The Korean Journal of Microbiology, Vol. 4, No. 4, December 006, p. 94-98 Copyright 006, The Microbiological Society of Korea Streptomyces phospholipase D w ey j m v Á½ Á Á k * w w w w Streptomyces somaliensisƒ w phospholipase D (PLD) w» w rp w w ey j m v e w. d v Streptomyces PLD X- w, S. somaliensis PLD 1 l w p š t ew 5 rp epitope w, w w ey j m v e w. e m g w y z SDS-PAGE Western blot, e w PLD 35 kda sww PLD z ey e w w p. ù PLD z PLD p y û. Key words ý immunoaffinity, phospholipase D, purification, Streptomyces Phospholipase D (PLD, EC 3.1.4.4) phosphodiester w ñ phosphatidic acid g yw z. w, PLD ƒ w p g phosphatidic acid w j y (phosphatidyl-x + Y-OH phosphatidyl-y + X-OH) ƒ š, mw phosphatidylcholine (PC) l phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine». ù PLD» s y», s ûš, w w. PLDƒ» w 1) z wš, ) t x» w hexane, ethylacetate, diethylether, butylacetate w z, 3) y z w. wr, S. antibioticus (9), S. cinnamoneus (13), S. acidmyceticus (5), S. halstedii (6), S. septatus (7), S. chromofuscus (17) ƒ y e Streptomyces (1) PLD s w š. ù w s PLD ³» PLD ³ w. w Streptomyces PLD rp w w w w rp w w, ELISA m w Streptomyces ³ PLD ³ w (16). *To whom correspondence should be addressed. Tel: 063-70-3439, Fax: 063-70-336 E-mail: tbuhm@chonbuk.ac.kr ƒ, S. somaliensis ³ PLDƒ butylacetate w 48 hr z y z ƒ š(90-9 %), Bennett's medium 7 hr w, z y 1- U/ml q (1). z ƒ w w w š z w. x ¾ Streptomyces PLD Sephadex, hydroxyapatite, phenyl Sepharose e w j m v (7, 13) š» š PLD û. y z p S. somaliensis PLD yw» w (1) š l epitope ww kw rp w wš, w w ey k w j m v w. Streptomycesƒ w PLD k w» w ƒ. PLD ³ m w S. somaliensis Bennett (10) 30 o C, 7 hr (80 rpm) w. ey j m v rp x ¾ w š Streptomyces sp. PMF PLD X- ray (1) w rp šw» w CLUSTALW program (15), PMF S. somaliensis PLD 94
Vol. 4, No. 4 Phospholipase D w ey j m v 95 1 w. ƒƒ, 506 505 PLD e 81.6%, w 7.71%, w 6.9%, 3.75% 1», Streptomyces sp. PMF PLD X-ray l S. somaliensis PLD 3 w w š q w. Cn3D viewer mw Streptomyces sp. PMF PLD X-ray t ƒ turn loop, 6-7 w e p ƒ w e w k w., 1 ü α-helix β-conformation x w w, w loop e»ƒ w. PMF PLD 1 w S. somaliensis PLD e p ùký w epitope w. ù S. somaliensis PLD 80% e ùkü w rp w» rp ƒ epitope w w v ƒ. S. somaliensis PLD 1 d v PHD (14), Jpred (3), ü w e dw antigenic v (11), t ew y d (4) w w x š, t ew loop w rp e l w epitope w. w l 5 epitope w, w w (Table 1). rp w conjugation w rp N- conjugation w cysteine residue ƒw. w z ƒ rp HPLC wš LC- MASS y w. Conjugation y yw keyhole limpet hemocyanin (KLH) /DMSO rp š 50 mm EDTAƒ w Na-phosphate buffer (ph 7.5) ƒ z hr g. Ellman SDS-PAGE conjugation y w z Sephadex G-5 column conjugate w. rp w KLH conjugate rp New Zealand white rabbit wš 3 e, x w x w. phosphate-buffered saline (PBS) sxy k Protein A column (Econo-Pac