15 6 1999 12 V o l. 15,N o. 6 Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy D ec., 1999 g P. p astoris 3 (, 100005), : 5 IGF2g ; ; Α P. p astoris, IGF2g ;, 60 m ggl P. p astoris rh IGF2g,,N g, P. p astoris,, H igh- level Express ion, Secretion and Character iza tion of Human In sulin- like Growth Factorg in M ethylotroph ic Y ea st P. p astoris L I J ingjun, HUAN G B ingren (Institu te of B asic M ed icine S ciences, CA M S & PUM C, T he N ational L aboratory of M ed ical M olecu lar B iology, B eij ing 100005) Abstract To increase the exp ression level of IGF2g, an exp ression vecto r con tain ing a tandem of 5 IGF2g exp ression cassettes w ith alcoho l ox idase (AOX1) p rom o ter w as con structed. T he gene encoding the m atu re fo rm of IGF2g w as fu sed in fram e to the S. cerev isiae Α2m ating facto r leader sequence, that is, the secretion of IGF2g depended on the secretion signal sequence of Α2facto ṙ A fter tran sfo rm ation of P. p astoris p ro teinase2deficien t strain w ith linearized exp ression vecto r, the tran sfo rm an ts exp ressed and secreted IGF2g to the grow th m edium w ere screened. T hen the cu ltu re of exp ression strain w as op tim ized and scaled up, the yield in the supernatan t of h igh b iom ass ferm en tation cu ltu re w as at the level of 60 m ggl. Fu rther analyses of rh IGF2g p roduced in P. p astoris show that it has co rrect M r,n 2term inal am ino acid sequence and h igh b ioactivity. Key words In su lin2like grow th facto r g ( IGF2g ), P. p astoris, H igh2level exp ression, Secretion g ( IGF2g ) Bell 1984 h IGF2g Cohn (Cohn fraction) cdna, rh IGF2g E. coli [ 1 ], 67, [ 5, 6 ], E. coli [ 7 62%, IGF2g ], H ansenu la p oly 2 [ 2 ] IGF2g IGF2g, N IH 3T 3 [ 9 ], [ 3 ], IGF2g IGF2g, [ 4 ], h IGF2g 3 T el: (010) 65296406, Fax: (010) 65240529 E2m ail: huangbr@cdm. im icam s. ac. cn, [ 8 m orp ha ] 700 ggl [ 7 ],, 1970 1,, : 1999201222, : 1999205212
894 15 h, 2% P. p astoris 116 rh IGF-g M illi2 h IGF2g, po re 0. 45 m HA, (N H 4) 2SO 4, P. p astoris rh IGF2g Phenyl2Sepharo se Fast F low, 60 m ggl (W aters 20g20) ; SP2, rh IGF2g Sepharo se H igh, IGF2g (XK16g15), ; IGF2g 1 111 IGF2g cdna, 5 Α2 (ΑF), SD S2PA GE [ 11 ], 3 c (cyct ) psk43 11713 N SB 2IGF2g, pao 815 T ricine2sd S2PA GE PVD F [ 12 ], K562 A Beckm an L F 3000 AB I491 P. p astoris SM D 1168 Invitrogen, h is4p ep 4 2 112 P rom ega T ricine, CA PS Sigm a h R &D,, HR P V ecto r P. p astoris pao 815 IGF2g Boeh ringer IGF2g S. cerev isiae Α PVD F Gelm an 0145 m HA M illipo re Α IGF 2g cdna Pharm acia 113 MM BM G BMM Invitrogen Α2F IGF2g cdna, E co R PTM 1 g B am H g pu C18, pu C182 [10 ] 114 Α - IGFg g, pu C182ΑF2IGF2g B am H g, : 5 2G GAA T TC A CCA T GA GA T T TCC K lenow E co R g T TCAA T T T T T 23 ( E cor I,, PCR 5 Α Kozak ) ; : 5 2 A T G Kozak E co R g T T TCCCA GTCA CGA CGT T 23 115 P. p astoris E co R g, pu C19, pu C192Α 2IGF2g : 20 m l BM G 24 h, 500 m l BM G 18 h515 L BM G, E co R g, pao 815 E co R g ; H, N H 4OH ph 5, 500 m l, 30, 40%,, B g l g 1 000 rgm in, 24 h ( B am H g 5 IGF2g ) 750 m l 50% 12 m lgl PTM 1, pao 81525 IGF2g, ph 5 315, F ig. 1, F ig. 2 pao 8152 12 m lgl PTM 1, 1% H it rap H igh Sub Perfo rm ance 117 P. p astoris rh IGF-g 11711 rh IGF2g [7 ] 11712 W estern T ricine2 211 pao815-5 IGF-g ( 85 ) pao 815 E cor g, E cor g B am H g psk43sb 2IGF2g ΑF2IGF2g IGF2g 3 E co R, 3 pu C18, Α, pao 8152IGF2g 5
