JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 5030-5034 0021-9193/90/095030-05$02.00/0
Vol. 172, No. 9
Copyright © 1990, American Society for Microbiology
Interaction between Heat Shock Protein DnaK and Recombinant Staphylococcal Protein A HALLDIS HELLEBUST,t* MATHIAS UHLtN, AND SVEN-OLOF ENFORS Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-10044 Stockholm, Sweden Received 6 March 1990/Accepted 2 July 1990 When a protein derived from the immunoglobulin G (IgG)-binding domains of staphylococcal protein A was expressed in Escherichia coil and recovered from cell extract by IgG affinity chromatography, the 69-kilodalton heat shock protein DnaK was found to be copurified. DnaK could be selectively eluted from the IgG column by ATP or by lowering the pH to 4.7. Protein A could subsequently be eluted by lowering the pH to 3.2. Thus, this procedure allows a one-step purification of both DnaK and protein A from cell extract. In vitro experiments with pure DnaK and protein A revealed that DnaK did not interfere with the IgG-binding properties of protein A but associated with its unfolded C-terminal in a salt-resistant manner. In addition, a specific interaction between DnaK and denaturated caseiI was found.
shock proteins and their role with respect to rDNA proteins expressed in bacteria. We have previously studied different recombinant DNA proteins derived from staphylococcal protein A expressed intracellularly in E. coli with respect to their stability against proteolytic degradation (11, 12). One of these protein A derivatives with a proteolytically sensitive C terminus was used as a model to study the effect of protein fusion on this stability. When this protein was purified by immunoglobulin G (IgG) affinity chromatography and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), it was found that the 69-kilodalton heat shock protein DnaK copurified with protein A (12). In this work, we studied the specific interaction in more detail and used different methods to selectively elute the DnaK from the IgG column. In vitro experiments with affinity-purified proteins show that DnaK interacts with the unfolded C-terminal region of protein A.
The heat shock response is one of the most conserved elements of biology; it is an inducible response to cellular stresses such as heat, exposure to ethanol, DNA damage, accumulation of denaturated or abnormal proteins, and nutrient starvation (18). In Escherichia coli, the response is an induction of at least 17 different proteins, but only for a few of the proteins is the physiological role known (20). Many of the heat shock proteins are also essential under normal growth conditions. (3, 19). DnaK is one of the most abundant heat shock proteins in E. coli; under normal growth at 37°C, it accounts for 1.4% of the total cellular protein (5, 10). It was originally identified as a host protein required for replication of bacteriophage lambda (20), and further studies of mutants have shown that DnaK is a modulator of the heat shock response (26). Purified DnaK binds tightly to ATP, has weak ATPase activity, and carries out autophosphorylation (28). A high degree of antigenic similarity has been found between DnaK and the corresponding components of yeast, Drosophila, chicken and human cells (1, 15). It has been found to associate in a salt-resistant manner with another heat shock protein, GrpE, both in vivo and in vitro, and it is released from this complex by ATP (14, 27). One function postulated for this protein is that it has an affinity for proteins that lack proper folding and releases itself from the substrate protein by hydrolysis of ATP (23). Recombinant DNA proteins often misfold when expressed in bacteria (4, 6), and one function of the heat shock proteins might be to contribute to the ability of the cells to degrade misfolded proteins. One of the heat shock proteins, the protease encoded by the lon gene, recognizes unfolded proteins and degrades them in an ATP-dependent manner (6, 7). Mutations in the dnaK, dnaJ, grpE, and groEL heat shock genes resulted in defective proteolysis (25). The GroEL protein has been shown to transiently associate with newly synthesized recombinant DNA (rDNA) proteins to promote their assembly or secretion, and its dissociation from the proteins is ATP dependent (2, 9). Thus, it is important to study the physiological role of different heat
MATERIALS AND METHODS Bacterial strains and plasmids. E. coli RRlAM15 (17, 24) containing plasmid pRITcI857 (L. Abrahmsen, Ph.D. thesis, The Royal Institute of Technology, Stockholm, Sweden, 1988) was used as the bacterial host. pRITcI857 encodes the temperature-sensitive lambda repressor cI857 and contains a kanamycin resistance gene. The plasmids encoding the protein A-derived proteins were pRIT2 (21) and pRIT31 (Abrahmsdn, Ph.D. thesis, 1988). Expression of the proteins is under control of the phage lambda PR promoter; these plasmids also contain a gene for ampicillin resistance. Cultivation. pRIT2 protein A was produced in a 5-liter fermentor on complex medium as described elsewhere (11). Plasmid-free cells or cells containing plasmid pRIT31 were grown in 1-liter baffled shaking flasks containing 100 ml of medium (12). The cells were harvested by centrifugation and stored at -80°C. Buffers. Buffers used were as follows: TM, 50 mM Tris hydrochloride (pH 7.4-10 mM MgCl2; TS, 50 mM Tris hydrochloride (pH 7.4)-150 mM NaCl; TSM, 50 mM Tris hydrochloride (pH 7.4)-150 mM NaCl-10 mM MgCl2; and TST, 50 mM Tris hydrochloride (pH 7.4)-S150 mM NaCl0.05% Tween 20. Protein purification. Protein A was purified from cell extracts by IgG affinity chromatography, using IgG-Sepha-
* Corresponding author. t Present address: Nycomed AS, Postboks 4220 Torshov, N-0401 Oslo 4, Norway.
