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R e f o l d i n g o f r e c o m b i n a n t p r o t e i n sEliana De Berna rdez ClarkE x p r e s s i o n o f r e c o m b i n a n t p r o te i n s a s i n c lu s i o n b o d i e si n b a c te r ia i s o n e o f t h e m o s t e f f ic i e n t w a y s t o p r o d u c ec loned p ro te ins , as l ong as the i nc lus ion body p ro te in canb e s u c c e s s fu l l y r e f o ld e d . A g g r e g a t i o n i s th e l e a d i n g c a u s eo f d e c r e a s e d r e fo l d i n g y i e l d s . D e v e l o p m e n ts d u r i n g t h e p a s ty e a r h a v e a d v a n c e d o u r u n d e r s ta n d i n g o f t h e m e c h a n i s mo f agg rega t i on i n i n v i t r o p r o te in f o l d i n g . N e w a d d i t i v e s top r e v e n t a g g r e g a t io n h a v e b e e n a d d e d t o a g r o w i n g l is t . Awea l th o f l ite ra tu re on the ro le o f chape rone s and fo lda ses i ni n v i v o p r o te in f o l d i n g h a s t r ig g e r e d t h e d e v e l o p m e n t o f n e wa d d i t i v e s a n d p r o c e s s e s t h a t m i m i c c h a p e r o n e a c t i v i t y i n v i t r o .

AddressesDepa rtment of C hem ical Engineering, Tufts University, Med ford,MA 02155, USA ; e-maih edeberna@tufts.eduCurrent Opinion in Biotechnology 1998, 9:1 57 -16 3htt p : / /b iomednet.com/e lecref/0958166900900157 Current Bio logy ISSN 0958-1669AbbreviationsDTT dithiotreitolGdm CI guanid in ium ch lor ide

I n t r o d u c t i o nEx p re ss io n o f c l o n ed g en es i n b ac t e r i a is w id e ly u sedb o th i n i n d u s t ry , f o r t h e p ro d u c t i o n o f p h arma ceu t i ca lp ro t e in s , an d i n r esea rch , for t h e p ro d u c t i o n o f p ro t e in sfo r s t ru c tu ra l an d /o r b io ch emica l s t u d i es . Bac t e r i a p ro d u cel a rg e q u an t i t i e s o f r eco mb in an t p ro t e in s i n r ap id , o f t eni n e x p e n s i v e , f e r m e n t a t i o n p r o c e ss e s ; h o w e v e r , t h e p r o d u c to f i n t e r es t is f r eq u e n t l y d ep o s i t ed i n i n so lu b l e i n ac t i v eag g reg a t es o r i n c lu s io n b o d i es . Th e g en era l s t r a t eg yu sed t o r eco v er ac t i v e p ro t e in f ro m in c lu s io n b o d i esinvolves th ree s teps: f i rs t ly , inclusion body i so lat ion andwash in g ; seco n d ly , so lu b i li za t i o n o f t h e ag g reg a t ed p ro t e in ,wh ich cau ses d e n a tu ra t i o n ; an d f i na ll y, r e fo ld in g o f t h eso lu b i l i zed p ro t e in . Wh i l e t h e e f f i c i en cy o f t h e f i r s t twos t ep s can b e r e l a t i v e ly h ig h , fo ld in g y i e ld s may b e l imi t edb y t h e p ro d u c t i o n o f i n ac t i v e mi s fo ld ed sp ec i es as we l las ag g reg a t es .W h en t h e fo rm at io n o f i n c lu s io n b o d i es was f ir s t o b se rv eda lmo s t two d ecad es ag o , ex i s t i n g p ro t e in fo ld in g p ro to co l swe re n o t , i n mo s t cases , ap p l i cab l e t o t h e fo ld in g o f r eco m-b in an t mammal i an p ro t e in s , wh ich a r e i n mo s t cases mu l -t i d o main , o l i g o mer i c , an d /o r d i su lp h id e b o n d ed p ro t e in s .E x i s t i n g p r o te i n f o l d i n g pr o t oc o l s h a d b e e n d e v e l o p e d t och arac t e r i ze fo ld in g i n t e rmed ia t es an d i n v es t i g a t e fo ld in gp a t h w a y s o f sm a l l, m o n o m e r i c p r o t e in s . W h e n a p p l i e d t oth e r e fo ld in g o f i n c lu s io n b o d y p ro t e in s , t h e se p ro to co l sf a i l ed t o p ro d u ce ac t i v e p ro t e in s w i th s i g n i f i can t y i e ld s .Ev en t o d ay, t h e l i t e r a tu re o n i d e n t i f i ca t i o n o f p ro t e in

