353-11(07-31).fm

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1 Kr. J. Micrbil. Bitechnl. Vl. 35, N. 3, (2007) ƒ w ³l Interfern α-1 œ e w ¼ÁÁ½Á 1 Á * ww œw» l, 1 vl Effect f Oxygen Supply n the Prductin f Interfern α-1 by Recmbinant Escherichia cli in Fedbatch Fermentatin. Yi, Jng-Ghil, Sek-Yung Mn, Yung-Jun Kim, Chul-S Shin 1, and Yn-M K*. Department f Bilgical Engineering and Center fr Advanced Biseparatin Technlgy, Inha University, Inchen , Krea, 1 Advanced Prtein Technlgies Crpratin, Yengtng-Dng. Suwn - In rder t achieve high-level expressin f interfern-α1 (IFN-α1) during fed-batch fermentatin f recmbinant E. cli, effects f xygen supply and inductin temperature n the expressin f recmbinant prteins were evaluated. Supplementatin f xygen and its transfer int cells is ne f the mst imprtant parameters invlved in the design and peratin f mixing-sparging equipment fr bireactrs. Generally, higher xygen supply stimulates cell grwth f aerbic micrrganism and cnsequently the amunt f prducts is increased. In this study, the ptimum aeratin strategy fr the higher prductin f IFN-α1 during fed-batch fermentatin f recmbinant E. cli was surveyed. The grwth f the cells was als mnitred with fur different cncentratins f disslved xygen (DO; limiting, 20%, 35%, 50%) cnditins. The DO was cntrlled by varying aeratin rates f air and pure xygen. Oxygen uptake rate (OUR) and specific xygen uptake rate (SOUR) were evaluated and cmpared fr the enhanced grwth and inductin f the cells and IFN-α1, respectively. We cnfirmed that increased DO by additinal xygen supply, up t 35%, can imprve the prductin f IFN-α1 during the fermentatin. Key wrds: Fed-batch fermentatin, xygen uptake rate, specific xygen uptake rates, interfern-α1 ƒ œ š s. ƒ z»» sw w w w, zƒ óú ¾ œ w. ³ w High Cell Density Culture(HCDC)» ply(3-hydrxy butyrate) zš ñþ[10]. w ³ w w w ƒ w š». š» yk š z z w ƒw zœ wš w s w ƒš [2, 4, 5, 7, 11]. *Crrespnding authr Tel: , Fax: ymk@inha.ac.kr ph w y v w œ w ³l interfern-α1 w z œ. w k š ph ƒ yw» š k» š sl ammnium in ƒ w ph ƒw» w» ƒ œ [6]. IFN-α1 interfern k I B-cell interfern, leukcyte interfern, lymphblast interfern, and ph2- stable interfern ù w e [1]. š s w w w» š»ƒ w w x, w ƒ w œ š [9, 13]., x ƒ mw w ³l IFN-α1 x» w, 4ƒ (DO; limiting, 20%, 35%, 50%) IFN-α1 x wifn-α1 x s Inductin w. w œ» œ. (OUR) p

