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Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells
Journal of Biotechnology, 5 (1987) 77-86
77
Elsevier JBT 00230
Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells Kazuaki Kitano a, Shigeru Fujimoto a, Masafumi N a k a o b, Takuya Watanabe a and Yoshio Nakao a a Applied Microbiology Laboratories and b Biology Laboratories, Central Research Division, Takeda Chemical h~dustries, Ltd., Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan
(Received 8 September1986; acceptedfor publication30 October 1986)
Summary Recombinant interferon-aA (rIFN-aA) accumulated in Escherichia coli 294 was rapidly degraded in vivo when glucose or oxygen in the medium was limited, whereas recombinant interferon-T (rIFN-T) and recombinant interleukin-2 (rIL-2) were stable under the same conditions. The degradation in vitro of rIL-2 by E. coil proteases was as rapid as that of rIFN-aA. On the other hand, rIFN-T maintained the same activity after proteolysis in vitro. Western blot analysis of the reaction mixture, however, showed that the rIFN-T molecule was converted into a smaller protein of about 15 kDa by losing the COOH-terminal portion of the peptide. Electron microscopic observations showed that rIFN-T and rIL-2 accumulated and formed inclusion bodies. No clear inclusion bodies were found in the cells accumulating rIFN-aA, wlfich is rapidly degraded in vivo. These results indicate that intracellular location and protein structure are related to the stability in vivo of recombinant proteins, rIFN-aA was also degraded rapidly in a i o n - mutant of the same organism, suggesting that protease La does not participate in this degradation process. Protein degradation; Recombinant protein; Escherichia coli; Inclusion body; Ion mutant
Introduction
Since the advent of recombinant DNA technology, a variety of eukaryotic genes including those of human insulin (Goeddel et al., 1979), human growth hormone 0168-1656/87/$03.50 © 1987 ElsevierSciencePublishersB.V. (BiomedicalDivision)
78 (Goeddel et al., 1979a), interferons (Goeddel et al., 1980; Taniguchi et al., 1980; Gray et al., 1982; Simons et al., 1984) and interleukin-2 (Taniguchi et al., 1983; Devos et al., 1983) have been cloned and expressed in E. coli. The expression of such genes, however, has often been limited by various factors: stability of the recombinant plasmid in the cells, the transcription efficiency of the coded genes, translation efficiency of the mRNA into proteins, and so on (Carrier et al., 1983). Intracellular degradation of the product is also an important subject for consideration, because eukaryotic foreign proteins are recognized as abnormal proteins in E. coli and consequently are degraded. For example, proteolytic degradation intermediates of the human growth hormone were detected in E. coli cells by immunoprecipitation (Goeddel et al., 1979a). Bacterially produced fibroblast interferon was rapidly degraded in a maxicell experiment (Taniguchi et al., 1980). Williams et al. (1982) reported that human insulin polypeptide (A chain, B chain or proinsulin) is accumulated in recombinant E. coli cells as inclusion bodies, probably because over produced proteins are precipitated and aggregated within the cell. The abundant expression of recombinant proteins often leads to the formation of such inclusion bodies (Kawaguchi et al., 1984; Schoemaker et al., 1985; Schoner et al., 1985). During the course of our studies on the production of recombinant proteins in E. coli, we found that recombinant interferon-aA (rlFN-aA) was degraded rapidly in vivo under certain conditions, whereas recombinant interferon-~, (rlFN-~,) and interleukin-2 (rlL-2) were stable under the same conditions. Here we describe the degradation in vivo and in vitro of these proteins by E. coli proteases in relation to intracellular location and protein structure.
Materials and Methods
Microorganisms Escherichia coli 294 (a derivative of K-12) harboring recombinant plasmids for the production of human rIFN-aA (pLIF-A-trp35), human rIFN-v (pHIT-trp2101), and human rIL-2 (pTB300) were used. All these plasmids are derivatives of pBR322 containing the trp promoter of E. coli and the cDNAs of human IFN-t~A (Goeddel et al., 1980), IFN-~, (Gray et ai., 1982) and IL-2 (Taniguchi et ai., 1983), respectively. A Ion- mutant, E. coli 294 lon/pLIF-A-trp35 was derived from 294/pLIFA-trp35 by P-1 mediated transduction (Lennox, 1955) of the Ion gene from E. coil AB1899 (Howard-Flanders et ai., 1964). Cultioation The inoculum was prepared by cultivating the organisms in the L-medium (10 g 1-1 Bacto tryptone, 5 g 1-1 Bacto yeast extract and 5 g 1-1 NaC1) supplemented with 10 mg 1-1 tetracycline hydrochloride at 37°C for 6-16 h. The cultivation was carried out in 5-liter jar fermenters containing 2.5 1 of the M-9 medium supplemented with 10 g 1-1 glucose and 10 g 1-1 casamino acids at an aeration rate of 1 w m and an agitation speed of 1100 rpm. The cultivation was started at 37°C and
79 the temperature was shifted down to 30°C at an optical density of 500 Klett unit (KU) and to 25°C at 1000 KU or carried out at 37°C throughout.