fo ld in g i n t e rm ed ia t es an d t h e e lu c id a t i o n o f fo ld in gp a th way s d ea l s mo s t l y wi th small mo n o m er i c p ro t e in sth a t h av e e i t h e r i n t ac t o r n o d i su lp h id e b o n d s [1 ] . F o rman y y ear s , eu k ary o t i c ex p ress io n h o s t s wh ich p ro d u ceds o l u b l e s e c r e t e d r e c o m b i n a n t p r o t e i n s b e c a m e f a v o r e do v er b ac t e r ia l h o s t s b ecau se o f t h e d i f f icu l t i e s en c o u n t e re dwh en r e fo ld in g i n c lu s io n b o d y p ro t e in s ; h o wev er , ca r e fu lex am in a t i o n o f th e fo ld in g co n d i t i o n s a l l o wed r esea rch er st o f i n d w a y s t o r e f o l d m u h i d o m a i n d i s u l p h i d e b o n d e dp ro t e in s wi th r e l a t i v e ly h ig h y i e ld s . M o s t o f t h e o r i g in a lwo rk o n i n c lu s io n b o d y p ro t e in fo ld in g can b e fo u n d i nth e p a t en t l i t e r a tu re s t a r t i n g a ro u n d 1 9 8 5 [2 ] .T h e r e c e n t l i t e r a t u r e i n c l u d e s m a n y e x a m p l e s i n w h i c hr e c o m b i n a n t p ro t e i n s h a v e b e e n p r o d u c e d b y r e f o l d -i n g f ro m in c lu s io n b o d i es . S o m e o f t h ese ap p l i ca t i o n sd em o n s t r a t e t h e u se o f su b o p t im al r e fo ld in g p ro to co l st o p ro d u ce smal l q u an t i t i e s o f p ro t e in fo r s tru c tu ra lan d /o r b io ch emica l s t u d i es . O th er ap p l i ca t i o n s d ea l w i thc o m m e r c i a l p r oc e s s e s. T o b e a c c e p t a b l e f o r c o m m e r c i a lap p l i ca t i o n s , r c fo ld in g p ro cesses mu s t b e f as t , i n ex p en s iv ean d h ig h ly e f f i c i en t . Th i s r ev i ew fo cu ses o n r ecen td e v e l o p m e n t s i n t h e o p t i m i z a t i o n o f r e fo l d i n g p ro c e s s e swi th emp h as i s o n meth o d o lo g i es ap p l i cab l e t o l a rg e - sca l ep ro t e in p ro d u c t i o n . S in ce mo s t p ro t e in s o f co mm erc i a lv a lu e a r c sec re t ed i n t h e i r n a tu ra l h o s t an d a r e l i k e ly t oc o n t a in d i s u l p h i d e b o n d s , t h i s r e v i e w e m p h a s i z e s r e c e n tp ro g ress i n p ro t e in r e fo ld in g wi th co n co mi t an t d i su lp h id eb o n d fo rmat io n , a l so ca l l ed o x id a t i v e p ro t e in r e fo ld in g .I n c l u s io n b o d y i s o l a t i o n a n d s o l u b i l i z a t io nEx p ress io n o f r eco m b in an t p ro t e in s as i n c lu s io n b o d i escan b e ad v an t ag e o u s d u e t o t h e v e ry h ig h lev e l s o fe n r i c h e d p r o t e i n p r o d u c e d a n d t h e p r o t e c t i o n o f t h ep ro t e in p ro d u c t f ro m p ro t co ly t i c d eg rad a t i o n . In ad d i t i o n ,w h e n p r o d u c i n g a r e c o m b i n a n t p r o d u c t w h i c h , w h e nact ive, can be tox ic o r le thal to the host cel l , inclusionb o d y p r o d u c t i o n m a y b e t h e b e s t a v a i la b l e m e t h o d . C e l l sco n t a in in g i n c lu s io n b o d i es a r e t y p i ca l l y d i s ru p t ed b y h ig hp ressu re h o m o g en i z a t i o n an d t h e r esu l t i n g su sp en s io n i scen t r i fu g ed t o r em o v e t h e so lu b l e f r ac t i o n. Occas io n a l l y al y ti c e n z y m e , s u c h a s l y so z y m e , m a y b e a d d e d b e f o r e c e lld i s ru p t i o n t o i n c rease e f f i c i en cy an d r ed u ce p o wer r eq u i r e -m e n t s . T h e r e s u l ti n g i n c l u si o n b o d y - c o n t a i n i n g p e l l e t isw a s h e d w i t h b u f f e r s c o n t a i n i n g e i t h e r l o w c o n c e n t r a t i o n so f ch ao t ro p i c ag en t s , su ch as u rea o r g u an id in iu m ch