2 (SOUR) ƒƒ IFN-α1 s w s Inductin sƒ. ³ ³ vl E.cli Rsetta BL21a v pet21a 6.1kb, fusin partner K6ub prtein lysine 6 ƒ N-terminal š ubiquitin 243 bp x ƒk sluble frm x ƒ g, w w. k6ub/ifn-α1 NdeI BamHI restrictin enzyme clning upstream tac prmter w ƒ. z Yeast extract 20 g/l, Glucse 20 g/l, KH 2 PO 4 5 g/l, K 2 HPO 4 3 g/l, Mg 2 SO g/l, ƒ»œ Yeast extract 211 g/l, glucse 274 g/l, (NH 4 ) 2 SO g/l, Mg 2 SO g/ L phƒ l ƒ œ. z x z 5.0 L (v 2L) z(gl, Krea)»,»,, ph s wš. ( rpm) m» œ»(1 vvm) w ü. ph 27% w 6.8 w. š z»s w g s PPG2000(sigma, USA) w. ƒ ³ w» w feeding ph-stat, k» š sl ammnium in ƒ w ph ƒw» w ph signal feeding pumpƒ. EFFECT OF OXYGEN SUPPLY ON THE PRODUCTION OF INTERFERON α albumin w 595 nm Ÿ dwš t w z w. k6ub/interfern-α1 d ƒ Interfern-α1 d w sampling z 10,000 rpm 5 z -20 C wƒ zƒ óùš w sample PBS z lysis buffer(30 mm NaPi mm NaCl + 5 mm EDTA + 0.5% tritn X-100) resuspensin z q sq» w s qz SDS PAGE z, Staining š standard marker w densitmetric scanning(fujifilm, FLA-5000) w Interfern-α1 dw. š z r y» ³ z k ü ƒ w w. z z m» w œ ³x w. j w wƒ e w ƒ [3]. w yw» w»x w. z» w r åƒ 0.5 vvm 1 vvm œ»œ w š, 200 rpm-1,000 rpm ƒ ƒ r 3 cm ³ d ³(dry cell weight, DCW) ³yw» ³ y e 10,000 rpm 5 w z yw w š w e z 105 C w w dw. z 10,000 rpm 5 w w Dinitrsalicyclic acid(dns) w y d w dw. Sigma (USA) Bradfrd assay kit w w. t bvine serum Fig. 1. Determinatin f KLa under different perating cnditin with water in 5 L-fermenter --; 0.5 vvm (impeller clearance 5 cm), --; 1GvvmG(impeller clearance 5Gcm), --; 1Gvvm (impeller clearance 4G cm), - -; 1G vvmg (impeller clearance 3 cm), --; 1vvm(impeller clearance 5cm)

3 228 LEE et al. œ»œ 1 vvm, 600 rpm 2.67(min ) ƒ -1 (Fig. 1). 600 rpm yw x sparger ù œ» Á r m ƒ». x m ƒ Agitatin speed 600 rpm, Aeratin rate 1 vvm, r 3 cm w z w ƒ œ œ» yww. ƒ k6ub/ifn-α1 e w Fig. 2 ƒ ƒ œ w ƒ 20% y k6ub/ifnα1 e w, Cell density, ttal prtein, k6ub/ifn-α1 37 C ƒƒ 55 g/l, mg/ml, 4.85mg/ml 30 C ƒƒ 53 g/l, mg/ml, 5.61 mg/mlš, 30 C s 25 C Inductin Cell density, ttal prtein, k6ub/ifn-α1 ƒƒ 53 g/ L, mg/ml, 6.43 mg/m Cell density 37 C ƒ target prtein k6ub/ifn-α1 30 C s 25 C Inductin 6.43 mg/ml ttal prtein 37%ƒ x. w k6ub/ifn-α1 w w wƒ û w. p sƒ ƒ œ w». ƒ w ƒ w w ƒ w [8]. Azterbacter vinelandii, 0.05 mg/l w ƒ w w» s û wš 50% w s ƒ 0.05 mg/l w. ƒ w s w [6]. Fig. 3 w» seed wš m» 2 L/min 200 rpm ww ƒ 8 z» š w phƒ w š ph» w feeding ƒ ƒ» wš rpm, m» 1 vvm 20% w» l (0.3 L/min)ƒ œ»(1 L/min) yw ƒ z 20% ƒ ƒ 23 Fig. 2. Cmparisn f k6ub/ifn-α1 prductin, dry cell weight and glucse cncentratin amng the 37 C, 30 C and 30 CG 25 C culture temperature at disslved xygen cncentratin f 20% in fed-batch fermentatin. ƒ (0.4 L/min)ƒ œ» (1 L/min) yw ƒ 20%. Fig. 4 m» w w ƒ z k6ub/ IFN-α1 e w ³wš 0%, 20%, 35%, 50% w x wwš, z 33 ttal prtein, k6ub/ifn-α1 x 0% ƒƒ 10.3 mg/ml, 3.0 mg/ml, 20% ƒƒ 16.2 mg/ml, 6.3 mg/mlš, 35% ƒƒ 21.8 mg/ml, 9.5 mg/ml, 50%