5030
VOL. 172, 1990
_~
5031
INTERACTION BETWEEN DnaK AND RECOMBINANT PROTEIN A
rose. The cells were suspended to 25% (wet weight) in TM, disintegrated in a French press, and clarified by centrifugation at 35,000 x g for 20 min. The cell extract was diluted 1:5 in TST before being loaded on the column. The column contained 2 ml of gel and was equilibrated with TST, loaded with 5 ml of sample, and washed with 50 ml of TST and 10 ml of 10 mM ammonium acetate (NH4Ac). Protein A was eluted with 0.2 M acetic acid (HAc) titrated to pH 3.2 with NH4Ac, and a 3-ml fraction was collected and lyophilized. The flow rate through the column was 2 ml min-', and the protein concentration was monitored by determining the A280. Three different elution methods were used to separate DnaK from the protein with which it interacts. Elution with ATP was performed with 3 mM ATP in either TM or TSM after washing with TST and elution buffer without ATP. Elution with salt was performed with 0.5, 1, or 2 M NaCl in 50 mM Tris hydrochloride (pH 7.4) after washing with TST and TS. For both ATP and salt elution, three fractions of 2.5 ml were collected, desalted on a Sephadex G-25 column equilibrated with TM (1:10), and lyophilized. The IgG column was further equilibrated with NH4Ac, and then the remaining protein was eluted with HAc (pH 3.2). Elution at lower pH was performed with 0.2 M HAc titrated with NH4Ac to pH 3.7, 4.2, or 4.7, respectively, after washing with TST and NH4Ac. Fractions of 2.5 ml were collected and lyophilized. The remaining protein was eluted with HAc (pH 3.2). In vitro studies. DnaK and protein A were purified from cell extracts by IgG affinity chromatography and eluted with HAc at pH 4.7 and 3.2, respectively. The HAc fractions were lyophilized, dissolved in TM, and clarified by centrifugation. The purified proteins were mixed and incubated at 37°C, and samples were removed for a second IgG purification. Analysis. Protein concentration was determined from the A280, using extinction coefficients of 0.21 ml mg-' cm-' for DnaK and 0.33 ml mg-' cm-' for pRIT2-encoded protein A. SDS-PAGE was performed with a 3.5% stacking gel and 11% separation gel (16). Native PAGE was performed with the Pharmacia Phast system, using a 10 to 15% gradient gel as recommended by the supplier. RESULTS Purification of DnaK. We had earlier observed that DnaK was recovered when protein A encoded by pRIT2 was purified by IgG affinity chromatography from cell extracts of E. coli (12). DnaK can interact with either IgG or protein A; to evaluate whether this interaction can be used for specific purification of DnaK, we decided to use the ATPase activity described for DnaK (28). A crude extract from E. coli cells containing pRIT2 was passed through an IgG-Sepharose column, and the effect of elution with ATP was investigated. When the protein was eluted with HAc (pH 3.2), both DnaK and protein A were released (Fig. 1, lane 1). A selective release of DnaK was achieved when ATP was used as an eluent (lanes 3 to 5). To exclude any effect of Mg or absence of salt in the elution buffer, the column was first washed with elution buffer without ATP (lane 2); no DnaK was released by this treatment. The remaining protein was eluted with HAc (pH 3.2) (lane 6). The desorption rate caused by ATP was higher when 150 mM NaCl was added to the elution buffer (lanes 3 to 5 compared with lanes 8 to 10). It has been reported that the pH optimum for ATPase activity is 8.8 (28), but the
933 66
*It'ho
4-
.--'
43
ts -4
22
}z.:~
_ M
1
2
3
4
5
6
7
8
9
10
FIG. 1. SDS-PAGE of IgG affinity-purified protein eluted with ATP. Crude cell extract was purified on IgG-Sepharose, and the bound protein was eluted with HAc (pH 3.2) (lane 1). A new sample was passed through the column; after washing, the column was equilibrated with elution buffer without ATP (lane 2), and the sample was eluted with 3 mM ATP in 50 mM Tris hydrochloride containing 10 mM MgCl2. Three fractions were collected (lanes 3 to 5), and the remaining protein was eluted with HAc (pH 3.2) (lane 6). Lanes 7 to 11, As lanes 2 to 6 except that the ATP-containing elution buffer contained 150 mM NaCl. Lane M, Marker proteins shown on the left as molecular sizes in kilodaltons. Arrows indicate DnaK (upper) and protein A (lower).
release of DnaK was slower at pH 8.8 than at pH 7.4 (data not shown). Buffer containing up to 2 M NaCl was also used as the eluent, but neither DnaK nor protein A was released (data not shown), suggesting that the binding of DnaK is salt resistant. DnaK could be quantitatively eluted from the IgG column by ATP (Fig. 1, lanes 8 to 10), but this method has the drawback that the sample must be desalted before lyophilization. A more convenient method of purifying DnaK was therefore tried. Both DnaK and protein A were eluted from the IgG column at pH 3.2; since the pH influences the stability of the interaction between proteins, a differential elution of DnaK using a higher pH was tried. When HAc (pH 3.7) was used as the eluent, both protein A and DnaK were released from the column, whereas elution at pH 4.2 yielded release of some of the protein A (data not shown). Elution with HAc at pH 4.7 gave a selective release of DnaK (Fig. 2, lanes 2 to 7). Protein A could then be released from IgG by HAc at pH 3.2 (lane 8). With this method, both protein A and DnaK could be purified by differential pH elution in a one-step procedure directly from the cell extract. In vitro studies with pure DnaK. Using affinity-purified pRIT2-encoded protein A and DnaK, eluted at different pHs, in vitro experiments were performed to investigate the interaction of DnaK with protein A and IgG. Purified samples of protein A and DnaK were mixed and incubated at 37°C. Samples were removed at different times for IgG purification, and the bound material was eluted at pH 3.2 and analyzed by SDS-PAGE. DnaK was recovered after the second IgG purification if it was mixed with protein A (Fig. 3, lanes 3 to 8) but was not recovered from the control sample without protein A (lane 9). This result shows that DnaK specifically interacts with protein A in vitro.
5032
J. BACTERIOL.
HELLEBUST ET AL.
3 _ 66
43
"
93
_0
66
fU*
43
%
_--_
_.
31
31
_WmmI _
222 MI
22
I1i
2
3
4
5
6
7
8
FIG. 2. SDS-PAGE of IgG affinity-purified protein eluted at different pHs. Crude cell extract was purified on IgG-Sepharose, and the bound protein was eluted with HAc (pH 3.2) (lane 1). A new sample was passed through the column; after washing, the protein was eluted with HAc (pH 4.7), and six fractions were collected (lanes 2 to 7). The remaining protein was eluted with HAc (pH 3.2) (lane 8). Lane M, Marker proteins shown on the left as molecular sizes in kilodaltons.