Degradation in vitro of recombinant proteins E. coli 294 was cultivated in M-9 medium supplemented with 10 g 1-1 glucose and 10 g 1-1 casamino acids at 37°C for 6 h. Cells were collected from 100 ml of medium by centrifugation, washed with and suspended in 50 ml distilled water, and sonicated for 3 rain with cooling in a Kubota Insonator 200 M. The sonicated cells (1 ml) were mixed with an equal volume of 1 mg ml- 1 solution of purified rlFN-aA, rlFN-T, and rlL-2 and incubated at 37°C for 4 h. Aliquots (200 /~1) were taken, mixed with an equal volume of 7 M guanidine hydrochloride and kept on ice for 30 min. The mixture was centrifuged at 15 000 rpm for 5 min and the activity in the supernatant was assayed. For Western blotting, the supernatant of the sonicated cells was used as the enzyme source and the reaction was stopped by adding 1% SDS. Western blot analysis Protein samples were electrophoresed on 15% polyacrylamide gels by the procedure of Laemmli (1970) and transferred to nitrocellulose filters. The transfer and staining of marker lanes was performed as described by Burnette (1981). The filters of the sample lanes were rinsed in Tris buffered saline (0.02 M Tris-HC1, pH 8.0, 0.5 M NaC1) containing 3% gelatine and incubated with a monoclonal antibody, MOT2-11.1 (Ichimori et al., 1985), against the COOH-terminal synthetic peptide (Lys13X-GluX46) or a monoclonal antibody, MoWN'I2-76.53 (Ichimori et al., 1986), against the NH2-terminal synthetic peptide (Gln4-Gly2X). The filters were washed, incubated with horseradish peroxidase-antimouse IgG conjugate, and washed again. The filters were developed with H202 and 4-chloro-l-naphthol to stain bound peroxidase. Electron microscopy Cells were pre-fixed with 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) for 3 h, and post-fixed with cacodylate-buffered 1% osmium tetroxide for 2 h. The fixed cells were dehydrated with a graded series of ethanol, and embedded in Epon 812. Ultrathin sections were prepared using an LKB Ultrotome III, double stained with uranyl acetate and lead citrate, and examined in a JEOL JEM-1200 EX transmission electron microscope. Assays rlFN-aA and rlFN-,/ were assayed by inhibition of the cytopathic effect of vesicular stomatitis virus on MDBK cells and Sindbis virus on FL ceils, respectively (Rubinstein et al., 1981). rIL-2 activity was determined by the ability to maintain an IL-2 dependent murine cell line, NKC3 (Tada et al., 1986). Cell growth was measured turbidimetrically by a Klett-Summerson colorimeter and expressed as KU. Glucose was analyzed by the YSI model 27 sugar analyzer.
80
Results Degradation in vivo of recombinant proteins in E. coli During the course of the growth studies using recombinant E. coli, we found that rlFN-aA was rapidly degraded in vivo when the glucose in the medium was used up (Fig. 1A). Once the degradation was triggered, it could not be suppressed by adding glucose. The half-life of the protein was about 100 min. The same phenomenon was also observed when dissolved oxygen in the medium was limited (data not shown). We next examined the effect of glucose deficiency on the synthesis of rlFN-y and rlL-2 using the same host organism. Degradation of these products was very slow compared to that of rlFN-aA (Fig. 1B,C). The half life of these proteins was estimated as 11.4 and 24 h, respectively. The degradation of rlFN-y and rlL-2 under limiting dissolved oxygen were also very slow (data not shown). Degradation in vitro of recombinant proteins by E. coil cell debris T o clarify the reason for the differences between rIFN-ctA and other recombinant products, we first compared the degradation in vitro of these proteins by the sonicated cells of the host organism. As is shown in Fig. 2, r I F N - a A was degraded rapidly as it was in vivo. The degradation of rIL-2 was almost comparable to that of rIFN-aA, whereas rIFN-y was quite stable against degradation in vitro and in vivo.
Analysis of degradation products of rlFN-y obtained in vitro by Western blotting As rIFN-y seemed to be tolerant against E. coli proteases, the reaction products
C 100
I I
O--&~
/
II !
x
i
Iz
/tx /
t
.g i 0
4
8
12
0
4
t 8 12 Cultivation p e r i o d (h)
n
0
4
8
12
Fig. 1. Effect of glucose deficiency on the production of recombinant proteins by E. coli 294. E. coli 294/pLIF-A-trp35 (A), 294/pHIT-trp2101 (B) and 294/pTB300 (C), producing rIFN-aA, rIFN-y and rIL-2, respectively, were cultivated at 37°C in 5-1 jars containing the supplemented M-9 medium under the aeration rate of 1 vvm and agitation speed of 1100 rpm. Growth (zx), glucose concentration (O) and activity (0) are expressed as relative values taking each maximum value as 100.
81
Fig. 2. Degradation solution of purified cells and incubated as 100.
in vitro of recombinant proteins by sonicated ceils of the host organism. 1 mg ml ’ rIFN-aA (A), rIFN-y (B) or rIL-2 (C) was mixed with an equal volume of sonicated at 37OC for 4 h. Activities were expressed as relative values taking the initial activity
obtained in vitro were analyzed in detail by Western blotting, using two kinds of monoclonal antibodies which recognize the COOH-terminal and NH,-terminal portion of rIFN-y, respectively (Fi g. 3). Mature rIFN-y was degraded rapidly and a
A 23456
B 7
0
123456
7
66, 200 45, 000 31, 000
21, 500 14, 400
Fig. 3. Western blot analysis of the reaction product of rIFN-y with E. co/i proteases. 1 mg ml-’ solution of rIFN-y was mixed with an equal volume of the supernatant of sonicatcd cells of the host organism and incubated at 37°C for 4 h. The reaction products were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose, incubated with monoclonal antibodies, (A) MoyZ-11.1, against the COOH-terminal synthetic peptide, and (B) MoWNyZ-76.53. against the NH?-terminal synthetic peptide, and detected as described in Materials and Methods. The samples arc as follows. Lane 0: Molecular weight markers: lysozyme, 14400; soybean trypsin inhibitor, 21500; carbonic anhydrase. 31000; ovalbumin, 45000; bovine serum albumin, 66200. Lane 1: 0 h sample (enzyme was boiled before mixing with the substrate). Lanes 2 to 6: reaction products of 0.5. 1 , 2, 3. and 4 h, respectively. Lane 7: mature rIFN-y (control).