lo r i d e(Gd m Cl ) , o r d e t e rg e n t s , su ch as T r i t o n X-1 0 0 [3 ,4 ,5 " ]an d so d iu m d eo x y ch o l a t e [4,6,7] . Th i s wash in g s t ep i sd es ig n ed t o r emo v e co n t amin an t s , e sp ec i a l l y p ro t e in s , t h a tm a y h a v e a d s o r b e d o n t o t h e h y d r o p h o b i c i n c l u s io n b o d i e sd u r in g p ro cess in g , an d co u ld a f f ec t p ro t e in r e fo ld in gy i e ld . A l te rn a t i v e ly , su c ro se g rad i en t c en t r i fu g a t i o n m ay b ep er fo rmed t o p u r i fy i n c lu s io n b o d i es an d sep ara t e t h emf ro m o th er ce l l u l a r co mp o n en t s [4 ,P 1 ] . Af t e r wash in g ,

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i n c lu s io n b o d ie s a re s o lu b i l i z e d u s in g s t ro n g d e n a tu ra n t s ,s u c h a s u re a , G d m C l , o r t h io c y a n a te s a l t s , o r d e t e rg e n t s ,s u c h a s S D S [ 8 ,P 1] , n - c e t y l t r i m e t h y l a m m o n i u m c h l o r i de[4] , s a rkosy l [6 ] , o r sod ium n-laurosy l sa rcos ine [7 ] , anda r e d u c in g a g e n t , s u c h a s [3 -m e rc a p to e th a n o l , d i th io t r e i to l( D T T ) , d i t h io e r y t h r it o l , o r c y s t e i n e . T e m p e r a t u r e s a b o v e3 0 0 C a re ty p ic a l ly u s e d to f a c i l i t a t e t h e s o lu b i l i z a t io np r o c e ss . A c h e l a t i n g a g e nt , s u c h a s E D T A o r E G T A , c a nb e in c lu d e d in th e s o lu b i l i z a t io n b u f fe r t o s c a v e n g e m e ta lio n s , w h ic h c o u ld c a u s e u n w a n te d o x id a t io n r e a c t io n s .S o l u b i l i z a t i o n c a n a l s o b e a c c o m p l i s h e d b y t h e a d d i t i o nof ac ids , such as 70% fo rm ic ac id [5] . Al te r na t ive ly , fo rp e r i p l a s m i c i n c l u s i o n b o d i e s t h e r e c o m b i n a n t p r o t e i n m a yb e r e c o v e r e d b y i n - s i t u s o lu b i l i z a t io n [P 2 ] i n w h ic h th ed e n a t u r a n t a n d r e d u c i n g a g e n t a r c a d d e d t o t h e b r o t h a tth e e n d o f t h e f e rm e n ta t io o p ro c e s s , a n d th e c e l l d e b r i s i ss e p a r a t e d f r o m t h e s o l u b l e m a t e r i a l b y a q u e o u s t w o - p h a s ee x t ra c t io n .

S o l u b i l i z e d i n c l u s i o n b o d y p r o t e i n s c a n b e c o n t a m i n a t e dw i th v a ry in g l e v e l s o f h o s t p ro te in s , n u c le i c a c id s , a n dc e l l m e m b r a n e c o m p o n e n t s . I t i s t h o u g h t t h a t t h ep r e s e n c e o f t h e s e m i c r o b i a l c o n t a m i n a n t s m a y i n d u c ea g g re g a t io n d u r in g r e fo ld in g , t h u s r e d u c in g o v e ra l l y i e ld s .M a a c h u p a l l i - R e d d y e t a l . [9 ] s h o w e d th a t w h e re a s n o n -p r o t e i n a c e o u s c o n t a m i n a n t s h a v e l i t tl e e f f e c t o n r e n a t -u r a t io n y i e ld s , a g g r e g a t io n o f p r o t e i n c o n t a m i n a n t s c a nre s u l t i n s ig n i f i c a n t l o s s e s b y t r ig g e r in g c o -a g g re g a t io n o ft h e d e s i r e d p r o t e i n . T h u s , s o m e i n c l u s io n b o d y p r o c e s s e sin c lu d e a p u r i f ic a t io n s t e p p r io r t o r e fo ld in g . T y p ic a l ly th iss t e p m a y b e io n e x c h a n g e [4, 10 ], s i z e e x c lu s io n [P 3 " , l 1 ],m e ta l a f f in i ty [1 2 ] , o r r e v e r s e p h a s e c h ro m a to g ra p h y [P 3 " ] .A c o m m o n f e a t u r e o f t h e s e c h r o m a t o g r a p h i c s t e p s i s t h a tt h e y a l l o p e r a t e w i t h b u f f e r s t h a t k e e p t h e p r o t e i n i n t h ed e n a tu re d r e d u c e d s t a t e . I f