The recovery of protein A was quantitative after the second IgG purification. The recovery of DnaK increased for up to 1 h of incubation time. A comparison of the amount of DnaK before and after the second IgG purification as judged by SDS-PAGE (Fig. 3, lanes 2 and 4) suggests that only a fraction (about 25%) of the protein was bound to protein A. When the concentration of DnaK was increased fourfold, a corresponding increase in binding to protein A was observed (lanes 6 to 8). Localization of the DnaK binding site. Since DnaK has an affinity for protein A encoded by pRIT2, we wished to identify the part of protein A that is responsible for the binding. It is clear that the interaction did not interfere with the IgG-binding properties of protein A, since IgG purification gave a quantitative recovery of protein A (Fig. 3). Another protein A-derived protein encoded by pRIT31 was therefore expressed in E. coli and purified by IgG affinity chromatography. The structures of pRIT2 and pRIT31 proteins A are schematically shown in Fig. 4A. The N terminus and the first four IgG-binding domains of pRIT2 and pRIT31 are identical, but they differ at the C terminus. The IgGbinding C domain of pRIT2 is truncated, whereas it is complete in pRIT31. The two proteins have different C-terminal regions of amino acids of unknown structure. The two proteins A were incubated with DnaK for 1 h at 37°C and subsequently purified by IgG affinity chromatography. The affinity between DnaK and pRIT2 protein A was much stronger than that between DnaK and pRIT31 protein A (Fig. 4B). This result suggests that DnaK interacts mainly with the C terminus of pRIT2 protein A. There was no interaction between DnaK and native staphylococcal protein A (Sigma Chemical Co.) (data not shown). Measuring heat shock induction. The affinity between
1
2
3
4
5
6
7
8
9
10
FIG. 3. SDS-PAGE of purified protein A and DnaK incubated in vitro. Protein A and DnaK (lanes 1 and 2, respectively) were mixed and incubated at 37°C, and samples were removed and subsequently purified by IgG affinity chromatography. The concentration of protein A was 1.2 mg ml-', and that of DnaK was either 0.4 or 1.6 mg ml-' (DnaK4x). Lanes 3 to 5, Protein A and DnaK incubated at 37°C for 0, 1, and 2 h, respectively; lanes 6 to 8, protein A and DnaK4x incubated at 37°C for 0, 1, and 2 h, respectively; lane 9, DnaK incubated at 37°C for 2 h; lane 10, protein A incubated at 37°C for 2 h. Each lane was loaded with an amount corresponding to 12 pug of protein A and 4 ,ug (lanes 2 to 5 and 9) or 16 ,ug (lanes 6 to 8) of DnaK before IgG purification. Lane M, Marker proteins shown on the left as molecular sizes in kilodaltons.
pRIT2-encoded protein A and DnaK could be used to measure the induction of the heat shock response in cells, even though only a fraction of DnaK bound to protein A in vitro. Cell extracts from heat-induced E. coli were incubated with protein A, and then the samples were purified by IgG affinity chromatography. DnaK bound specifically to protein A in vitro, and the amount of DnaK complexed to protein A increased after heat shock induction (Fig. 5, lanes 1 to 4). It has previously been shown that protein A is degraded in the C terminus by cytoplasmic proteases when produced in E. coli (12). The results presented in Fig. 5 suggest that the protease(s) responsible for this degradation was present in all of the cell extracts, since protein A was extensively degraded in these samples (lanes 1 to 4) as compared with the control in which protein A was incubated with buffer (lane 5). Interaction between DnaK and casein. It has been postulated that DnaK binds to denaturated proteins (23). One method of analyzing the interaction between proteins in vitro is native electrophoresis. The complex between pRIT2encoded protein A and DnaK could be separated from free protein A and DnaK by native PAGE (Fig. 6, lanes 1 to 3). This method was used to study the interaction between DnaK and heat-denaturated casein. There was a strong association between DnaK and casein (Fig. 6, lane 4). DISCUSSION It has earlier been postulated that one function of DnaK in the cell is to bind to unfolded proteins and that the protein releases itself from the substrate by hydrolysis of ATP (23). The experiments presented here support and extend this
INTERACTION BETWEEN DnaK AND RECOMBINANT PROTEIN A
VOL. 172, 1990
A APRIT2
E'] DI Al B IC'ti
pRIT31
E
Df A B
93
-*
66
t
43
_
31
_
5033
..... 4-
B 66
43 1s
_ _
22 31
M
-
22 M
_06
1
2
FIG. 4. SDS-PAGE of protein A encoded by pRIT2 and pRIT31 incubated in vitro with DnaK. (A) Schematic structure of the proteins. From the N-terminus, they contain 12 amino acids from lambda Cro protein, followed by the IgG-binding domains E', D, A, and B from staphylococcal protein A. The truncated C domain of the gene product of pRIT2 (C') contains 37 of the 58 amino acids in the complete domain, followed by a 14-amino-acid tail at the C terminus (L'). pRIT31 contains the complete C domain, followed by a 24-amino-acid tail at the C terminus (L). (B) SDS-PAGE analysis. Proteins A encoded by pRIT2 and pRIT31 (1.2 mg ml-'), respectively, were incubated with DnaK (0.4 mg ml-1) for 1 h at 37°C and purified by IgG affinity chromatography. Lane 1, pRIT2 protein A incubated with DnaK; lane 2, pRIT31 protein A incubated with DnaK. Each lane was loaded with an amount corresponding to 12 ,ug of protein A and 4 ,ug of DnaK before the second IgG purification. Lane M, Marker proteins shown on the left as molecular sizes in kilodaltons.
hypothesis. DnaK has affinity for the C terminus of the recombinant protein encoded by pRIT2 and dissociates from it in the presence of ATP. The truncated IgG-binding C domain of the pRIT2 gene product probably lacks proper folding (12). This protein will therefore have a large unfolded tail of 51 amino acids. In comparison, the pRIT31 protein, which has considerably less affinity for DnaK, has only 24 amino acids after the last complete IgG-binding domain. A difference between pRIT2- and pRIT31-encoded proteins with respect to proteolytic degradation has earlier been shown (12). The pRIT2 protein was degraded in the C terminus included in the truncated C domain, whereas the complete C domain of pRIT31 was stable and degradation proceeded only in the 24-amino-acid tail. It is therefore possible that the mechanism of recognition is the same for DnaK and the protease. Interestingly, DnaK also has a strong affinity for denaturated casein, which also is known to be a good substrate for several E. coli proteases (8, 13). It has been shown elsewhere that dnaK mutants have a re-
1
.4,in .x_.._
2
3
_
4
_
5
FIG. 5. SDS-PAGE of protein A incubated with heat-shocked cells. E. coli RR1AM15 without plasmids was grown at 30°C and then switched to 42°C, and samples were removed 0, 15, 30, and 60 min after the temperature switch. The samples were concentrated to 12.5% (wet weight), disintegrated, and clarified by centrifugation. These cell extracts were incubated with protein A (0.6 mg ml-') for 2 h at 37°C, and then the samples were purified by IgG affinity chromatography. Lanes: 1 to 4, cell extract from 0, 15, 30, and 60 min, respectively, after the temperature switch, incubated with protein A; 5, protein A incubated with buffer; M, marker proteins shown on the left as molecular sizes in kilodaltons. Arrow indicates DnaK.
duced ability to degrade abnormal proteins (25), which suggests that DnaK binding could be a signal for proteolysis. Protein A encoded by pRIT2 could be a good model protein with which to study whether DnaK protects unfolded re-
1 2 3 4 5 FIG. 6. Native PAGE of protein A, DnaK, and casein. Protein A casein was mixed with DnaK and separated by native PAGE. Lanes: 1, protein A; 2, protein A plus DnaK; 3, DnaK; 4, casein plus DnaK; 5, casein. The complex bands are indicated by arrows. The final concentrations of protein in the samples were 1.5 mg ml-1 for protein A, 2 mg ml-1 for denaturated casein, and 1 mg ml-' for DnaK. Each lane was loaded with 1 ,ul of sample. or denaturated
5034
J. BACTERIOL.
HELLEBUST ET AL.
gions of proteins against proteolysis or whether it directs them toward proteolysis. Such experiments were performed, but only a fraction of the protein A molecules was bound to DnaK, and the protease responsible for degradation of protein A was rapidly inactivated in cell extract (12); as a result, no conclusion could be drawn from those experiments. It is therefore desirable to purify the protease and perform in vitro experiments with protein A, DnaK, and protease.