82 smaller protein of about 15 kDa, which is recognized by the monoclonal antibody against the NHz-terminal peptide but not by that against the C O O H - t e r m i n a l peptide, increased; this protein was stable against further proteolysis. The molecular weight of rlFN-~, purified from the cells after glucose starvation in vivo was, on the other hand, mostly mature 18 kDa (data not shown).
Intracellular localization of the recombinant proteins Recombinant proteins often accumulate and form inclusion bodies. The intracellular distribution of these proteins in E. coli cells was examined next. The cultivated cells, whose contents of rIFN-o~A, rIFN-'y, and rlL-2 were about 20, 15, and 15% of the cellular soluble proteins (estimated by SDS-polyacrylamide gel electrophoresis analysis), respectively, were fixed with glutaraldehyde, thin sectioned, and observed by a transmission electron microscope. Under production conditions the cells of all the recombinant strains were longer than the host cells. Although r I F N - a A was accumulated at the highest concentration, no clear inclusion bodies were observed
It
°
i
t""
Fig. 4. Transmission electron micrographs of thin sections of recombinant E. co/i. The organisms, (A) host, (B) rIFN-aA producer, (C) rIFN-y producer, and (D) rIL-2 producer, were cultivated under the conditions described in Materials and Methods. The cells were fixed with glutaraldehyde and osmium tetroxide, ultrathin-sectioned, and double stained with uranyl acetate and lead citrate. The contents of recombinant proteins were about 20% (rIFN-aA), 15% (rIFN-3,) and 15% (rIL-2) of cellular soluble proteins.
83 inside the cells (Fig. 4B). Therefore, rlFN-aA, which is easily degraded in vivo, seemed to be accumulated mostly as a soluble form in the cytoplasm. On the other hand, clear inclusion bodies were observed in the cells of rlFN-T and rlL-2 producers (Fig. 4C,D). The inclusions generally were localised at the polar or sub-polar regions as had already been reported (Schoner et al., 1985). Effect of Ion protease on the in vivo degradation of rlFN-aA The Ion gene product, protease La in E. coli plays an important role in degrading
abnormal proteins intracellularly (Mount, 1980). Its effect on the degradation in vivo of rlFN-aA was therefore investigated. The Ion- gene of E. coli AB1899 was introduced into the producer of rlFN-aA by P-1 mediated transduction, and the degradation in vivo of rlFN-aA under glucose starvation was checked. (Fig. 5). The deficiency of the Ion protease did not affect the degradation rate of rlFN-ctA.
Discussion
A variety of proteins encoded by eukaryotic genes cloned in E. coli appear to be labile, possibly because they are degraded by E. coli proteases (Itakura et al., 1977; Goeddel et al., 1979a; Taniguchi et al., 1980). In the studies reported here we found that rlFN-aA accumulated intracellularly in E. coli 294 was rapidly degraded when glucose or dissolved oxygen was limited in the medium, whereas rlFN-T and rlL-2 accumulated in the cells were hardly degraded under the same conditions. To explore the cause of this difference, we took two approaches: we examined the degradation in vitro and the intracellular localization of the recombinant products. Various recombinant proteins including polypeptide-proinsulin chimeric protein (Williams et al., 1982), human interferon-,/ (Simons et al., 1984), bovine growth hormone (Schoner et al., 1985), and prochymosin (Kawaguchi et al., 1984; Schoemaker et al., 1985) accumulate as inclusion bodies or aggregates in E. coli cells. We found that rlFN-T and rlL-2 also accumulate as inclusion bodies in E. coli 294 cells. Clear inclusion bodies, however, were not observed for rlFN-aA accumulated at concentrations as high as 20% of soluble proteins. As rlFN-aA is very soluble in water, it seems to accumulate in solution in the cytoplasm and to be attacked easily by proteases. On the other hand, although rlL-2 in solution was degraded rapidly like rlFN-aA (Fig. 2), it accumulated in the cells was hardly degraded. Thus the inclusion bodies are likely to protect rlL-2 from contact with cellular proteases in the cytoplasm. Cheng et al. (1981) also found that while the X90 protomer synthesized at single-copy gene levels is rapidly degraded, the overproduced protein, which formed proteinaceous aggregates in plasmid-containing strains, is completely stable. rlFN-T did not lose its activity even after being incubated with E. coli cell debris for 4 h (Fig. 2). During this period, the mature rlFN-T was, however, rapidly converted into a smaller protein of 15 kDa (Fig. 3). Accordingly, the 15 kDa protein seemed to have the same antiviral activity as that of mature rlFN-T. As the 15 kDa protein bound to a monoclonal antibody that recognizes the NH2-terminus portion
84
100,
== >o ._>e50
8 Cultivation period (h)
10
Fig. 5. Effect of glucose deficiencyon the production of rlFN-aA by a Ion- mutant of E. coli 294. E. coli 294 Ion/pLIF-A-trp35 was cultivated under the same conditions as those in Fig. 1. Growth (/,), glucose concentration (O) and rlFN-aA activity (I) are expressed as relative values taking each maximum value as 100.