6 : g P. p astoris 895 F ig. 1 Construction of the exp ression vecto r 212 rh IGF-g H is + M u t s h is4 ( ) IGF2g AOX1 ( ) [ 7, 8 ]. P. p astoris SM D 1168, IGF2g,, rh IGF2g S a l g B g l g, P. 715 kd (F ig. 3) p astoris h is4 AOX1 P. p astoris rh IGF2, S a l g g, DNA, H is + M u t +, 13%B g l g M u t + M u t s 3 4 d, 612 kd M u t + M u t s H is +, 811 kd, h IGF2g 5 AOX1 h IGF2g 3 PCR
896 15, 570 bp ( ), h IGF2g F ig. 3 Screening fo r the po sitive transfo rm ants of h igh2 level yield 1:M ut + SM D 1168gpAO 815; 2-4:M ut + po sitive transfo rm ants; 5:M ut s SM D 1168gpAO 815; 6-7:M ut s po sitive transfo rm ants; 8: L ow 2range p ro tein mo lecular w eigh t m arker F ig. 2 Restriction endonuclease analyses of pao 81525 IGF2 g digested w ith B g l g and B am H g (A ) and S al g (B) (A )D igested w ith B g l g and B am H g ; 1: pao 81525 IGF2g gb g l g + B am H g (2. 4 kb, 4. 0 kb, 9. 0 kb) ; 2: pao 81523 IGF2g gb g l g + B am H g (2. 4 kb, 4. 0 kb, 5. 4 kb) ; 3: pao 8152IGF2g gb g l g + B am H g (1. 8 kb, 2. 4 kb, 4. 0 kb) ; 4: pao 815gB g l g + B am H g (1. 3 kb, 2. 4 kb, 4. 0 kb) ; 5: ΚgH in d g (B)D igested w ith S al g ; 1: pao 815gS al g (7. 7 kb) ; 2: pao 8152IGF2g gs al g (8. 2 kb) ; 3: pao 81523 IGF2g gs al g (11. 7 kb) ; 4: pao 81525 IGF2g gs al g (15. 3 kb) ; 5: ΚgH in d g SD S2PA GE (F ig. 3), M u t + M u t s M u t + phy, rh IGF2g P. p astoris 1%, 3 4 d 213 hr IGF-g, 6 L ( 220 ggl ), rh IGF2g 60 m ggl H it rap, SD S2PA GE 90% (F ig. 4) F ig. 4 Purification of rh IGF2g 1: Supernatant of ferm entation culture; 2: Samp le purified by hydrophobic interaction chrom atogra2 phy and cation exchange chrom atography; 3: Samp le purified by hydrophobic interaction chrom atogra2, M u t s, 214 P. p astoris rh IGF-g, M u t + 21411
6 : g P. p astoris 897 F ig. 5 B ioactivity assay of P. p astoris2derived h r IGF2g g Samp le, Standard F ig. 6 W estern2blo t analysis of rh IGF2g 1: L ow 2range p ro tein mo lecular w eigh t m arker; 2: Pep tide m arker; 3: P. p astoris2derived rh IGF2g ; 4: E. coli2derived rh IGF2g ; 5: Culture supernatant of SM D 1168gpAO 815 2,, P. p astoris rh IGF2g 10 nggm l : B ip, PD I, Cy2 K562, cloph ilin s Calnex in [ 14 ],, (F ig. 5) ; P. p astoris SM D 1168gpAO 815 K562 rh IGF2g, rh IGF2g 21412 W estern T ris2 T ricine2sd S2PA GE, W estern, 13 C F ig. 6, P. p astoris rh IGF2g, 13 C IGF2g, 715 kd;, NM R rh IGF2g T ricine2sd S2PA GE P. p astoris 21413 N N 15, A YR PSETL 2, N h IGF2g 3 IGF2g, IGF2g,, IGF2g P. p astoris,,,, PAOX1,, SM D 1168, IGF2g,,, IGF2g IGF2g [ 13 ], ph,, ph 315, 1% 2% IGF2g 3, E. coli,, IGF2g SD S PA GE CGGELVD Α 2h IGF2g, P. p astoris (KEX2 ) T ricine2sd S2PA GE
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