We have also found that another rDNA protein, protein disulfide isomerase, associated with the heat shock protein GroEL when expressed in E. coli (L. Cedergren and M. Uhldn, unpublished results). Thus, the different elements of the heat shock response seem to play an important role in the production of rDNA proteins. This heat shock response must be considered in analysis of rDNA protein with respect to both stability and the state of folding. The approach described here offers a simple method of purifying DnaK in a one-step procedure from a crude cell extract. Such purified DnaK can be used in in vitro experiments with different proteins to study the interaction between structure, folding, and heat shock protein binding. Recently, the dnaK gene was cloned and expressed as a fusion protein to the human serum albumin-binding domains of streptococcal protein G (22). Thus, the DnaK fusion protein can be purified and immobilized in a one-step procedure by using human serum albumin affinity chromatography (A. Hagman and M. Uhlen, unpublished results). The interaction between the immobilized DnaK and unfolded polypeptide chains may provide a method of separating proteins that lack proper folding from native proteins.
8. 9.
10. 11.
12. 13.
14.
15. 16. 17.
18.
ACKNOWLEDGMENTS We thank Alfred Goldberg (Harvard Medical School) as well as Bjcwn Nilsson, Tomas Moks, and Maria Murby for useful discus-
19. 20.
sions.
We thank the Swedish Board for Technical Development for financial support.
LITERATURE CITED 1. Bardwell, J. C. A., and E. A. Craig. 1984. Major heat shock gene of Drosophila and Escherichia coli heat-inducible dnaK gene are homologous. Proc. Natl. Acad. Sci. USA 81:848-852. 2. Bochkareva, E. S., N. M. Lissin, and A. S. Girshovich. 1988. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature (London) 336:254257. 3. Bukau, B., and G. C. Walker. 1989. Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism. J. Bacteriol. 171: 2337-2346. 4. Chesshyre, J. A., and A. R. Hipkiss. 1989. Low temperatures stabilize interferon alpha-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli. Appl. Microbiol. Biotechnol. 31:158-162. 5. Georgopoulos, C., K. Tilly, D. Drahos, and R. Hendrix. 1982. The B66.0 protein of Escherichia coli is product of the dnaK+ gene. J. Bacteriol. 149:1175-1177. 6. Goff, S. A., and A. L. Goldberg. 1985. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell 41:587-595. 7. Goff, S. A., R. Voellmy, and A. L. Goldberg. 1988. Protein
21. 22. 23.
breakdown and the heat-shock response, p. 207-238. In M. Rechsteiner (ed.), Ubiquitin. Plenum Publishing Corp., New York. Goldberg, A. L., K. H. S. Swamy, C. H. Chung, and F. Larimore. 1982. Proteinases in Escherichia coli. Methods Enzymol. 80:680-702. Goloubinoff, P., A. A. Gatenby, and G. H. Lorimer. 1989. GroE heat-shock proteins promote assembly of foreign procaryotic ribulose biphosphate carboxylase oligomers in Escherichia coli. Nature (London) 337:44 47. Harendeen, S. L., R. A. vanBogelen, and F. C. Neidhardt. 1979. Levels of major proteins of Escherichia coli during growth at different temperatures. J. Bacteriol. 139:185-194. Hellebust, H., M. Murby, L. Abrahmsen, M. Uhlen, and S. 0. Enfors. 1989. Different approaches to stabilize a recombinant fusion protein. Bio/Technology 7:165-168. Hellebust, H., M. Uhl6n, and S.-O. Enfors. 1989. Effect of protein fusion on the stability of proteolytically sensitive sites in recombinant DNA proteins. J. Biotechnol. 12:275-284. Hwang, B. J., W. J. Park, C. H. Chung, and A. L. Goldberg. 1987. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La. Proc. Natl. Acad. Sci. USA 84:5550-5554. Johnson, C., G. N. Chandrasekhar, and C. Georgopoulos. 1989. Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro. J. Bacteriol. 171:1590-1596. Kelley, P. M., and M. J. Schlesinger. 1982. Antibodies to two major chicken heat shock proteins cross-react with similar proteins in widely divergent species. Mol. Cell. Biol. 2:267-274. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Langley, K. E., M. R. Villarejo, A. V. Fowler, P. J. Zamenhof, and I. Zabin. 1975. Molecular basis of ,B-galactosidase alphacomplementation. Proc. Natl. Acad. Sci. USA 72:1254-1257. Lindquist, S. 1986. The heat-shock response. Annu. Rev. Biochem. 55:1151-1191. Lindquist, S., and E. A. Craig. 1988. The heat shock proteins. Annu. Rev. Genet. 22:631-677. Neidhardt, F. C., R. A. VanBogelen, and V. Vaughn. 1984. The genetics and regulation of heat-shock proteins. Annu. Rev. Genet. 18:295-329. Nilsson, B., L. Abrahmsen, and M. Uhlen. 1985. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 4:1075-1080. Nygren, P.-A., M. Eliasson, L. Abrahmsen, and M. Uhlen. 1988. Analysis and use of the serum albumin binding domains of streptococcal protein G. J. Mol. Recognition 1:69-74. Pelham, H. R. B. 1986. Speculations on the functions of the major heat-shock protein and glucose-regulated proteins. Cell
46:959-961.
24. Ruther, U. 1982. pUR 250 allows rapid chemical sequencing of both DNA strands of its insert. Nucleic Acids Res. 10:57655772. 25. Straus, D. B., W. A. Walter, and C. A. Gross. 1988. Escherichia coli heat shock gene mutants are defective in proteolysis. Genes Dev. 2:1851-1858. 26. Tilly, K., N. McKittrick, M. Zylicz, and C. Georgopoulos. 1983. The dnaK protein modulates the heat-shock response of Escherichia coli. Cell 34:641-646. 27. Zylicz, M., D. Ang, and C. Georgopoulos. 1987. The grpE protein of Escherichia coli. J. Biol. Chem. 262:17437-17442. 28. Zylicz, M., J. H. LeBowitz, R. McMacken, and C. Georgopoulos. 1983. The dnaK protein of Escherichia coli processes an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system. Proc. Natl. Acad. Sci. USA 80:6431-6435.