of rlFN-T but did not bind to one that recognizes the COOH-terminus portion of the molecule, it is clear that the protein is missing the COOH-terminus portion of rlFN-7. Honda et al. (1986) obtained two lower molecular weight derivatives of rIFN-T, 15 kDa and 17 kDa proteins, in addition to mature 18 kDa protein, when purified from the lysozyme-EDTA extract of recombinant E. coli cells. These derivatives were missing 15 and 4 amino acids from the COOH-terminus of mature rlFN-T, respectively: they had same antiviral activity as the mature protein. Thus our 15 kDa protein seemed to correspond to that of Honda et al. (1986). The 15 kDa protein is very stable against further proteolysis by E. coli proteases as is clear from Fig. 3. Natural IFN-7 exists as a dimer (Yip et al., 1982) and rlFN-y was also found to behave as a non-covalent dimer on Sephadex G100 gel filtration under non-denaturing conditions. The dimer form may, therefore, prevent the rlFN-y from further degradation. This fact suggests that protein structure also affects the stability of the molecule against proteases. T h e E. coli Ion protease, a DNA-binding protein with ATP-dependent proteolytic activity (Charette et al., 1981; Chung et al., 1981), is known to recognize and degrade abnormal proteins for the cells; the degradation of abnormal proteins occurs 2 - 4 times more slowly in Ion- mutants (Bukhari and Zipser, 1973; Kowit and Goldberg, 1977; Gottesman and Zipser, 1978). Figure 5, however, shows that the Ion protease does not participate in the degradation of r l F N - a A under glucose or dissolved oxygen deficiency. Although the Ion protease requires ATP for its activity, the intracellular degradation of r I F N - - A is triggered under energy deficiency (glucose or dissolved oxygen deficiency). As E. coli is known to have several kinds of proteases (Goldberg et al., 1981), it is probable that another enzyme participates in this degradation process.
85
Acknowledgements O u r appreciation is extended to Dr. Barbara J. B a c k m a n n , who provided us E. coli AB1899, a n d to Drs. Y. Sugino, A. K a k i n u m a , S. A k i y a m a , a n d A. I m a d a for
their s t i m u l a t i n g discussions a n d e n c o u r a g e m e n t t h r o u g h o u t this work. W e also appreciate the d e t e r m i n a t i o n of rlL-2 activity b y Dr. O. Shiho, a n d the technical assistance of Messrs. M. N a k a t s u a n d S. Hikita.
References
Bukhari, A.J. and Zipser, D. (1973) Mutants of Escherichia coli with a defect in the degradation of nonsense fragments. Nat. New Biol. 243, 238-241. Bumette, W.N. (1981) Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate~polyacrylamidegels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195-203. Carrier, M.J., Nugent, M.E., Tacon, W.C.A. and Primrose, S.B. (1983) High expression of cloned genes in E. coil and its consequences. Trends Biotechnol. 1, 109-113. Charette, M.F., Henderson, G.W. and Markovitz, A. (1981) ATP hydrolysis-dependent protease activity of the Ion (capR) protein of Escherichia coil K-12. Proc. Natl. Acad. Sci. USA 78, 4728-4732. Cheng, Y.E., Kwoh, D.Y., Kwoh, T.J., Soltvedt, B.C. and Zipser, D. (1981) Stabilization of a degradable protein by its overexpression in Escherichia coli. CJene 14, 121-130. Chung, C.H. and Goldberg, A.L. (1981) The product of the Ion (capR) gene in Escherichia coil is the ATP-dependent protease, prot~ase L& Proc. Nail. Acad. Sci. USA 78, 4931-4935. Devos, R., Plaetinck, G., Cheroutre, H., Simons, G., Degrave, W., Tavemier, J., Remant, E. and Fiers, W. (1983) Molecular cloning of human interleukin-2 cDNA and its expression. Nucleic Acids Res. 11, 4307-4323. Goeddel, D.V., Heyneker, H.L., Hozumi, T., Arentzen, R., Itakura, K., Yansura, D.G., Ross, M.J., Miozzari, G., Crea, R. and Seeburg, P.H. (1979a) Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature 281, 544-548. Goeddel, D.V., Kleid, D.G., Bolivar, F., Heyneker, H.L., Yansura, D.G., Crea, R., Hirose, T., Kxaszewski, A., Itakura, K. and Riggs, A.D. (1979b) Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc. Nail. Acad. Sci. USA 76, 106-110. Goeddel, D.V., Yelverton, E., Ullrich, A., Heyneker, H.L., Miozzari, G., Holmes, W., Sceburg, P.H., Dull, T., May, L., Stebbing, N., Crea, R., Maeda, S., McCandliss, R., Sloma, A., Tabor, J.M., Gross, M., Familletti, P.C. and Pestka, S. (1980) Human leukocyte interferon produced by E. coli is biologically active. Nature 287, 411-416. Goldberg, A.L., Swamy, K.H.S., Chung, C.H. and Larimore, F.S. (1981) Proteases in Escherichia coll. Methods Enzymol. 80, 680-702. Gottesman, S. and Zipser, D. (1978) Deg phenotype of Escherichia coil Ion mutants. J. Bacteriol. 133, 844-851. Gray, P.W., Leung, D.W., Pennica, D., Yelverton, E., Najarian, R., Simousen, C.C., Derynck, R., Sherwood, P.J., Wallace, D.M., Berger, S.L., Levinson, A.D. and Goeddel, D.V. (1982) Expression of human immune interferon cDNA in E. coil and monkey cells. Nature 295, 503-508. Honda, S., Asano, T., Kajio, T., Nakagawa, S., Ikeyama, S., Ichimori, Y., Sugino, H., Nara, K. and Kakinuma, A. (1986) Differential purification by immunoaffinity chromatography of two COOHterminal portion-deleted derivatives of recombinant human interferon-y from Escherichia coli, to be submitted. Howard-Flanders, P., Simson, E. and Theriot, L. (1964) A locus that controls filament formation and sensitivity to radiation in Escherichia coli K-12. Genetics 49, 237-246.