Protein cartridge, Bio-Rad, USA) PBS 10 k x š z, 0.1 M glycine-hcl (ph.5) immunoglobin G (IgG) w. ƒ IgG SDS-PAGE band w y w š, ELISA w w rp w titer ƒƒ 1 10-5. ey j m v 1 ml y y N-hydrosuccinimide (NHS) Sepharose (10 µmol NHS/ml) w w IgG ƒƒ 1 hr g. NHS Sepharose w w z (100 w w w / k w w ) 45-6% š ml 3.4-5.4 mg IgGƒ ww (Table ). ey j m v w PLD S. somaliensis 70% ammonium sulfate e w z wš e. n k z w e (0.75.5 cm) 30 sxy g. e w 0.1 M glycine-hcl (ph.5) PLD y z š SDS- PAGE y w. PLD Western blot Western blot 1 w -3 1 10 z w w z horse radish peroxidase w. z y d PLD ƒ w y choline oxidase-peroxidase (8) x, z PLD y y w EDTA ƒw 100 C o 5 ƒ w. ƒ w y 1 unit» 1µmol yg w z w. Bradford () BSA t w w. š ey e PLD w PLD w ey e w» w Table 1. Amino acid sequence of synthetic peptides a of Streptomyces somaliensis phospholipase D expected as suitable epitopes from protein structural prediction programs and X-ray crystal information Epitope Amino acid sequence of synthetic peptide Predicted structure Residue number PLD1 CPDKTNADRDYDTVN-NH loop 99-11 PLD CNSWKGDYLDTSHPV-NH β β loop 183-196 PLD3 CRSSPGATWADGKPY-NH β β loop 43-436 PLD4 CLALVTGDQQKA-NH loop 399-409 PLD5 CDPANRGAVGSGGYSQIKS-NH β α loop 370-387 a For conjugation with keyhole limpet hemocyanin, a cysteine residue was added to the N-terminal of epitopes.
96 In Sun Park et al. Kor. J. Microbiol Affi-Gel Hz Hydrizide (Bio-Rad, Hercules, USA), vinylsulfoneactivated agarose (Sigma, St. Louis, USA), EAH-Sepharose (Amersham, Uppsala, Sweden), ECH-Sepharose (Amersham), NHS- Sepharose (Amersham), epoxy-activated Sepharose (Amersham) antipld3 IgG w g e wš PLD y w. w w NHS-Sepharoseƒ ƒ w. NHS-Sepharose ƒ rp w w g ey e w š, antipld4 IgG-Sepharose PLD w w. 0.6-3. mg w w 1 ml column ƒƒ w sx yw wš 0.1 M glycine-hcl (ph.5) PLD w. PLDw e w PLD 60 µg ¾ ƒw (Fig. 1). 1 ml e w 3.4 mg IgG w 1.8% (w/w)ƒ PLD w -w w w (Table ). ey j m v w PLD k k z ƒ ey e w Table 3 š, antipld3 IgG- Sepharose PLD j m Fig.. e w Fig. w j m. ƒ e PLD z 7-11% š 0.8-1.6 Fig. 1. Binding capacity of Streptomyces somaliensis phospholipase D on the immunoaffinity gel as a function of concentration of protein loaded on the antipld4 IgG-Sepharose. Table. Coupling efficiency of antipeptide antibodies on NHSactivated Sepharose Immunoaffinity matrix Bound IgG mg/ml gel Coupling efficiency % antipld1 IgG-Sepharose 4.6 56 antipld IgG-Sepharose 4.7 5 antipld3 IgG-Sepharose 4.0 6 antipld4 IgG-Sepharose 3.4 45 antipld5 IgG-Sepharose 5.4 58 û ùkû. û ƒ ey e z y w» w z z SDS-PAGE Western blot w. SDS- PAGE antipld1 IgG-Sepharose antipld3 IgG-Sepharose z y z S. somaliensis PLD (MW 53,635) ew 55 kda e band ùkþ, Western blot band PLD ùkþ (Fig. 3). wr, AntiPLD, 4, 5 e SDS-PAGE 55 kda PLD band 35 kda bandƒ ùk û» antipld1 