t h e s o lu b i l i z e d p ro te in i s t ob e s to re d fo r l a t e r u s e , i t m a y ty p ic a l ly b e e x c h a n g e din to a n a c id i c b u f fe r , s u c h a s 1 0 % a c e t i c a c id o r 5 -1 0 m MH C I [ P 3 , 13 ] a n d f r e e z e - d r i e d . E x p o s u r e t o l o w p H m a yre s u l t , fo r s o m e p ro te in s , i n t h e fo rm a t io n o f p a r t i a llyf o l d e d i n t e r m e d i a t e s u n a b l e t o r e f o l d t o t h e n a t i v eac t ive configura t ion [14] . In th is case , the lyoph i l izedp ro te in s h o u ld b e r e s o lu b i l i z e d u s in g c h a o t ro p ic a g e n t s o rd e t e r g e n t s , b e f o r e r e f o l d i n g i s a t t e m p t e d .R e n a t u r a t i o n o f t h e s o l u b i l i z e d p r o t e i nS e v e ra l m e th o d s , i n c lu d in g d i lu t io n , d i a ly s i s , d i a f i l t r a t io n ,g e l f i l t r a t io n , a n d im m o b i l i z a t io n o n to a s o l id s u p p o r t , m a yb e e m p l o y e d t o r e m o v e o r r e d u c e e x c e s s d e n a t u r i n g a n dre d u c in g a g e n t s , a l l o w in g p ro te in s to r e n a tu re . D i lu t io no f t h e d e n a t u r e d s o l u t io n d i r e c t l y i n to r e n a t u r a t i o n b u f f e ri s t h e e a s i e s t p ro c e s s . In d i a ly s i s , t h e d e n a tu re d p ro te ins o lu t io n i s d i a ly z e d a g a in s t r e n a tu ra t io n b u f fe r . B e c a u s ed ia ly s is i s b a s e d o n th e d i f fu s io n o f s m a l l e r m o le c u le sa n d i o n s t h r o u g h m e m b r a n e s , i t m a y b e t o o s l o w t ob e u s e d i n c o m m e r c i a l s c al e p r o d u c t i o n o f p r o te i n s .I n a d d i ti o n , e x p o s u r e o f t h e p r o t e i n t o i n t e r m e d i a t ec o n c e n t r a t i o n o f d e n a t u r a n t s f o r a p r o l o n g e d p e r i o d o f t i m em a y c a u s e a g g re g a t io n . D ia f i l t r a t io n i s a f a s t e r , t h e re fo re ,

m o r e p r a c t i c a l m e m b r a n e - b a s e d a l t e r n a t i v e b e c a u s e t h era t e o f d e n a tu ra n t r e m o v a l i s n o t d i f fu s io n l im i t e d , t h ed r iv in g fo rc e b e in g p re s s u re d i f f e re n c e ; h o w e v e r , a s t h ed r iv in g fo rc e fo r b u f fe r e x c h a n g e i s t h e p re s s u re d ro pa c ro s s t h e m e m b r a n e , a c c u m u l a t i o n o f d e n a t u r e d p r o t e i no n t h e m e m b r a n e m a y l i m i t i t s a p p l i c a t i o n d u e t oe x c e s s i v e f o u li n g . G e l f i lt r a ti o n c h r o m a t o g r a p h y h a s b e e ns u c c e s sf u l l y u se d t o r e n a t u r e s e c r e t o r y l e u k o c y t e p r o t e a s ein h ib i to r , c a rb o n ic a n h y d ra s e a n d ly s o z y m e [P 4 , 1 5 -1 7 ] ;h o w e v e r , p r o b l e m s i n fl o w t h r o u g h t h e c o l u m n m a y a ri s ed u e t o p r o t e i n a g g r e g a t i o n u p o n b u f f e r e x c h a n g e . A g g r e -g a t i o n i n a c h r o m a t o g r a p h i c c o l u m n c a n b e p r e v e n t e db y i m m o b i l i z i n g i n d i v i d u a l p o l y p e p t i d e c h a i n s o n t o t h em a t r ix [12 ,1 3 ,1 8 ]. P o te n t i a l c o m p l i c a t io n s m a y a r i s e i ffo ld in g o f t h e p ro te in i s i n h ib i t e d b y b in d in g to th es o li d s u p p o r t, w h i c h c o u l d b e p r e v e n t e d b y u s i n g f u s io np ro te in s [19 ,2 0 ] . In a d d i t io n to b u f f e r e x c h a n g e , c o lu m nc h r o m a t o g r a p h y a l l o w s f o r so m e d e g r e e o f p u ri f ic a t io n o fth e d e s i r e d p ro d u c t .I n t h e c a s e o f d i s u l p h i d e b o n d e d p r o t e in s , r e n a t u r a t io