86 lchimori, Y., Kurokawa, T., Honda, S., Suzuki, N., Wakimasu, M. and Tsukamoto, K. (1985) Monoclonal antibodies to human interferon-3,. I. Antibodies to a synthetic carboxyl-terminal peptide. J. Immunol. Methods 80. 55-66. lchimori, Y., Suzuki, N., Kitada, C. and Tsukamoto, K. (1986) Monoclonal antibodies to human interferonq,. II. Antibodies with neutralizing activity. Hybridoma, in press. ltakura, K., Hirose. T.. Crea, R., Riggs, A.D., Heyneker, H.L., Bolivar, F. and Boyer, H.W. (1977) Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin. Science 198, 1056-1063. Kawaguchi, Y., Shimizu. N., Nishimori. K.. Uozumi, T. and Beppu. T. (1984) Renaturation and activation of calf prochymosin produced in a soluble form in Escherichia coli. J. Biotechnol. 1, 307-315. Kowit, J.D and Goldberg, A.L. (1977) Intermediate steps in the degradation of a specific abnormal protein in Escherichia coli. L Biol. Chem. 252, 8350-8357. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lennox, E.S. (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1,190-206. Mount, D.W. (1980) The genetics of protein degradation in bacteria Annu. Rev. Genet. 14, 279-319. Rubinstein, S., Familletti, P.C. and Pestka, S. (1981) Convenient assay for interferons. J. Virol. 37, 755-758. Schoemaker, J.M., Brasnett, A.H. and Marston, F.A.O. (1985) Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies. EMBO J. 4, 775-780. Schoner, R.G., Ellis, L.F. and Schoner, B.E. (1985) Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Bio/Technology 3, 151-154. Simons, G., Remaut, E., Ailet, B., Devos, R. and Fiers, W. (1984) High-level expression of human interferon gamma in Escherichia coli under the control of the PL promoter of bacteriophage lambda. Gene 28, 55-64. Tada, H., Shiho, O., Kuroshima, K., Koyama, M. and Tsukamoto, K. (1986) An improved colorimetric assay for interleukin 2. J. Immunol. Methods, in press. Taniguchi, T., Guarente, L., Roberts, T.M, Kimelman, D., Douhan 111, J. andPtashne, M. (1980) Expression of the human fibroblast interferon gene in Escherichia coli. Proc. Natl. Acad. Sci. USA 77, 5230-5233. Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N., Yoshimoto, R. and Hamuro, J. (1983) Structure and expression of a cloned eDNA for human interleukin-2. Nature 302, 305-310. Williams, D.C., Van Frank, ILM., Muth, W.L. and Burnett, LP. (1982) Cytoplasmic inclusion bodies in Escherichia coil producing biosynthetic human insulin proteins. Science 215, 687-689. Yip, Y.K., Barrowclough, B.S., Urban, C. and Vilcek, .I. (1982) Molecular weight of human gamma interferon is similar to that of other human interferons. Science 215, 411-413.
77
Elsevier JBT 00230
Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells Kazuaki Kitano a, Shigeru Fujimoto a, Masafumi N a k a o b, Takuya Watanabe a and Yoshio Nakao a a Applied Microbiology Laboratories and b Biology Laboratories, Central Research Division, Takeda Chemical h~dustries, Ltd., Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan
(Received 8 September1986; acceptedfor publication30 October 1986)
Summary Recombinant interferon-aA (rIFN-aA) accumulated in Escherichia coli 294 was rapidly degraded in vivo when glucose or oxygen in the medium was limited, whereas recombinant interferon-T (rIFN-T) and recombinant interleukin-2 (rIL-2) were stable under the same conditions. The degradation in vitro of rIL-2 by E. coil proteases was as rapid as that of rIFN-aA. On the other hand, rIFN-T maintained the same activity after proteolysis in vitro. Western blot analysis of the reaction mixture, however, showed that the rIFN-T molecule was converted into a smaller protein of about 15 kDa by losing the COOH-terminal portion of the peptide. Electron microscopic observations showed that rIFN-T and rIL-2 accumulated and formed inclusion bodies. No clear inclusion bodies were found in the cells accumulating rIFN-aA, wlfich is rapidly degraded in vivo. These results indicate that intracellular location and protein structure are related to the stability in vivo of recombinant proteins, rIFN-aA was also degraded rapidly in a i o n - mutant of the same organism, suggesting that protease La does not participate in this degradation process. Protein degradation; Recombinant protein; Escherichia coli; Inclusion body; Ion mutant
Introduction
Since the advent of recombinant DNA technology, a variety of eukaryotic genes including those of human insulin (Goeddel et al., 1979), human growth hormone 0168-1656/87/$03.50 © 1987 ElsevierSciencePublishersB.V. (BiomedicalDivision)
78 (Goeddel et al., 1979a), interferons (Goeddel et al., 1980; Taniguchi et al., 1980; Gray et al., 1982; Simons et al., 1984) and interleukin-2 (Taniguchi et al., 1983; Devos et al., 1983) have been cloned and expressed in E. coli. The expression of such genes, however, has often been limited by various factors: stability of the recombinant plasmid in the cells, the transcription efficiency of the coded genes, translation efficiency of the mRNA into proteins, and so on (Carrier et al., 1983). Intracellular degradation of the product is also an important subject for consideration, because eukaryotic foreign proteins are recognized as abnormal proteins in E. coli and consequently are degraded. For example, proteolytic degradation intermediates of the human growth hormone were detected in E. coli cells by immunoprecipitation (Goeddel et al., 1979a). Bacterially produced fibroblast interferon was rapidly degraded in a maxicell experiment (Taniguchi et al., 1980). Williams et al. (1982) reported that human insulin polypeptide (A chain, B chain or proinsulin) is accumulated in recombinant E. coli cells as inclusion bodies, probably because over produced proteins are precipitated and aggregated within the cell. The abundant expression of recombinant proteins often leads to the formation of such inclusion bodies (Kawaguchi et al., 1984; Schoemaker et al., 1985; Schoner et al., 1985). During the course of our studies on the production of recombinant proteins in E. coli, we found that recombinant interferon-aA (rlFN-aA) was degraded rapidly in vivo under certain conditions, whereas recombinant interferon-~, (rlFN-~,) and interleukin-2 (rlL-2) were stable under the same conditions. Here we describe the degradation in vivo and in vitro of these proteins by E. coli proteases in relation to intracellular location and protein structure.