3 e w e» k. 35 kda j» S. somaliensis ƒ Western blot ùkù PLD w ƒ q. l z PLD y û ƒ ey e q. w e m g PLD band j m v» w. ù antipld1 3 w e m z PLDƒ band, Streptomyces PLD 1,, 3 w x ey e w š q. wš û z y ƒ z w. x S. somaliensis PLD k 4 o C» y j m v ww y w., Q-, SP-, CM-. DEAE-Sepharose y e, octyl-, Table 3. Purification summary of phospholipase D from Streptomyces somaliensis a Purification Volume (ml) Total activity (units) Protein (µg) Specific activity (units/mg) Recovery (%) Purification (fold) Culture supernatant 6.70 6.70 308. 1.7 100.0 1.0 Ammonium sulfate 5.40 1.91 113.4 16.9 8.7 0.8 antipld1 Sepharose 0.55 0.48 14.3 33.6 7. 1.6 antipld Sepharose 0.60 0.53 4.0.1 8.0 1.0 antipld3 Sepharose 0.50 0.75 6.5 8.3 11. 1.3 antipld4 Sepharose 0.65 0.65.7 8.6 9.7 1.3 antipld5 Sepharose 0.9 0.58 3. 17.4 8.7 0.8 a Dialyzed solution (5.4 ml) of the ammonium sulfate precipitate was loaded on each antipld IgG-Sepharose column.
Vol. 4, No. 4 Phospholipase D w ey j m v 97 Fig.. A typical chromatogram of Streptomyces somaliensis phospholipase D on antipld3 IgG-Sepharose. Arrow represent elution start of 0. M glycine-hcl buffer, ph.5 and bar represent purified phospholipase D. Fig. 4. Performance of the antipld3 IgG-Sepharose column during chromatographic runs. After each consecutive run, the 1 ml column was washed with about 30 ml of distilled water at a flow rate of 1 ml for the next run. Fig. 3. (A) SDS-PAGE of Streptomyces somaliensis phospholipase D purified by immunoaffinity chromatography on antipeptide IgG- Sepharose and (B) its Western blot diagram. (A) Molecular weights of protein markers in lane 1 are indicated to the left: lane, ammonium sulfate precipitate of culture supernatant; lanes 3 to 7, phospholipase D after chromatography on antipld1 IgG-Sepharose to antipld5 IgG-Sepharose, respectively. (B) lane 1, ammonium sulfate precipitate of culture supernatant; lanes to 6, phospholipase D after chromatography on antipld1 IgG-Sepharose to antipld5 IgG-Sepharose, respectively. phenyl-, butyl-sepharose w e, HPLC e, hydroxyapatite e, prep gel» ph, protease inhibitor ƒw w PLDƒ x ww, z PLD y y. w x w e w ùkû, w e z PLD z û y wì kw d. z, Glycine-HCl w (ph.5) ¼ š w PLD y z 1 M Tris-HCl buffer (ph 7.5) yw» y ph w š ƒw. w, w proteases š w protease inhibitor cocktail (Roche, Mannheim, Germany), e ƒw» PLD proteases w w ƒ. PLD j» w Tween, Triton X, octylglucoside, Nonidet P40 y CHAPS, SDS y z w w z ù y w. w, sucrose, maltose, glycerol ƒ PLD y z w. w l Streptomyces PLD j w w., z kw wì PLD y w x l ƒ PLD ƒ w š PLD ö ƒ. x, PLD y z w ƒ z wš. e PLD w e z w w. w e IgGƒ ph.5 w PLD w w» IgG ƒ š w w v ƒ. ey e PLD w y» w antipld3 IgG-Sepharose 5 ww Fig. 4 z e š. z ±10% y yƒ e w ƒ e l PLD z y. w w w w ( y:d008) w w.
