. B. Primrose, and W. C. A. Tacon. 1983. The stability of recombinant DNA. Dev.Ind. Microbiol. 24:271-285. 14. Oka, K., S. Sumi, T. Fuwa, K. Yoda, M. Yamasaki, T. Tamura, and T. Miyake. 1987. Efficacies of different secretion vectors for secretion of human epidermal growth factor by Escherichia coli. Agric. Biol. Chem. 51:1099-1104. 15. Samuni, A. 1975. A direct spectrophotometric assay and determination of Michaelis constant for the 3-lactamase reaction. Anal. Biochem. 63:17-26. 16. Schein, C., and M. Noteborn. 1988. Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Bio/Technology 6:291-294. 17. Sharma, S. K. 1986. Recovery of soluble human renin from inclusion bodies produced in recombinant Escherichia coli. J. Biotechnol. 4:119-124. 18. Sharma, S. K., D. B. Evans, C. S. C. Tomich, J. C. Cornette, and R. G. Ulnich. 1987. Folding and activation of recombinant human prorenin. Biotechnol. Appl. Biochem. 9:181-193. 19. Tacon, W., N. Carey, and S. Emtage. 1980. The construction and characterisation of plasmid vectors suitable for the expression of all DNA phases under the control of the E. coli tryptophan promoter. Mol. Gen. Genet. 177:427-438. 20. Taniguchi, T., L. Guarente, T. M. Roberts, D. Kimelman, J. Douhan HI, and M. Ptashne. 1980. Expression of the human
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fibroblast interferon gene in Escherichia coli. Proc. Natl. Acad. Sci. USA 77:5230-5233. 21. Tommassen, J., J. Leunissen, J. M. Damma, and P. Overduin. 1985. Failure of E. coli K-12 to transport phoE-lacZ hybrid proteins out of the cytoplasm. EMBO J. 4:1041-1047. 22. Tsuji, T., R. Nakagawa, N. Sugamoto, and K. Fukuhara. 1987. Characterization of disulfide bonds in recombinant proteins:
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ill
reduced human interleukin 2 in inclusion bodies and its oxidative refolding. Biochemistry 26:3129-3134. 23. Weir, M. P., and J. Sparks. 1987. Purification and renaturation of recombinant human interleukin-2. Biochem. J. 245:85-91. 24. Williams, D. C., R. M. Van Frank, W. L. Muth, and J. P. Burnett. 1982. Cytoplasmic inclusion bodies in Escherichia coli producing human insulin proteins. Science 215:687-689.