nb u f f e r s m u s t p r o m o t e d i s u l p h i d e b o n d f o r m a t i o n (o x i -d a ti o n) . T h e m o s t c o m m o n m e t h o d s u s e d t o p r o m o t eo x id a t io n d u r in g r e fo ld in g a re : a i r o x id a t io n ; t h e o x id os h u f f l in g s y s t e m ; th e u s e o f m ix e d d i s u lp h id e s ; a n d o x i -d a t i o n o f s u l p h o n a t e d p r o t e i n s. A l t h o u g h , o x i d a ti o n w i t ha ir o r o x y g e n i n t h e p r e s e n c e o f t ra c e a m o u n t s o f m e t a lio n s i s s im p le a n d in e x p e n s iv e [P 2 , 2 1 ] , r e n a tu ra t io n r a t e sa n d y ie ld s c a n b e lo w . H ig h e r o x id a t io n r a t e s a n d y i e ld sc a n b e o b ta in e d b y u t i l i z in g ' o x id o s h u f f l in g ' r e a g e n t s ,l o w m o l e c u l a r w e i g h t t h io l s i n r e d u c e d a n d o x i d i z e dfo rm s , w h ic h a l lo w fo r b o th fo rm a t io n a n d r e s h u f f l in go f d i s u lp h id e b o n d s , w h ic h c a n a l t e r c o n f ig u ra t io n s . T h em o s t c o m m o n o x i d o s h u f f l i n g r e a g e n t s a r e r e d u c e d a n do x i d i z e d g l u t a t h i o n e ( G S H / G S S G ) , b u t t h e p a i r s c y s -t e i n e / c y s t i n e , c y s t e a m i n e / c y s t a m i n e , D T T / o x i d i z e d g l u -t a th io n e , a n d d i th io e ry th r i to l /o x id iz e d g lu t a th io n e h a v ea l s o b e e n u t i l i z e d . T y p ic a l ly a 1 -3 m M re d u c e d th io l a n da 1 0 :1 to 5 :1 r a t io o f r e d u c e d to o x id iz e d th io l a re u s e d top r o m o t e p r o p e r d i s u l p h i d e b o n d i n g [ 21 ] . M o r e r e c e n tl y ,w e h a v e s h o w n t h a t o p t i m u m r e n a t u r a t i o n y i e l d s a r eo b ta in e d w h e n th e r a t io o f r e d u c e d to o x id iz e d th io l isa n y w h e re b e tw e e n 3 :1 a n d 1 :1 [22 ] . A d i s a d v a n ta g e o fth e o x id o s h u f f l in g s y s t e m o v e r th e u s e o f a i r o x id a t io n , i sth e h ig h c o s t o f s o m e o f t h e r e a g e n t s , p a r t i c u la r ly o x id iz e dg lu ta th io n e .A n o t h e r s t r a t e g y e m p l o y e d t o o x i d i z e p r o t e i n s d u r i n gf o l d i n g i s t h e f o r m a t i o n o f m i x e d d i s u l p h i d e s b e t w e e no x i d i z e d g l u t a t h i o n e a n d r e d u c e d p r o t e i n b e f o r e r e n a t -u ra t io n [3 ] . F o r m a t io n o f m ix e d d i s u lp h id e s in c re a s e st h e s o l u b i l it y o f t h e d e n a t u r e d p r o t e i n b y i n c r e a s i ng t h eh y d r o p h i l ic c h a r a c t e r o f t h e p o l y p e p t i d e c h a in . D i s u l p h i d eb o n d f o r m a t i o n is t h e n p r o m o t e d b y a d d i n g c a t al y t ica m o u n t s o f a r e d u c i n g a g e n t i n t h e r e n a t u r a t i o n s t e p . As im i l a r p ro te c t io n o f t h io l g ro u p s d u r in g s o lu b i l i z a t io n c a nb e a c h i e v e d b y s u l p h o n a t i o n o f t h e d e n a t u r e d p r o t e in ,i n w h i c h a r e d u c i n g a g e n t a n d s o d i u m s u l p h i t e a r e u s e d

8/3/2019 Refolding of ant Proteins

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Refolding of rec omb inantproteins De BernardezClark 159

t o c l eav e d i su lp h id e b o n d s an d p ro t ec t t h e r esu l t i n gth io l g ro u p s as su lp h o n a t es [P 3 " ,5 " ] . Un d er r en a tu ra t i o nc o n d i t io n s , t h e p r o t e c t i o n g r o u p s a r e r e m o v e d b y o x i d a t io ni n t h e p r e s e n c e o f s m a l l a m o u n t s o f a re d u c i n g a g e n t t op r o m o t e d i s u l p h i d e b o n d r e s h uf f li n g .