4 EFFECT OF OXYGEN SUPPLY ON THE PRODUCTION OF INTERFERON α Fig. 3. Time curse prfiles f dry cell weight by cntrlling DO, agitatin speed, aeratin rate in fed-batch fermentatin. DO was maintained at abut 20%. Fig. 4. Cmparisn f k6ub/ifn-α1 prductin, dry cell weight and glucse cncentratin amng the 0%, 20%, 35%, 50% disslved xygen cncentratin in fed-batch fermentatin. 각각 22.1 mg/ml, 9.14 mg/ml target prtein인 k6ub/ifnα1 발현량이 가장 높은 것을 알 수 있었다. 용존산소량은 산 소소비 속도를 측정함으로써 정확한 임계점을 찾을 수 있었 는데 추가적인 산소공급이 없는 상태에서는 산소전달 속도 보다 산소소비 속도가 더 커서 세포성장 및 단백질의 발현 에도 저해를 가져오는 것을 알 수 있었다. 그러나 오히려

5 230 LEE et al. Table 1. Fed-batch cultures fr prductin f k6ub/ifn-α1 at different culture cnditin. Culture & Inductin cnditin Final dry cell weight (g/l) Ttal Prtem (mg/ml) Vlumetric yields (g/l) Specific yields (mg/g CDW) IFN-α cntent per ttal prtein (%) Maximum OUR (mm O 2 /h) Limited-xygen Saturated-Oxygen 20% Saturated-Oxygen 35% Saturated-Oxygen 50% Maximum KLa with water: (mmlo 2 /1 h) target prtein x w 50% 35% ƒ x (Table 1). w zœ ƒ w ƒ. š z, z sparging hle l r œ»œ 1 vvm, ƒ 600 rpm (K L a) 2.67(min -1 ) ƒ. 20% g k6ub/ifn-α1 30 C s wš 25 C IPTG Inductin w x 6.43mg/ml ttal prtein 37% ƒ x. k6ub/ifn-α1 x ƒ 35% ƒ ùkþ. dw yw 35% w ƒ w œ yw.»l w,. w ³ œw APtech. REFERENCES 1. Babu, R. and S. Swaminathan prductin f interfern-a in high cell density culture f recmbinant Escherichia cli and its single step purificatin frm reflded inclusin bdy prteins, Micrbil Bitechnl, 53: Chen, C. G Autmated fed-batch fermentatin with feed-back cntrls based n disslved xygen (DO) and ph fr prductin f DNA vaccines, Jurnal f Industrial Micrbilgy & Bitechnlgy, 18: Emili Gmez, Victria E. Sants, and Almudena Alcn Oxygen-Uptake and Mass-Transfer Rates n the Grwth f Pseudmnas putida CECT5279: Influence n Bidesulfurizatin (BDS) Capability, Energy & Fuels, 20: Jeng, K. J. and S. Y. Lee High-level prductin f human leptin by fed-batch cultivatin f recmbinant Escherichia cli and its puri.catin. Appl. Envirn. Micrbil. 65: Kim, B. S. and L. Seung Chu High cell density fedbatch cultivatin f Escherichia cli using expnential feeding cmbined with ph-stat, Biprcess. Bisyst. Eng., 26: Kim, S. S Effects f Specific Grwth rate n the High Cell Density Cultivatin f Lipase-prducing Recmbinant Escherichia cli, M. S. Thesis, Dept. f Micrbilgy, Inha University, Inchen. 7. Lee, S. Y High-cell-density culture f Escherichia cli, TIBTECH., 14: Mtarjemi, M. and G. J. Jamesn Mass transfer frm very small bubbles-the ptimum bubble size fr aeratin, Chem. Eng. Sci. 33: Rabhi-Essafi, I. and A. Sadk A strategy fr highlevel expressin f sluble and functinal human interfern a as a GST-fusin prtein in E.cli, Prtein Engineering, Design & Selectin, pp Risenberg, D., B. Schulz, W.A. Knrre, and H.-D. Phl High-cell-density cultivatin f Escherichia cli at cntrlled specific grwth rate, J. Bitechnl., 20: Shin, C. -S., H, Min Sen Enhanced Prductin f Human Mini-Prinsulin in Fed-Batch Cultures at High Cell Density f Escherichia cli BL21(DE3)[pET-3aT2M2], Bitechnl. Prg, 13: S. Shams Yazdani, and A. Rushdi Shakri A high cell density fermentatin strategy t prduce recmbinant malarial antigen in E. cli, Bitechnlgy, Letters 26: Yee, L. and H. W. Banch Recmbinant trypsin prductin in high cell density fed-batch cultures in Escherichia cli, Bitechnl, and Bieng., 41: (Received July 3, 2007/Accepted Sep. 6, 2007)