Materials and Methods
Microorganisms Escherichia coli 294 (a derivative of K-12) harboring recombinant plasmids for the production of human rIFN-aA (pLIF-A-trp35), human rIFN-v (pHIT-trp2101), and human rIL-2 (pTB300) were used. All these plasmids are derivatives of pBR322 containing the trp promoter of E. coli and the cDNAs of human IFN-t~A (Goeddel et al., 1980), IFN-~, (Gray et ai., 1982) and IL-2 (Taniguchi et ai., 1983), respectively. A Ion- mutant, E. coli 294 lon/pLIF-A-trp35 was derived from 294/pLIFA-trp35 by P-1 mediated transduction (Lennox, 1955) of the Ion gene from E. coil AB1899 (Howard-Flanders et ai., 1964). Cultioation The inoculum was prepared by cultivating the organisms in the L-medium (10 g 1-1 Bacto tryptone, 5 g 1-1 Bacto yeast extract and 5 g 1-1 NaC1) supplemented with 10 mg 1-1 tetracycline hydrochloride at 37°C for 6-16 h. The cultivation was carried out in 5-liter jar fermenters containing 2.5 1 of the M-9 medium supplemented with 10 g 1-1 glucose and 10 g 1-1 casamino acids at an aeration rate of 1 w m and an agitation speed of 1100 rpm. The cultivation was started at 37°C and
79 the temperature was shifted down to 30°C at an optical density of 500 Klett unit (KU) and to 25°C at 1000 KU or carried out at 37°C throughout.
Degradation in vitro of recombinant proteins E. coli 294 was cultivated in M-9 medium supplemented with 10 g 1-1 glucose and 10 g 1-1 casamino acids at 37°C for 6 h. Cells were collected from 100 ml of medium by centrifugation, washed with and suspended in 50 ml distilled water, and sonicated for 3 rain with cooling in a Kubota Insonator 200 M. The sonicated cells (1 ml) were mixed with an equal volume of 1 mg ml- 1 solution of purified rlFN-aA, rlFN-T, and rlL-2 and incubated at 37°C for 4 h. Aliquots (200 /~1) were taken, mixed with an equal volume of 7 M guanidine hydrochloride and kept on ice for 30 min. The mixture was centrifuged at 15 000 rpm for 5 min and the activity in the supernatant was assayed. For Western blotting, the supernatant of the sonicated cells was used as the enzyme source and the reaction was stopped by adding 1% SDS. Western blot analysis Protein samples were electrophoresed on 15% polyacrylamide gels by the procedure of Laemmli (1970) and transferred to nitrocellulose filters. The transfer and staining of marker lanes was performed as described by Burnette (1981). The filters of the sample lanes were rinsed in Tris buffered saline (0.02 M Tris-HC1, pH 8.0, 0.5 M NaC1) containing 3% gelatine and incubated with a monoclonal antibody, MOT2-11.1 (Ichimori et al., 1985), against the COOH-terminal synthetic peptide (Lys13X-GluX46) or a monoclonal antibody, MoWN'I2-76.53 (Ichimori et al., 1986), against the NH2-terminal synthetic peptide (Gln4-Gly2X). The filters were washed, incubated with horseradish peroxidase-antimouse IgG conjugate, and washed again. The filters were developed with H202 and 4-chloro-l-naphthol to stain bound peroxidase. Electron microscopy Cells were pre-fixed with 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) for 3 h, and post-fixed with cacodylate-buffered 1% osmium tetroxide for 2 h. The fixed cells were dehydrated with a graded series of ethanol, and embedded in Epon 812. Ultrathin sections were prepared using an LKB Ultrotome III, double stained with uranyl acetate and lead citrate, and examined in a JEOL JEM-1200 EX transmission electron microscope. Assays rlFN-aA and rlFN-,/ were assayed by inhibition of the cytopathic effect of vesicular stomatitis virus on MDBK cells and Sindbis virus on FL ceils, respectively (Rubinstein et al., 1981). rIL-2 activity was determined by the ability to maintain an IL-2 dependent murine cell line, NKC3 (Tada et al., 1986). Cell growth was measured turbidimetrically by a Klett-Summerson colorimeter and expressed as KU. Glucose was analyzed by the YSI model 27 sugar analyzer.