C o m p e t i t i o n b e t w e e n f o l d i n g a n d a g g r e g a t i o nF o rm at io n o f o f f -p a th way sp ec i es , su ch as i n co r r ec t l yfo ld ed sp ec i es an d ag g reg a t es , a r e t h e cau se o f d ec reasedren a tu ra t i o n y i e ld s . Becau se ag g reg a t i o n i s an i n t e rmo lecu -l a r p h e n o m e n o n , i t is h i g h ly p r o t e i n c o n c e n t r a t i o n d e p e n -d e n t . T h e m o s t d i r e c t m e a n s o f m i n im i z i n g a g g r e g a ti o n isb y d e c r e a s i n g p r o t e i n c o n c e n t r a t i o n . I t h as b e e n s u g g e s t e dt h a t o p t i m u m r e c o v e r y y i e l ds c a n b e e x p e c t e d i f t h epro tein co nce nt ra t ion i s in the ra nge of 10 -50 I .tg /ml [21"] .Ren a tu ra t i o n a t su ch l o w p ro t e in co n cen t r a t i o n s r eq u i r esl a rg e v o lu mes o f r e fo ld in g b u f f e r , d r i v in g p ro d u c t i o nco s t s u p ward .T h e k e y t o a s u c c e s s f u l c o m m e r c i a l r e f o l d i n g p r o c e s sl i e s i n ach i ev in g h ig h y i e ld s wh i l e r e fo ld in g a t h ig hp ro t e in co n cen t r a t i o n s . On e so lu t i o n i n v o lv es u s in g e i t h e rs l ow c o n t i n u o u s o r d i s c o n t in u o u s a d d i t io n o f d e n a t u r e dp ro t e in t o r e fo ld in g b u f f e r [3 * ] . En o u g h t ime i s a l lo wedb e tw een ad d i t i o n s fo r t h e p ro t e in t o fo ld p as t t h e ea r l ys t ag es i n t h e fo ld in g p a th way , wh en i t i s su scep t i b l e t oa g g re g a t io n . T h e c o m p o n e n t s o f t h e s o l u t io n c o n t a i n in gt h e d e n a t u r e d p r o t e i n m u s t b e c a r e f u l l y e x a m i n e d t oav o id d e t r imen ta l e f f ec t s d u e t o t h e i r accu mu la t i o n i n t h ere fo ld in g so lu t i o n a f t e r mu l t i p l e ad d i t i o n s t ep s . An o th era l t e rn a t i v e fo r d ec reas in g p ro t e in ag g reg a t i o n wh i l e fo ld in ga t r e l a t iv e ly h ig h p ro t e in co n c en t r a t i o n s (u p t o 4 mg /m lfo r ca rb o n i c an h y d rase I I ) i s t o u se t h e t em p era tu re - l ea ptact ic [23] , in which the p ro tein i s a l lowed to refo ld atl o w t emp era tu res , t o min imize ag g reg a t i o n , an d t h en t h et emp era tu re i s r ap id ly r a i sed t o p ro mo te f as t f o ld in g a f t e rt h e i n t e r m e d i a t e s r e s p o n s ib l e f o r a g g re g a t io n h a v e b e e nd ep l e t ed . A t h i rd meth o d i n v o lv es fo ld in g b y d i l u t i o n t of i n a l d en a tu ran t co n cen t r a t i o n s t h a t a r e h ig h en o u g h t oso lu b i l i ze ag g reg a t es b u t l o w en o u g h t o p ro mo te p ro p erfo ld in g . We h av e sh o wn th a t t h e o x id a t i v e r en a tu ra t i o no f l y so zy me can b e ca r r i ed o u t a t p ro t e in co n c en t r a t i o n so f u p t o 5 mg /m l wi th v e ry h ig h y i e ld s i n t h e p resen ceo f 1 -2 M Gd m C1 [2 2 "] . An a l t e rn a t i v e m eth o d w h ich a l soe x p o s e s t h e r e f o l d i n g p r o t e i n t o i n t e r m e d i a t e d e n a t u r a n tc o n c e n t r a t i o n s w a s d e v e l o p e d b y M a e d a e t a l . [24]. In thismeth o d , r en a tu ra t i o n i s s t a r t ed b y d i a ly s i s ag a in s t a b u f f e rco n t a in in g h ig h d en a tu ran t co n cen t r a t i o n (8 M u rea ) an dt h i o l / d i s u l p h i d e e x c h a n g e r e a g e n t s , a n d t h e d e n a t u r a n tco n cen t r a t i o n i n t h e d i a ly s i s b u f f e r i s g r ad u a l l y d i l u t edu s i n g b u f f e r w i t h o u t d e n a t u r a n t . U s i n g t h is m e t h o d ,M a e d a e t a t [2 4 ] were ab l e t o r e fo ld immu n o g lo b u l i n Ga t co n ce n t r a t i o n s a b o v e 1 mg /m l wi th y i e ld s as h ig h as7 0 % . F o r p ro t e in s t h a t d o n o t t en d t o ag g reg a t e a ti n t e rmed ia t e d en a tu ran t co n cen t r a t i o n s , t h e s l o w d i a ly s i sm e t h o d c a n s u c c e s s f u l ly p r e v e n t a g g r e g a ti o n b y e x p o s i n gt h e p r o t e i n t o a s l ow d e c r e a s e i n d e n a t u r a n t c o n c e n t r a t io n .F o r p r o t e i n s t h a t a g g r e g a t e a t i n t e r m e d i a t e d e n a t u r a n t