Vol. 172, No. 9
Copyright © 1990, American Society for Microbiology
Interaction between Heat Shock Protein DnaK and Recombinant Staphylococcal Protein A HALLDIS HELLEBUST,t* MATHIAS UHLtN, AND SVEN-OLOF ENFORS Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-10044 Stockholm, Sweden Received 6 March 1990/Accepted 2 July 1990 When a protein derived from the immunoglobulin G (IgG)-binding domains of staphylococcal protein A was expressed in Escherichia coil and recovered from cell extract by IgG affinity chromatography, the 69-kilodalton heat shock protein DnaK was found to be copurified. DnaK could be selectively eluted from the IgG column by ATP or by lowering the pH to 4.7. Protein A could subsequently be eluted by lowering the pH to 3.2. Thus, this procedure allows a one-step purification of both DnaK and protein A from cell extract. In vitro experiments with pure DnaK and protein A revealed that DnaK did not interfere with the IgG-binding properties of protein A but associated with its unfolded C-terminal in a salt-resistant manner. In addition, a specific interaction between DnaK and denaturated caseiI was found.
shock proteins and their role with respect to rDNA proteins expressed in bacteria. We have previously studied different recombinant DNA proteins derived from staphylococcal protein A expressed intracellularly in E. coli with respect to their stability against proteolytic degradation (11, 12). One of these protein A derivatives with a proteolytically sensitive C terminus was used as a model to study the effect of protein fusion on this stability. When this protein was purified by immunoglobulin G (IgG) affinity chromatography and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), it was found that the 69-kilodalton heat shock protein DnaK copurified with protein A (12). In this work, we studied the specific interaction in more detail and used different methods to selectively elute the DnaK from the IgG column. In vitro experiments with affinity-purified proteins show that DnaK interacts with the unfolded C-terminal region of protein A.
The heat shock response is one of the most conserved elements of biology; it is an inducible response to cellular stresses such as heat, exposure to ethanol, DNA damage, accumulation of denaturated or abnormal proteins, and nutrient starvation (18). In Escherichia coli, the response is an induction of at least 17 different proteins, but only for a few of the proteins is the physiological role known (20). Many of the heat shock proteins are also essential under normal growth conditions. (3, 19). DnaK is one of the most abundant heat shock proteins in E. coli; under normal growth at 37°C, it accounts for 1.4% of the total cellular protein (5, 10). It was originally identified as a host protein required for replication of bacteriophage lambda (20), and further studies of mutants have shown that DnaK is a modulator of the heat shock response (26). Purified DnaK binds tightly to ATP, has weak ATPase activity, and carries out autophosphorylation (28). A high degree of antigenic similarity has been found between DnaK and the corresponding components of yeast, Drosophila, chicken and human cells (1, 15). It has been found to associate in a salt-resistant manner with another heat shock protein, GrpE, both in vivo and in vitro, and it is released from this complex by ATP (14, 27). One function postulated for this protein is that it has an affinity for proteins that lack proper folding and releases itself from the substrate protein by hydrolysis of ATP (23). Recombinant DNA proteins often misfold when expressed in bacteria (4, 6), and one function of the heat shock proteins might be to contribute to the ability of the cells to degrade misfolded proteins. One of the heat shock proteins, the protease encoded by the lon gene, recognizes unfolded proteins and degrades them in an ATP-dependent manner (6, 7). Mutations in the dnaK, dnaJ, grpE, and groEL heat shock genes resulted in defective proteolysis (25). The GroEL protein has been shown to transiently associate with newly synthesized recombinant DNA (rDNA) proteins to promote their assembly or secretion, and its dissociation from the proteins is ATP dependent (2, 9). Thus, it is important to study the physiological role of different heat
MATERIALS AND METHODS Bacterial strains and plasmids. E. coli RRlAM15 (17, 24) containing plasmid pRITcI857 (L. Abrahmsen, Ph.D. thesis, The Royal Institute of Technology, Stockholm, Sweden, 1988) was used as the bacterial host. pRITcI857 encodes the temperature-sensitive lambda repressor cI857 and contains a kanamycin resistance gene. The plasmids encoding the protein A-derived proteins were pRIT2 (21) and pRIT31 (Abrahmsdn, Ph.D. thesis, 1988). Expression of the proteins is under control of the phage lambda PR promoter; these plasmids also contain a gene for ampicillin resistance. Cultivation. pRIT2 protein A was produced in a 5-liter fermentor on complex medium as described elsewhere (11). Plasmid-free cells or cells containing plasmid pRIT31 were grown in 1-liter baffled shaking flasks containing 100 ml of medium (12). The cells were harvested by centrifugation and stored at -80°C. Buffers. Buffers used were as follows: TM, 50 mM Tris hydrochloride (pH 7.4-10 mM MgCl2; TS, 50 mM Tris hydrochloride (pH 7.4)-150 mM NaCl; TSM, 50 mM Tris hydrochloride (pH 7.4)-150 mM NaCl-10 mM MgCl2; and TST, 50 mM Tris hydrochloride (pH 7.4)-S150 mM NaCl0.05% Tween 20. Protein purification. Protein A was purified from cell extracts by IgG affinity chromatography, using IgG-Sepha-
* Corresponding author. t Present address: Nycomed AS, Postboks 4220 Torshov, N-0401 Oslo 4, Norway.