98 In Sun Park et al. Kor. J. Microbiol š x 1.,, k. 004. Streptomyces somaliensisƒ w s phospholipase D transphosphatidylationy p. wz 40, 11-16.. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7, 48-54. 3. Cuff, J.A., M.E. Clamp, A.S. Siddiqui, M. Finlay, and G.J. Barton. 1998. Jpred: A Consensus Secondary Structure Prediction Server. Bioinformatics 14, 89-893. 4. Emini, E.A., J.V. Hughes, D.S. Perlow, and J. Boger. 1985. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J. Virol. 55, 836-839. 5. Hasegawa, M. and N. Ota. 199. Production of phospholipase D- K. Japan Patent. JP199088981-A. 6. Hatanaka, T., M. Kubota-Akizawa, T. Negish, and T. Hagishita. 00. Study on thermostability of phospholipase D from Streptomyces sp. Biochim. Biophys. Acta 1598, 156-164. 7. Hatanaka, T., T. Negish, M. Kubota-Akizawa, and T. Hagishita. 00. Purification, characterization, cloning and sequencing of phospholipase D from Streptomyces septatus TH-. Enzyme Microb. Thechnol. 31, 33-41. 8. Imamura, S. and Y. Horiuti. 1978. Enzymatic determination of phospholipase D activity with choline oxidase. J. Biochem. 83, 677-680. 9. Iwasaki, Y., H. Nakano, and T. Yamane. 1994. Phospholipase D from Streptomyces antibioticus: cloning, sequencing, expression, and relationship to other phospholipases. Appl. Microbiol. Biotechnol. 4, 90-99. 10. Jone, K.L. 1949. Fresh isolation of Actinomycetes in which the presence of sporogenous aerial mycelia is a fluctuating characteristic. J. Bacteriol. 57, 141-145. 11. Kolaskar, A.S. and P.C. Tongaonkar. 1990. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett. 76, 17-174. 1. Leiros, I., F. Secundo, C. Zambonelli, S. Servi, and E. Hough. 000. The first crystal structure of a phospholipase D. Structure 8, 655-667. 13. Ogino, C., Y. Negi, T. Matsumiya, K. Nakaoka, A. Kondo, S. Kuroda, S. Tokuyama, S. kikkawa, T. Yamane, and H. Fukuda. 1999. Purification, characterization, and sequence determination of phospholipase D secreted by Streptoverticillium cinnamoneum. J. Biochem. 15, 63-69. 14. Rost, B. 1996. PHD: predicting one-dimensional protein structure by profile based neural networks. Method Enzymol. 66, 55-539. 15. Thompson J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res., 4673-4680. 16. Uhm, T.B. and S.H. Lee. 003. A peptide antibody for rapid screening of Streptomyces species producing phospholipase D. Biotechnol. Lett. 5, 883-886. 17. Yang, H. and M.F. Roberts. 00. Cloning, overexpression, and characterization of a bacterial Ca + -dependent phospholipase D. Protein Sci. 11, 958-968. (Received October 7, 006/Accepted November 7, 006) ABSTRACT : Purification of Streptomyces Phospholipase D by Immunoaffinity Chromatography using Peptide Antibodies In Sun Park, Young Ah Kim, SuJin Jeong, and Tai-Boong Uhm* (Division of Biological Sciences, Chonbuk National University, Jeonju 561-756, Korea) An immunoaffinity chromatography for the specific binding of Streptomyces somaliensis phospholipase D (PLD) that is considered as an industrially potential enzyme was developed. By using the protein structure prediction programs and the X-ray crystal structure of a Streptomyces PLD, 5 different epitopes with high antigenicity that are predicted to locate on the surface of the S. somaliensis PLD were selected and then synthesized for the preparation of antipeptide antibodies. Each purified rabbit IgG was coupled with NHS-activated Sepharose to prepare the immunoaffinity resins. After one-step purification of the culture concentrate on the antipeptide IgG-coupled Sepharose column, SDS-PAGE and the Western blot analysis of the purified samples showed that purification of PLD on the affinity columns was satisfactory, indicating that the peptide design using the structural information of Streptomyces PLDs was rational. However, the purified PLD in the solution aggregated rapidly, which resulted in poor specific activity and low purification yield.