co n cen t r a t i o n s , f a s t o r s l o w d i l u t i o n o f d en a tu red p ro t e inin to r en a tu ra t i o n b u f f e r , r a t h e r t h an s l o w d i a ly s i s , i s t h er e f o ld i n g m e t h o d o f c h o ic e .As ag g reg a t i o n i s t h e majo r cau se b eh in d l o w r en a tu ra t i o ny i e ld s , e l u c id a t i n g t h e ag g reg a t i o n p a th way may h o ld t h ek ey t o su ccess fu l p ro t e in r e fo ld in g a t mo d era t e t o h ig hp r o t e i n c o n c e n t r a t i o n s . I n t e r m e d i a t e s w i t h h y d r o p h o b i cp a t ch es ex p o sed t o t h e so lv en t p l ay a c ru c i a l r o l e i n t h ep ar t i t i o n b e tween n a t i v e an d ag g reg a t ed co n fo rmat io n s .F o ld in g i n t e rmed ia t es a r e b e l i ev ed t o p o ssess s i g n i f i can te l e m e n t s o f s e c o n d a r y s tr u c t u r e b u t l i tt l e o f th e n a t i vet e r ti a r y s t r u c tu r e . D u e t o th e e x p a n d e d v o l u m e o f t h e s ei n t e r m e d i a t e s , h y d r o p h o b i c p a t c h e s , w h i c h m a y n o r m a l l yb e b u r i ed i n t h e n a t i v e s t a t e , a r e ex p o sed t o t h e so lv en t .W h e n h y d r o p h o b i c r e g io n s o n s e p a r a t e p o l y p e p t i d e c h a in sin t e r ac t , i n t e rmed ia t e s a r e d iv e r t ed o f f t h e co r r e c t fo ld in gp a th w ay i n to ag g reg a t es . Th e so ca l l ed 'mo l t e n g lo b u l e 'i n t e rmed ia t e i s b e l i ev ed t o p l ay a majo r ro l e i n t h ek inet ics o f fo ld ing [25] and pr oba bly p lays a ro le inag g reg a t io n . De sp i t e t h e c o n t ro v e r sy o v er t h e n a tu re o ft h i s i n t e rmed ia t e (o n -p a th way v er su s o f f -p a th way ) [2 6 " ]f ro m a k in e t i c p o in t o f v i ew, i n t e rm o lecu l a r a s so c i a t i o no f m o l t e n g l o b u l e - li k e i n t e r m e d i a t e s m a y b e t h e s t a rt i ngp o in t o f t h e ag g reg a t i o n p a th way . On t h e o th e r h an d , Yo n[2 7 ] su g g es t s t h a t i n t e rmo lecu l a r a s so c i a t i o n s r esp o n s ib l efo r ag g reg a t e fo rmat io n may a r i se f ro m f l u c tu a t i n g sp ec i est h a t p r e c e d e t h e m o l t e n - g l o b u l e s t a t e .P i o n e e r w o r k b y G o l d b e r g e t a l . [28] shed l igh t in toth e n a tu re o f i n t e r ac t i o n s r esp o n s ib l e fo r ag g reg a t io nd u r i n g f o ld i ng . T h e y s h o w e d t h a t i n c o r r e c t d is u l p h id eb o n d i n g m a y n o t b e t h e m a j o r c au s e o f a g g re g a t io nb e c a u s e a g g r e g a t e s w e r e f o r m e d e v e n w h e n a c a r -b o x y meth y l a t ed p ro t e in was fo ld ed , t h a t i s , a l l cy s t e in esa r e b l o c k e d f r o m f o r m i n g d i s u l p h i d e b o n d s . T h e y a l s os h o w e d t h a t a g g r e g at i o n is a n o n - sp e c i f ic p h e n o m e n o n . O nt h e o t h e r h a n d , S p e e d e t a l . [2 9 ] r ece n t l y r ep o r t e d t h a ti n mix e d fo ld in g ex p e r im en t s u s in g t h e P 2 2 t a i l sp ik e an dco a t p ro t e in s , f o ld in g i n t e rm ed ia t e s o f t h e two p ro t e in sd id n o t co ag g reg a t e , b u t r a t h e r t h a t t h ey p re fe r r ed t ose l f - as so c i a t e , su g g es t i n g t h a t ag g reg a t i o n i s a sp ec i f i cp h e n o m e n o n . S i n c e t h e y o n l y a n a l y z e d s o l u b l e a g g r eg a te s ,S p e e d e t a l . [29 ] sugges t that i t i s possib le that largerag g reg a t es co u ld g ro w b y a d i f f e r en t mech an i sm in v o lv in gn o n - sp ec i f i c i n t e r ac t i o n s .M o r e r e c e n tl y , M a a c h u p a l l i - R e d d y e t a l . [9" ] p ro v id ed n ewev id en c e o f t h e n o n - sp ec i f i c n a tu re o f t h e ag g reg a t i o nr e a c t i o n b y c o n d u c t i n g m i x e d o x i d a t i v e r e n a t u r a t i o ns t u d ie s w i t h h e n e g g - w h i t e l y s o z y m e a n d t h r e e o t h e rpro teins: 13-galactosidase, bov ine serum albumin , andr ib o n u c l ease A . Th ey fo u n d t h a t fo r e ig n p ro t e in s t h a th av e a t en d en cy t o ag g reg a t e wh en fo ld ed i n i so l a t i o n ,such as 13-galactosidase and bovine serum albumin ,s i g n i f i can t l y d ecreased l y so zy me r en a tu ra t i o n y i e ld s b yp r o m o t i n g a g g r e g a t i o n i n m i x e d f o l d i n g e x p e r i m e n t s .On t h e o th e r h an d , r i b o n u c l ease A , wh ich d o es n o t