5030
VOL. 172, 1990
_~
5031
INTERACTION BETWEEN DnaK AND RECOMBINANT PROTEIN A
rose. The cells were suspended to 25% (wet weight) in TM, disintegrated in a French press, and clarified by centrifugation at 35,000 x g for 20 min. The cell extract was diluted 1:5 in TST before being loaded on the column. The column contained 2 ml of gel and was equilibrated with TST, loaded with 5 ml of sample, and washed with 50 ml of TST and 10 ml of 10 mM ammonium acetate (NH4Ac). Protein A was eluted with 0.2 M acetic acid (HAc) titrated to pH 3.2 with NH4Ac, and a 3-ml fraction was collected and lyophilized. The flow rate through the column was 2 ml min-', and the protein concentration was monitored by determining the A280. Three different elution methods were used to separate DnaK from the protein with which it interacts. Elution with ATP was performed with 3 mM ATP in either TM or TSM after washing with TST and elution buffer without ATP. Elution with salt was performed with 0.5, 1, or 2 M NaCl in 50 mM Tris hydrochloride (pH 7.4) after washing with TST and TS. For both ATP and salt elution, three fractions of 2.5 ml were collected, desalted on a Sephadex G-25 column equilibrated with TM (1:10), and lyophilized. The IgG column was further equilibrated with NH4Ac, and then the remaining protein was eluted with HAc (pH 3.2). Elution at lower pH was performed with 0.2 M HAc titrated with NH4Ac to pH 3.7, 4.2, or 4.7, respectively, after washing with TST and NH4Ac. Fractions of 2.5 ml were collected and lyophilized. The remaining protein was eluted with HAc (pH 3.2). In vitro studies. DnaK and protein A were purified from cell extracts by IgG affinity chromatography and eluted with HAc at pH 4.7 and 3.2, respectively. The HAc fractions were lyophilized, dissolved in TM, and clarified by centrifugation. The purified proteins were mixed and incubated at 37°C, and samples were removed for a second IgG purification. Analysis. Protein concentration was determined from the A280, using extinction coefficients of 0.21 ml mg-' cm-' for DnaK and 0.33 ml mg-' cm-' for pRIT2-encoded protein A. SDS-PAGE was performed with a 3.5% stacking gel and 11% separation gel (16). Native PAGE was performed with the Pharmacia Phast system, using a 10 to 15% gradient gel as recommended by the supplier. RESULTS Purification of DnaK. We had earlier observed that DnaK was recovered when protein A encoded by pRIT2 was purified by IgG affinity chromatography from cell extracts of E. coli (12). DnaK can interact with either IgG or protein A; to evaluate whether this interaction can be used for specific purification of DnaK, we decided to use the ATPase activity described for DnaK (28). A crude extract from E. coli cells containing pRIT2 was passed through an IgG-Sepharose column, and the effect of elution with ATP was investigated. When the protein was eluted with HAc (pH 3.2), both DnaK and protein A were released (Fig. 1, lane 1). A selective release of DnaK was achieved when ATP was used as an eluent (lanes 3 to 5). To exclude any effect of Mg or absence of salt in the elution buffer, the column was first washed with elution buffer without ATP (lane 2); no DnaK was released by this treatment. The remaining protein was eluted with HAc (pH 3.2) (lane 6). The desorption rate caused by ATP was higher when 150 mM NaCl was added to the elution buffer (lanes 3 to 5 compared with lanes 8 to 10). It has been reported that the pH optimum for ATPase activity is 8.8 (28), but the
933 66
*It'ho
4-
.--'
43
ts -4
22
}z.:~
_ M
1
2
3
4
5
6
7
8
9
10
FIG. 1. SDS-PAGE of IgG affinity-purified protein eluted with ATP. Crude cell extract was purified on IgG-Sepharose, and the bound protein was eluted with HAc (pH 3.2) (lane 1). A new sample was passed through the column; after washing, the column was equilibrated with elution buffer without ATP (lane 2), and the sample was eluted with 3 mM ATP in 50 mM Tris hydrochloride containing 10 mM MgCl2. Three fractions were collected (lanes 3 to 5), and the remaining protein was eluted with HAc (pH 3.2) (lane 6). Lanes 7 to 11, As lanes 2 to 6 except that the ATP-containing elution buffer contained 150 mM NaCl. Lane M, Marker proteins shown on the left as molecular sizes in kilodaltons. Arrows indicate DnaK (upper) and protein A (lower).
release of DnaK was slower at pH 8.8 than at pH 7.4 (data not shown). Buffer containing up to 2 M NaCl was also used as the eluent, but neither DnaK nor protein A was released (data not shown), suggesting that the binding of DnaK is salt resistant. DnaK could be quantitatively eluted from the IgG column by ATP (Fig. 1, lanes 8 to 10), but this method has the drawback that the sample must be desalted before lyophilization. A more convenient method of purifying DnaK was therefore tried. Both DnaK and protein A were eluted from the IgG column at pH 3.2; since the pH influences the stability of the interaction between proteins, a differential elution of DnaK using a higher pH was tried. When HAc (pH 3.7) was used as the eluent, both protein A and DnaK were released from the column, whereas elution at pH 4.2 yielded release of some of the protein A (data not shown). Elution with HAc at pH 4.7 gave a selective release of DnaK (Fig. 2, lanes 2 to 7). Protein A could then be released from IgG by HAc at pH 3.2 (lane 8). With this method, both protein A and DnaK could be purified by differential pH elution in a one-step procedure directly from the cell extract. In vitro studies with pure DnaK. Using affinity-purified pRIT2-encoded protein A and DnaK, eluted at different pHs, in vitro experiments were performed to investigate the interaction of DnaK with protein A and IgG. Purified samples of protein A and DnaK were mixed and incubated at 37°C. Samples were removed at different times for IgG purification, and the bound material was eluted at pH 3.2 and analyzed by SDS-PAGE. DnaK was recovered after the second IgG purification if it was mixed with protein A (Fig. 3, lanes 3 to 8) but was not recovered from the control sample without protein A (lane 9). This result shows that DnaK specifically interacts with protein A in vitro.
5032
J. BACTERIOL.
HELLEBUST ET AL.
3 _ 66
43
"
93
_0
66
fU*
43
%
_--_
_.
31
31
_WmmI _
222 MI
22
I1i
2
3
4
5
6
7
8
FIG. 2. SDS-PAGE of IgG affinity-purified protein eluted at different pHs. Crude cell extract was purified on IgG-Sepharose, and the bound protein was eluted with HAc (pH 3.2) (lane 1). A new sample was passed through the column; after washing, the protein was eluted with HAc (pH 4.7), and six fractions were collected (lanes 2 to 7). The remaining protein was eluted with HAc (pH 3.2) (lane 8). Lane M, Marker proteins shown on the left as molecular sizes in kilodaltons.
