An improved bulk purification method for Escherichia coli elongation factor, Ts

ANALYTICAL

BIOCHEMISTRY

141, 351-354 (1984)

An Improved

Bulk Purification Method for Escherichia Elongation Factor, Ts’

co/i

MARILYNYODER,JOYCELENNANE,ANDFRANCESJURNAK Department of Biochemistry, University of California. Riverside, California 92521 Received October 26, 1983 A bulk purification procedure has been designed to maximize the yield of Escherichia co/i elongation factor, Ts, with a minimum of effort and time. The enzyme purification is achieved by DEAE-Sepharose and elongation factor Tu-affinity chromatographies. The typical yield is 150 m&kg of E. coli (B) cells. KEY WORDS: elongation factor Ts, EF-Tu resin.

Ts* is one of three cytoplasmic factors which are essential for the elongation of a polypeptide chain during protein synthesis in Escherichia co/i (for review, see Ref. (1)). Ts catalyzes the exchange of GDP for GTP on another elongation factor, Tu, in order to convert Tu to a form which recognizes aminoacyl-tRNA. The intermediate formed in the exchange process, a Tu-Ts binary complex, is relatively stable in the absence of GDP and has a dissociation constant of 7.7 X lop9 M (2). The basic molecular properties of Ts have been reported (3-6) but very little is known about the molecular nature of the Tu-Ts interaction. Although small crystals of the Tu-Ts complex have been grown (7,8), attempts to further characterize the Tu-Ts complex by three-dimensional X-ray diffraction studies have been restricted by the limited quantities of Ts from previous purification schemes (3,7,9). To obtain larger quantities of homogenous Ts, we redesigned the purification protocol to incorporate more efficient ’ This investigation was supported by grants from the American Philosophical Society (Penrose 9272) and the United States Public Health Service (GM26895). * Abbreviations used: Tu and Ts, cytoplasmic protein synthesis elongation factors from E. coli; Tu resin, TuGDP&aminohexanoic acid-activated Sepharose 4B; SDS, sodium dodecyl sulfate: PMSF, phenylmethylsulfonyl fluoride.

affinity chromatographic methods ( 10) and to maximize the yield of very pure Ts with minimum effort. With our new scheme, we routinely obtain yields of 150 mg of Ts/kg of E. coli cells. MATERIALS

Unless otherwise noted all biochemicals, including the 6-aminohexanoic acid-activated Sepharose 4B, were purchased from Sigma. For protein purification, E. cofi (B) cells (3/4 log) grown on enriched medium were obtained from Grain Processing Corporation, DEAE-Sepharose CG6B was from Pharmacia Fine Chemicals, and ultrapure ammonium sulfate was from Schwarz/Mann. For the enzymatic assays, [3H]GDP (7 mCi/lrmol) was purchased from Schwarz/Mann, the nitrocellulose filters (HA type) were from Millipore Corporation, and Bray’s scintillation cocktail was from ICN. For the polyacrylamide gels, electrophoresis-grade reagents, including acrylamide, NJV-methylenebisacrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate, and glycine, were purchased from Bio-Rad Laboratories. METHODS

Ts assay. The unit activity of Ts is defined as the amount of Ts which catalyzes the 351

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352

YODER,

LENNANE,

exchange of 1 pmol of t3H]GDP with TuGDP in 5 min at 0°C. The Ts assay is based on a standard nitrocellulose filter assay as previously described (1 I, 12). As noted by other investigators (7,9), the assay must be carried out under conditions in which the added Ts is proportional to the amount of [3H]GDP exchange on Tu-GDP. The correct relationship is maintained if the molar ratio for Tu/Ts is greater than 15. Protein concentration. Purified Ts and total protein concentrations were determined by the method of Bradford (13) as modified by Sedmak and Grossberg ( 14) using bovine serum albumin as a standard. Polyacrylamide gel electrophoresis. For the SDS-polyacrylamide gel, the method of Laemmli (15) was utilized. Molecular weights were determined by a comparison of migration distances to standards (16). Preparation of Tu resin. E. coli (B) TuGDP was prepared as previously described (17). For coupling to the 6-aminohexanoic acid-activated Sepharose 4B resin, Tu-GDP was dialyzed against Buffer H (50 IIIM 4 - (2 - hydroxyethyl) - I- piperazineethanesulfonic acid (Hepes), pH 7.5, 100 mM KCI, 10 InM 2-mercaptoethanol, 0.1 mM PMSF, and 10e5 M GDP). The activated resin was preswollen in 1 mM HCI and rinsed with 2 vol of Buffer H. Approximately 30 mg of Tu-GDP was added to each milliliter of prepared resin and mixed for 1 h at 20°C. After centrifugation, the excess Tu-GDP in the supematant was removed. The resin was washed twice with 2 vol of Buffer H and mixed for 1 h at 20°C with 2 vol of 100 mM Tris-HCI, pH 7.5, to block any remaining active resin sites. The TABLE