80
Results Degradation in vivo of recombinant proteins in E. coli During the course of the growth studies using recombinant E. coli, we found that rlFN-aA was rapidly degraded in vivo when the glucose in the medium was used up (Fig. 1A). Once the degradation was triggered, it could not be suppressed by adding glucose. The half-life of the protein was about 100 min. The same phenomenon was also observed when dissolved oxygen in the medium was limited (data not shown). We next examined the effect of glucose deficiency on the synthesis of rlFN-y and rlL-2 using the same host organism. Degradation of these products was very slow compared to that of rlFN-aA (Fig. 1B,C). The half life of these proteins was estimated as 11.4 and 24 h, respectively. The degradation of rlFN-y and rlL-2 under limiting dissolved oxygen were also very slow (data not shown). Degradation in vitro of recombinant proteins by E. coil cell debris T o clarify the reason for the differences between rIFN-ctA and other recombinant products, we first compared the degradation in vitro of these proteins by the sonicated cells of the host organism. As is shown in Fig. 2, r I F N - a A was degraded rapidly as it was in vivo. The degradation of rIL-2 was almost comparable to that of rIFN-aA, whereas rIFN-y was quite stable against degradation in vitro and in vivo.
Analysis of degradation products of rlFN-y obtained in vitro by Western blotting As rIFN-y seemed to be tolerant against E. coli proteases, the reaction products
C 100
I I
O--&~
/
II !
x
i
Iz
/tx /
t
.g i 0
4
8
12
0
4
t 8 12 Cultivation p e r i o d (h)
n
0
4
8
12
Fig. 1. Effect of glucose deficiency on the production of recombinant proteins by E. coli 294. E. coli 294/pLIF-A-trp35 (A), 294/pHIT-trp2101 (B) and 294/pTB300 (C), producing rIFN-aA, rIFN-y and rIL-2, respectively, were cultivated at 37°C in 5-1 jars containing the supplemented M-9 medium under the aeration rate of 1 vvm and agitation speed of 1100 rpm. Growth (zx), glucose concentration (O) and activity (0) are expressed as relative values taking each maximum value as 100.
81
Fig. 2. Degradation solution of purified cells and incubated as 100.
in vitro of recombinant proteins by sonicated ceils of the host organism. 1 mg ml ’ rIFN-aA (A), rIFN-y (B) or rIL-2 (C) was mixed with an equal volume of sonicated at 37OC for 4 h. Activities were expressed as relative values taking the initial activity
obtained in vitro were analyzed in detail by Western blotting, using two kinds of monoclonal antibodies which recognize the COOH-terminal and NH,-terminal portion of rIFN-y, respectively (Fi g. 3). Mature rIFN-y was degraded rapidly and a
A 23456
B 7
0
123456
7
66, 200 45, 000 31, 000
21, 500 14, 400
Fig. 3. Western blot analysis of the reaction product of rIFN-y with E. co/i proteases. 1 mg ml-’ solution of rIFN-y was mixed with an equal volume of the supernatant of sonicatcd cells of the host organism and incubated at 37°C for 4 h. The reaction products were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose, incubated with monoclonal antibodies, (A) MoyZ-11.1, against the COOH-terminal synthetic peptide, and (B) MoWNyZ-76.53. against the NH?-terminal synthetic peptide, and detected as described in Materials and Methods. The samples arc as follows. Lane 0: Molecular weight markers: lysozyme, 14400; soybean trypsin inhibitor, 21500; carbonic anhydrase. 31000; ovalbumin, 45000; bovine serum albumin, 66200. Lane 1: 0 h sample (enzyme was boiled before mixing with the substrate). Lanes 2 to 6: reaction products of 0.5. 1 , 2, 3. and 4 h, respectively. Lane 7: mature rIFN-y (control).
82 smaller protein of about 15 kDa, which is recognized by the monoclonal antibody against the NHz-terminal peptide but not by that against the C O O H - t e r m i n a l peptide, increased; this protein was stable against further proteolysis. The molecular weight of rlFN-~, purified from the cells after glucose starvation in vivo was, on the other hand, mostly mature 18 kDa (data not shown).
Intracellular localization of the recombinant proteins Recombinant proteins often accumulate and form inclusion bodies. The intracellular distribution of these proteins in E. coli cells was examined next. The cultivated cells, whose contents of rIFN-o~A, rIFN-'y, and rlL-2 were about 20, 15, and 15% of the cellular soluble proteins (estimated by SDS-polyacrylamide gel electrophoresis analysis), respectively, were fixed with glutaraldehyde, thin sectioned, and observed by a transmission electron microscope. Under production conditions the cells of all the recombinant strains were longer than the host cells. Although r I F N - a A was accumulated at the highest concentration, no clear inclusion bodies were observed
It
°
i
t""
Fig. 4. Transmission electron micrographs of thin sections of recombinant E. co/i. The organisms, (A) host, (B) rIFN-aA producer, (C) rIFN-y producer, and (D) rIL-2 producer, were cultivated under the conditions described in Materials and Methods. The cells were fixed with glutaraldehyde and osmium tetroxide, ultrathin-sectioned, and double stained with uranyl acetate and lead citrate. The contents of recombinant proteins were about 20% (rIFN-aA), 15% (rIFN-3,) and 15% (rIL-2) of cellular soluble proteins.