The recovery of protein A was quantitative after the second IgG purification. The recovery of DnaK increased for up to 1 h of incubation time. A comparison of the amount of DnaK before and after the second IgG purification as judged by SDS-PAGE (Fig. 3, lanes 2 and 4) suggests that only a fraction (about 25%) of the protein was bound to protein A. When the concentration of DnaK was increased fourfold, a corresponding increase in binding to protein A was observed (lanes 6 to 8). Localization of the DnaK binding site. Since DnaK has an affinity for protein A encoded by pRIT2, we wished to identify the part of protein A that is responsible for the binding. It is clear that the interaction did not interfere with the IgG-binding properties of protein A, since IgG purification gave a quantitative recovery of protein A (Fig. 3). Another protein A-derived protein encoded by pRIT31 was therefore expressed in E. coli and purified by IgG affinity chromatography. The structures of pRIT2 and pRIT31 proteins A are schematically shown in Fig. 4A. The N terminus and the first four IgG-binding domains of pRIT2 and pRIT31 are identical, but they differ at the C terminus. The IgGbinding C domain of pRIT2 is truncated, whereas it is complete in pRIT31. The two proteins have different C-terminal regions of amino acids of unknown structure. The two proteins A were incubated with DnaK for 1 h at 37°C and subsequently purified by IgG affinity chromatography. The affinity between DnaK and pRIT2 protein A was much stronger than that between DnaK and pRIT31 protein A (Fig. 4B). This result suggests that DnaK interacts mainly with the C terminus of pRIT2 protein A. There was no interaction between DnaK and native staphylococcal protein A (Sigma Chemical Co.) (data not shown). Measuring heat shock induction. The affinity between
1
2
3
4
5
6
7
8
9
10
FIG. 3. SDS-PAGE of purified protein A and DnaK incubated in vitro. Protein A and DnaK (lanes 1 and 2, respectively) were mixed and incubated at 37°C, and samples were removed and subsequently purified by IgG affinity chromatography. The concentration of protein A was 1.2 mg ml-', and that of DnaK was either 0.4 or 1.6 mg ml-' (DnaK4x). Lanes 3 to 5, Protein A and DnaK incubated at 37°C for 0, 1, and 2 h, respectively; lanes 6 to 8, protein A and DnaK4x incubated at 37°C for 0, 1, and 2 h, respectively; lane 9, DnaK incubated at 37°C for 2 h; lane 10, protein A incubated at 37°C for 2 h. Each lane was loaded with an amount corresponding to 12 pug of protein A and 4 ,ug (lanes 2 to 5 and 9) or 16 ,ug (lanes 6 to 8) of DnaK before IgG purification. Lane M, Marker proteins shown on the left as molecular sizes in kilodaltons.
pRIT2-encoded protein A and DnaK could be used to measure the induction of the heat shock response in cells, even though only a fraction of DnaK bound to protein A in vitro. Cell extracts from heat-induced E. coli were incubated with protein A, and then the samples were purified by IgG affinity chromatography. DnaK bound specifically to protein A in vitro, and the amount of DnaK complexed to protein A increased after heat shock induction (Fig. 5, lanes 1 to 4). It has previously been shown that protein A is degraded in the C terminus by cytoplasmic proteases when produced in E. coli (12). The results presented in Fig. 5 suggest that the protease(s) responsible for this degradation was present in all of the cell extracts, since protein A was extensively degraded in these samples (lanes 1 to 4) as compared with the control in which protein A was incubated with buffer (lane 5). Interaction between DnaK and casein. It has been postulated that DnaK binds to denaturated proteins (23). One method of analyzing the interaction between proteins in vitro is native electrophoresis. The complex between pRIT2encoded protein A and DnaK could be separated from free protein A and DnaK by native PAGE (Fig. 6, lanes 1 to 3). This method was used to study the interaction between DnaK and heat-denaturated casein. There was a strong association between DnaK and casein (Fig. 6, lane 4). DISCUSSION It has earlier been postulated that one function of DnaK in the cell is to bind to unfolded proteins and that the protein releases itself from the substrate by hydrolysis of ATP (23). The experiments presented here support and extend this
INTERACTION BETWEEN DnaK AND RECOMBINANT PROTEIN A
VOL. 172, 1990
A APRIT2
E'] DI Al B IC'ti
pRIT31
E
Df A B
93
-*
66
t
43
_
31
_
5033
..... 4-
B 66
43 1s
_ _
22 31
M
-
22 M
_06
1
2
FIG. 4. SDS-PAGE of protein A encoded by pRIT2 and pRIT31 incubated in vitro with DnaK. (A) Schematic structure of the proteins. From the N-terminus, they contain 12 amino acids from lambda Cro protein, followed by the IgG-binding domains E', D, A, and B from staphylococcal protein A. The truncated C domain of the gene product of pRIT2 (C') contains 37 of the 58 amino acids in the complete domain, followed by a 14-amino-acid tail at the C terminus (L'). pRIT31 contains the complete C domain, followed by a 24-amino-acid tail at the C terminus (L). (B) SDS-PAGE analysis. Proteins A encoded by pRIT2 and pRIT31 (1.2 mg ml-'), respectively, were incubated with DnaK (0.4 mg ml-1) for 1 h at 37°C and purified by IgG affinity chromatography. Lane 1, pRIT2 protein A incubated with DnaK; lane 2, pRIT31 protein A incubated with DnaK. Each lane was loaded with an amount corresponding to 12 ,ug of protein A and 4 ,ug of DnaK before the second IgG purification. Lane M, Marker proteins shown on the left as molecular sizes in kilodaltons.
hypothesis. DnaK has affinity for the C terminus of the recombinant protein encoded by pRIT2 and dissociates from it in the presence of ATP. The truncated IgG-binding C domain of the pRIT2 gene product probably lacks proper folding (12). This protein will therefore have a large unfolded tail of 51 amino acids. In comparison, the pRIT31 protein, which has considerably less affinity for DnaK, has only 24 amino acids after the last complete IgG-binding domain. A difference between pRIT2- and pRIT31-encoded proteins with respect to proteolytic degradation has earlier been shown (12). The pRIT2 protein was degraded in the C terminus included in the truncated C domain, whereas the complete C domain of pRIT31 was stable and degradation proceeded only in the 24-amino-acid tail. It is therefore possible that the mechanism of recognition is the same for DnaK and the protease. Interestingly, DnaK also has a strong affinity for denaturated casein, which also is known to be a good substrate for several E. coli proteases (8, 13). It has been shown elsewhere that dnaK mutants have a re-
1
.4,in .x_.._
2
3
_
4
_
5
FIG. 5. SDS-PAGE of protein A incubated with heat-shocked cells. E. coli RR1AM15 without plasmids was grown at 30°C and then switched to 42°C, and samples were removed 0, 15, 30, and 60 min after the temperature switch. The samples were concentrated to 12.5% (wet weight), disintegrated, and clarified by centrifugation. These cell extracts were incubated with protein A (0.6 mg ml-') for 2 h at 37°C, and then the samples were purified by IgG affinity chromatography. Lanes: 1 to 4, cell extract from 0, 15, 30, and 60 min, respectively, after the temperature switch, incubated with protein A; 5, protein A incubated with buffer; M, marker proteins shown on the left as molecular sizes in kilodaltons. Arrow indicates DnaK.
duced ability to degrade abnormal proteins (25), which suggests that DnaK binding could be a signal for proteolysis. Protein A encoded by pRIT2 could be a good model protein with which to study whether DnaK protects unfolded re-
1 2 3 4 5 FIG. 6. Native PAGE of protein A, DnaK, and casein. Protein A casein was mixed with DnaK and separated by native PAGE. Lanes: 1, protein A; 2, protein A plus DnaK; 3, DnaK; 4, casein plus DnaK; 5, casein. The complex bands are indicated by arrows. The final concentrations of protein in the samples were 1.5 mg ml-1 for protein A, 2 mg ml-1 for denaturated casein, and 1 mg ml-' for DnaK. Each lane was loaded with 1 ,ul of sample. or denaturated
5034
J. BACTERIOL.
HELLEBUST ET AL.
gions of proteins against proteolysis or whether it directs them toward proteolysis. Such experiments were performed, but only a fraction of the protein A molecules was bound to DnaK, and the protease responsible for degradation of protein A was rapidly inactivated in cell extract (12); as a result, no conclusion could be drawn from those experiments. It is therefore desirable to purify the protease and perform in vitro experiments with protein A, DnaK, and protease.