AND

JURNAK

Tu-GDP resin was stored in Buffer Tu (50 mM Tris-HCI, pH 7.5, 10 mM MgQ, 10 mM 2-mercaptoethanol, and 1 X lo-’ M GDP) until required. The excess Tu-GDP was dialyzed against Buffer Tu and quantitated spectrophotometrically and by the standard [3H]GDP-binding assay (11). The difference between the initial and the recovered Tu-GDP was the amount of Tu-GDP coupled to the aflinity resin. In repeated preparations, 18-27 mg of Tu-GDP was coupled per milliliter of resin. RESULTS

Several schemes for the purification of Ts from E. coli or Bacillus stearothermophilus have been reported (3,7,9,11). We have designed a more efficient, higher yielding, rapid purification scheme which incorporates our modifications as well as the best of previously published protocols. A summary of our protocol is shown in Table 1. Electrophoretic analysis of the sample pool following each step is shown in Fig. 1. All work and conductivity measurements were carried out at 10°C. Cell lysis. One kilogram of frozen E. coli (B) paste was thawed in 1700 ml of Buffer A (20 IYIM Tris-HCl, pH 7.6, 10 InM MgClz, 10 mM 2-mercaptoethanol, 0.1 mM PMSF, and 5 X IO-’ M GDP) containing 50 mM NH&l and lysed in a Gaulin mill at 8000 psi. After removal of the cell debris by centrifugation, the supematant was diluted with water to a conductivity of 2.3 mmho and adjusted to a pH of 7.6. Assays of the diluted supematant indicated 1.54 X IO* units of Ts activity. 1

SUMMARYOFPIJRFICATION OFTSFROM1 kgo~ Escherichia coli (B)CELLPASTE Volume (ml)

Step Cell extract DEAE-Sepharose Tu resin

CL6B

8ooO 2550 174

Total

protein but)

70,400 5,621 154.1

Total (units

activity X 10e6) 154.6 152.0 80.9

Specific activity (units/mg) 2,196 27,041 525,000

Yield 6) 100 98.3 52.3

CHROMATOGRAPHIC

PURIFICATION

OF ELONGATION

FACTOR

-

I

2

3

Ts

353

18,500 12,000

4

FIG. 1. Electrophoretic analysis of Ts samples. Ts samples following each chromatographic step were electrophoresed on a 12.5% polyacrylamide slab gel using a sodium dodecyl sulfate, Tris/glycine buffer system (15). The protein samples in each of the lanes are: 1, post-DEAE-Sepharose sample of Ts pool; 2 and 3, post-Tu resin sample of Ts pool; and 4, protein markers bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (29,000), r!Slactoglobulin (l&500), and cytochrome c (12,ooO).

Ion-exchange chromatography. The supernatant was applied to a DEAE-Sepharose column (7.5 X 75 cm) equilibrated in Buffer A with a conductivity of 1.7 mmho. The column was washed with 8 liters of Buffer A at a flow rate of 150 ml/h until the absorbance at 280 nm reached background levels. The column was developed with a 1Zliter linear gradient from 0.0 to 0.15 M KC1 in Buffer A. Fractions of 20 ml were collected and assayed for Ts activity. By including all fractions with any measurable Ts activity, more than 98% of the Ts activity was recovered, precipitated with 80% ammonium sulfate, and stored at -80°C until required. The increase in specific activity of Ts indicated a 20-fold purification. It should also be noted that the binding of Ts to the DEAE-Sepharose resin is extremely sensitive to the conductivity and elutes at 3.9 mmho at 8°C. AfJinity chromatography. Jacobson and Rosenbusch ( 10) first suggested the use of a Tu resin for the purification of Ts and demonstrated its feasibility for the preparation of very small quantities of Ts. We have simplified their initial procedure as described under

Methods and have adapted the procedure to the routine purification of considerably larger quantities of Ts. The ammonium sulfate pellet of Ts was resuspended in Buffer Ts (50 mM Tris-HCl, pH 7.5, 0.15 M KCl, 10 mM 2-mercaptoethanol, and 0.1 mM PMSF). Approximately 1.52 X lo* units of crude Ts were loaded at a flow rate of 180 ml/h onto 10 ml of Tu resin packed into a column and equilibrated in Buffer TuTs (50 tIIM Tris-HCl, pH 7.5, 5 ItIM EDTA, 10 tItM 2-mercaptoethanol, and 0.1 tIIM PMSF). The Tu resin was washed extensively with 300 ml of Buffer TuTs until the absorbance at 280 nm reached background levels for at least 50 ml. When less extensive washing was utilized, a few proteins appeared to be retarded by the Tu resin and contaminated the final product. The Ts was eluted from the Tu resin with Buffer Tu containing 10 mM GDP. The affinity step was repeated until all the Ts activity had been isolated from the initial sample. Approximately 8.09 X IO’ units of Ts with an overall specific activity of 525,000 units/mg were recovered. Each application of the Tu-