83 inside the cells (Fig. 4B). Therefore, rlFN-aA, which is easily degraded in vivo, seemed to be accumulated mostly as a soluble form in the cytoplasm. On the other hand, clear inclusion bodies were observed in the cells of rlFN-T and rlL-2 producers (Fig. 4C,D). The inclusions generally were localised at the polar or sub-polar regions as had already been reported (Schoner et al., 1985). Effect of Ion protease on the in vivo degradation of rlFN-aA The Ion gene product, protease La in E. coli plays an important role in degrading
abnormal proteins intracellularly (Mount, 1980). Its effect on the degradation in vivo of rlFN-aA was therefore investigated. The Ion- gene of E. coli AB1899 was introduced into the producer of rlFN-aA by P-1 mediated transduction, and the degradation in vivo of rlFN-aA under glucose starvation was checked. (Fig. 5). The deficiency of the Ion protease did not affect the degradation rate of rlFN-ctA.
Discussion
A variety of proteins encoded by eukaryotic genes cloned in E. coli appear to be labile, possibly because they are degraded by E. coli proteases (Itakura et al., 1977; Goeddel et al., 1979a; Taniguchi et al., 1980). In the studies reported here we found that rlFN-aA accumulated intracellularly in E. coli 294 was rapidly degraded when glucose or dissolved oxygen was limited in the medium, whereas rlFN-T and rlL-2 accumulated in the cells were hardly degraded under the same conditions. To explore the cause of this difference, we took two approaches: we examined the degradation in vitro and the intracellular localization of the recombinant products. Various recombinant proteins including polypeptide-proinsulin chimeric protein (Williams et al., 1982), human interferon-,/ (Simons et al., 1984), bovine growth hormone (Schoner et al., 1985), and prochymosin (Kawaguchi et al., 1984; Schoemaker et al., 1985) accumulate as inclusion bodies or aggregates in E. coli cells. We found that rlFN-T and rlL-2 also accumulate as inclusion bodies in E. coli 294 cells. Clear inclusion bodies, however, were not observed for rlFN-aA accumulated at concentrations as high as 20% of soluble proteins. As rlFN-aA is very soluble in water, it seems to accumulate in solution in the cytoplasm and to be attacked easily by proteases. On the other hand, although rlL-2 in solution was degraded rapidly like rlFN-aA (Fig. 2), it accumulated in the cells was hardly degraded. Thus the inclusion bodies are likely to protect rlL-2 from contact with cellular proteases in the cytoplasm. Cheng et al. (1981) also found that while the X90 protomer synthesized at single-copy gene levels is rapidly degraded, the overproduced protein, which formed proteinaceous aggregates in plasmid-containing strains, is completely stable. rlFN-T did not lose its activity even after being incubated with E. coli cell debris for 4 h (Fig. 2). During this period, the mature rlFN-T was, however, rapidly converted into a smaller protein of 15 kDa (Fig. 3). Accordingly, the 15 kDa protein seemed to have the same antiviral activity as that of mature rlFN-T. As the 15 kDa protein bound to a monoclonal antibody that recognizes the NH2-terminus portion
84
100,
== >o ._>e50
8 Cultivation period (h)
10
Fig. 5. Effect of glucose deficiencyon the production of rlFN-aA by a Ion- mutant of E. coli 294. E. coli 294 Ion/pLIF-A-trp35 was cultivated under the same conditions as those in Fig. 1. Growth (/,), glucose concentration (O) and rlFN-aA activity (I) are expressed as relative values taking each maximum value as 100.
of rlFN-T but did not bind to one that recognizes the COOH-terminus portion of the molecule, it is clear that the protein is missing the COOH-terminus portion of rlFN-7. Honda et al. (1986) obtained two lower molecular weight derivatives of rIFN-T, 15 kDa and 17 kDa proteins, in addition to mature 18 kDa protein, when purified from the lysozyme-EDTA extract of recombinant E. coli cells. These derivatives were missing 15 and 4 amino acids from the COOH-terminus of mature rlFN-T, respectively: they had same antiviral activity as the mature protein. Thus our 15 kDa protein seemed to correspond to that of Honda et al. (1986). The 15 kDa protein is very stable against further proteolysis by E. coli proteases as is clear from Fig. 3. Natural IFN-7 exists as a dimer (Yip et al., 1982) and rlFN-y was also found to behave as a non-covalent dimer on Sephadex G100 gel filtration under non-denaturing conditions. The dimer form may, therefore, prevent the rlFN-y from further degradation. This fact suggests that protein structure also affects the stability of the molecule against proteases. T h e E. coli Ion protease, a DNA-binding protein with ATP-dependent proteolytic activity (Charette et al., 1981; Chung et al., 1981), is known to recognize and degrade abnormal proteins for the cells; the degradation of abnormal proteins occurs 2 - 4 times more slowly in Ion- mutants (Bukhari and Zipser, 1973; Kowit and Goldberg, 1977; Gottesman and Zipser, 1978). Figure 5, however, shows that the Ion protease does not participate in the degradation of r l F N - a A under glucose or dissolved oxygen deficiency. Although the Ion protease requires ATP for its activity, the intracellular degradation of r I F N - - A is triggered under energy deficiency (glucose or dissolved oxygen deficiency). As E. coli is known to have several kinds of proteases (Goldberg et al., 1981), it is probable that another enzyme participates in this degradation process.
85
Acknowledgements O u r appreciation is extended to Dr. Barbara J. B a c k m a n n , who provided us E. coli AB1899, a n d to Drs. Y. Sugino, A. K a k i n u m a , S. A k i y a m a , a n d A. I m a d a for
their s t i m u l a t i n g discussions a n d e n c o u r a g e m e n t t h r o u g h o u t this work. W e also appreciate the d e t e r m i n a t i o n of rlL-2 activity b y Dr. O. Shiho, a n d the technical assistance of Messrs. M. N a k a t s u a n d S. Hikita.
References
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