We have also found that another rDNA protein, protein disulfide isomerase, associated with the heat shock protein GroEL when expressed in E. coli (L. Cedergren and M. Uhldn, unpublished results). Thus, the different elements of the heat shock response seem to play an important role in the production of rDNA proteins. This heat shock response must be considered in analysis of rDNA protein with respect to both stability and the state of folding. The approach described here offers a simple method of purifying DnaK in a one-step procedure from a crude cell extract. Such purified DnaK can be used in in vitro experiments with different proteins to study the interaction between structure, folding, and heat shock protein binding. Recently, the dnaK gene was cloned and expressed as a fusion protein to the human serum albumin-binding domains of streptococcal protein G (22). Thus, the DnaK fusion protein can be purified and immobilized in a one-step procedure by using human serum albumin affinity chromatography (A. Hagman and M. Uhlen, unpublished results). The interaction between the immobilized DnaK and unfolded polypeptide chains may provide a method of separating proteins that lack proper folding from native proteins.
8. 9.
10. 11.
12. 13.
14.
15. 16. 17.
18.
ACKNOWLEDGMENTS We thank Alfred Goldberg (Harvard Medical School) as well as Bjcwn Nilsson, Tomas Moks, and Maria Murby for useful discus-
19. 20.
sions.
We thank the Swedish Board for Technical Development for financial support.
LITERATURE CITED 1. Bardwell, J. C. A., and E. A. Craig. 1984. Major heat shock gene of Drosophila and Escherichia coli heat-inducible dnaK gene are homologous. Proc. Natl. Acad. Sci. USA 81:848-852. 2. Bochkareva, E. S., N. M. Lissin, and A. S. Girshovich. 1988. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature (London) 336:254257. 3. Bukau, B., and G. C. Walker. 1989. Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism. J. Bacteriol. 171: 2337-2346. 4. Chesshyre, J. A., and A. R. Hipkiss. 1989. Low temperatures stabilize interferon alpha-2 against proteolysis in Methylophilus methylotrophus and Escherichia coli. Appl. Microbiol. Biotechnol. 31:158-162. 5. Georgopoulos, C., K. Tilly, D. Drahos, and R. Hendrix. 1982. The B66.0 protein of Escherichia coli is product of the dnaK+ gene. J. Bacteriol. 149:1175-1177. 6. Goff, S. A., and A. L. Goldberg. 1985. Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes. Cell 41:587-595. 7. Goff, S. A., R. Voellmy, and A. L. Goldberg. 1988. Protein
21. 22. 23.
breakdown and the heat-shock response, p. 207-238. In M. Rechsteiner (ed.), Ubiquitin. Plenum Publishing Corp., New York. Goldberg, A. L., K. H. S. Swamy, C. H. Chung, and F. Larimore. 1982. Proteinases in Escherichia coli. Methods Enzymol. 80:680-702. Goloubinoff, P., A. A. Gatenby, and G. H. Lorimer. 1989. GroE heat-shock proteins promote assembly of foreign procaryotic ribulose biphosphate carboxylase oligomers in Escherichia coli. Nature (London) 337:44 47. Harendeen, S. L., R. A. vanBogelen, and F. C. Neidhardt. 1979. Levels of major proteins of Escherichia coli during growth at different temperatures. J. Bacteriol. 139:185-194. Hellebust, H., M. Murby, L. Abrahmsen, M. Uhlen, and S. 0. Enfors. 1989. Different approaches to stabilize a recombinant fusion protein. Bio/Technology 7:165-168. Hellebust, H., M. Uhl6n, and S.-O. Enfors. 1989. Effect of protein fusion on the stability of proteolytically sensitive sites in recombinant DNA proteins. J. Biotechnol. 12:275-284. Hwang, B. J., W. J. Park, C. H. Chung, and A. L. Goldberg. 1987. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La. Proc. Natl. Acad. Sci. USA 84:5550-5554. Johnson, C., G. N. Chandrasekhar, and C. Georgopoulos. 1989. Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro. J. Bacteriol. 171:1590-1596. Kelley, P. M., and M. J. Schlesinger. 1982. Antibodies to two major chicken heat shock proteins cross-react with similar proteins in widely divergent species. Mol. Cell. Biol. 2:267-274. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Langley, K. E., M. R. Villarejo, A. V. Fowler, P. J. Zamenhof, and I. Zabin. 1975. Molecular basis of ,B-galactosidase alphacomplementation. Proc. Natl. Acad. Sci. USA 72:1254-1257. Lindquist, S. 1986. The heat-shock response. Annu. Rev. Biochem. 55:1151-1191. Lindquist, S., and E. A. Craig. 1988. The heat shock proteins. Annu. Rev. Genet. 22:631-677. Neidhardt, F. C., R. A. VanBogelen, and V. Vaughn. 1984. The genetics and regulation of heat-shock proteins. Annu. Rev. Genet. 18:295-329. Nilsson, B., L. Abrahmsen, and M. Uhlen. 1985. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 4:1075-1080. Nygren, P.-A., M. Eliasson, L. Abrahmsen, and M. Uhlen. 1988. Analysis and use of the serum albumin binding domains of streptococcal protein G. J. Mol. Recognition 1:69-74. Pelham, H. R. B. 1986. Speculations on the functions of the major heat-shock protein and glucose-regulated proteins. Cell
46:959-961.
24. Ruther, U. 1982. pUR 250 allows rapid chemical sequencing of both DNA strands of its insert. Nucleic Acids Res. 10:57655772. 25. Straus, D. B., W. A. Walter, and C. A. Gross. 1988. Escherichia coli heat shock gene mutants are defective in proteolysis. Genes Dev. 2:1851-1858. 26. Tilly, K., N. McKittrick, M. Zylicz, and C. Georgopoulos. 1983. The dnaK protein modulates the heat-shock response of Escherichia coli. Cell 34:641-646. 27. Zylicz, M., D. Ang, and C. Georgopoulos. 1987. The grpE protein of Escherichia coli. J. Biol. Chem. 262:17437-17442. 28. Zylicz, M., J. H. LeBowitz, R. McMacken, and C. Georgopoulos. 1983. The dnaK protein of Escherichia coli processes an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system. Proc. Natl. Acad. Sci. USA 80:6431-6435.