354

YODER, LENNANE,

affinity procedure was accomplished in 3 h. The final protein product migrated as a single band on SDS-polyacrylamide gels as shown in Fig. 1. DISCUSSION

We have redesigned the purification protocol for Ts in order to maximize the yield of the purified enzyme. Using DEAE-Sepharose and Tu-affinity chromatographies, we routinely obtain a yield of 52% or 150 mg of Ts/kg of E. coli (B) cells. The specific activity of the purified Ts generally exceeds 500,000 units/mg. In previously published protocols, the purification procedure involved three or four chromatographic steps and the overall yield of Ts, with a specific activity of 500,000 units/mg or greater, was much lower: 6% or 15 mg/kg of E. coli (B) cells (3) and 13% or 12.5 mg/kg of B. stearothermophilus cells (9). Although there is one report of a Ts purification scheme based on two chromatographic steps in which the yield is higher, 23-45%, the specific activity of the purified enzyme is lower, 350,000 units/mg (7). We conclude that the scheme presented herein has the advantage of a greater yield of enzymatically homogeneous Ts with a shorter protocol. The protocol may also be applicable for purifying Ts or Ts-like proteins from other organisms. To determine the feasibility of the Tuaffinity procedure for routine bulk purification of Ts, we measured the activity of the Tu-affinity resin and determined that lo15% of the immobilized protein is available for complexation to Ts. In practical terms, we obtain a yield of 2 1 mg of Ts/ 10 ml of resin in each 3-h purification. Because the Sepharose bead of the affinity resin tolerates a high flow rate, scaling the Tu-affinity method is straightforward, limited only by the availability of Tu-GDP. We have also found that the resin can be recycled innumerable times and remains active toward Ts complexation for months at 4’C. Apparently,

AND JURNAK

the immobilization of Tu-GDP on the resin stabilizes the elongation factor, whereas the unbound protein looses activity in a few weeks under the same conditions. In summary, we have redesigned the purification scheme for Ts to achieve greater efficiency and a significantly improved yield. ACKNOWLEDGMENTS We thank Estaban Masuda, Lillian McCollum, and Teri Tarabino for technical assistanceduring the duration of the research.

REFERENCES 1. Miller, D. L., and Weissbach, H. (1977) in Molecular Mechanisms of Protein Biosynthesis (Pestka, S., and Weissbach, H., eds.), pp. 323-327, Academic Press, New York. 2. Arai, K., Kawakita, M., and Kaziro, Y. (1974) J. Biochem. 76, 293-306. 3. Hackmann, J., Miller, D. L., and Weissbach, H. (1971) Arch. Biochem. Biophys. 147,457-466. 4. Arai, K., Kawakita, M., Kaziro, Y., Kondo, T., and Ui, N. (1973) J. Biochem. 73, 1095-I 105. 5. Miller, D. L., Hackmann, J., and Weissbach, H. (197 I) Arch. B&hem. Biophys. 144, 115- I2 I. 6. Arai, K., Kawakita, M., Nakamura, S., Ishikawa, I., and Kaziro, Y. (1974) J. Biochem. 76, 523-534. 7. Arai, K., Kawakita, M., and Kaziro, Y. (1972) J. Biol. Chem. 247(21), 7029-7037. 8. Leberman, R., Schulz, G. E., and Suck, D. (198 I) FEBS Lett. 124(2), 279-28 1. 9. Wittinghofer, A., and Leberman, R. (1976) Eur. J. B&hem. 62, 373-382. 10. Jacobson, G. R., and Roynbusch, J. P. (1977) FEBS Lett. 71(l), 8-10. Il. Miller, D. L., and Weissbach, H. (1974) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 30, Part F, pp. 2 19-232, Academic Press, New York. 12. Miller, D. L., and Weissbach, H. (1970) B&hem. Biophys. Res. Commun. 38(6), 1016-1022. 13. Bradford, M. M. (1976) Anal. Biochem. 72, 248254.

14. Sedmak, J. J., and Grossberg, S. E. (1977) Anal. Biochem. 79, 544-552. 15. Laemmli, U. K. (1970) Nature (London) 227,680685.

16. Weber, K., and Osbom, M. (1969) J. Biol. Chem. 244,4406.

17. Lottie, A., Ribeiro, S., Reid, B. R., and Jumak, F. (1984) J. Biol. Chem. 259, 5010-5016.