8/3/2019 Refolding of ant Proteins

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160 Biochem ical ngineering

s ig n i f i c a n t ly a g g re g a te u p o n fo ld in g in i s o la t io n , d id n o ta f f e c t l y s o z y m e r e n a t u r a t io n y i e l d s i n m i x e d f o l d i n ge x p e r i m e n t s . W e h a v e r e c e n t l y c o n d u c t e d e x p e r i m e n t st r y i n g t o u n d e r s t a n d t h e r o l e th a t d i s u l p h i d e b o n d i n gp la y s in th e a g g re g a t io n p a th w a y [3 0 ] . We fo u n d th a ta g g re g a t io n i s f a s t a n d th a t a g g re g a te c o n c e n t ra t io n d o e sn o t s ig n i f i c a n t ly in c re a s e b e y o n d th e f i r s t m in u te o fr e n a t u r at i o n . H y d r o p h o b i c i n t e r a c ti o n s , a n d n o t d i s u l p h i d eb o n d i n g , w e r e f o u n d t o b e t h e m a j o r c a u s e o f a g gr e g a t io n .U n d e r r e n a t u r a t i o n c o n d i t i o n s t h a t p r o m o t e d i s u l p h i d eb o n d in g , h o w e v e r , a g g re g a te s i z e , b u t n o t c o n c e n t ra t io n ,w a s f o u n d t o i n c r e a s e d u e t o d i s u l p h i d e b o n d f o r m a t i o n ,r e s u l t in g i n c o v a l e n t l y b o n d e d a g g r e g a te s . B a s e d o nth e s e r e s u l t s , i t i s p o s s ib l e to s p e c u la t e th a t i n m ix e df o l d i n g e x p e r i m e n t s , i n w h i c h t w o o r m o r e p r o t e i n s a r es i m u l t a n e o u s l y r e f o l d e d , s m a l l s o l u b l e a g g r e g a t e s m a yfo rm d u e to s p e c i f i c i n t e ra c t io n s th a t a re h y d ro p h o b ic inn a t u r e , a n d l a rg e h e t e r o g e n e o u s a g g r e g a t e s m a y g r o w v iad i s u l p h i d e b o n d i n g o f u n p a i r e d c y s t e i n e s , t h u s r e c o n c il i n gt h e c o n f l i c t i n g o b s e r v a t i o n s o f S p e e d e t a l . [29"] andM a a c h u p a l l i - R e d d y e t a l . [9 " ] o n th e s p e c i f i c /n o n - s p e c i f i cn a tu re o f a g g re g a te s .A n e x a m i n a t i o n o f a g g r e g a t i o n d a t a f o r th e P 2 2 t a i l s p i k ep r o t e i n , c o m b i n e d w i t h t h e p o s t u l a t io n o f t h r e e p o s s i b l em a t h e m a t i c a l m o d e l s t o d e s c r i b e t h e a g g r e g a t i o n p r o c e s s ,l e d S p e e d e t a l . [3 1 ] to c o n c lu d e th a t a g g re g a te s g ro wv i a a c l u s t e r - c l u s t e r m u l t i m e r i z a t i o n m e c h a n i s m i n w h i c hm u h im e rs o f a n y s i z e a s s o c ia t e to fo rm a l a rg e r a g g re g a te .A g g r e g a t i o n i s n o t m e d i a t e d b y t h e s e q u e n t i a l a d d i t i o no f m o n o m e r i c su b u n i t s a n d d o e s n o t s t o p w h e n t h ec o n c e n t r a t i o n o f m o n o m e r i c s u b u n i t s i s d e p l e t e d . T h i sc o n f i rm s th e o b s e rv a t io n [3 0 ] th a t a g g re g a t io n i s f a s t ,a n d th a t a g g re g a te s i z e , r a th e r t h a n to t a l a g g re g a tec o n c e n t ra t io n , i n c re a s e s a s t im e p ro g re s s e s .B a s e d o n th e h y p o th e s i s t h a t a g g re g a t io n i s c a u s e d b yi n t e r a c t i o n s b e t w e e n h y d r o p h o b i c p a t c h e s i n p a r t i a l l yfo ld e d p o ly p e p t id e c h a in s , i t i s p o s s ib l e to e n v i s io ns t r a t e g ie s to d e c re a s e a g g re g a te fo rm a t io n . A c a re fu le x a m i n a t i o n o f s t r u ct u r a l a n d a m i n o a c i d s e q u e n c e d a t ac a n l e a d to th e id e n t i f i c a t io n o f h y d r o p h o b ic p a t c h e sw i t h i n t h e p r o t e i n m o l e c u l e t h a t c o u l d p a r t i c i p a t e i n i n t e r -m o l e c u l a r i n t er a c t io n s . M u t a t i o n s c a u s i n g t h e d i s r u p t io n o fs u c h h y d r o p h o b i c p a t c h e s m a y r e d u c e a g g r e g a ti o n . T h i ss t r a t e g y w a s t e s t e d b y P l t i c k th u n a n d c o - w o rk e r s [3 2, 33 ]w h o i d e n t i fi e d m u t a t i o n s l o c a t e d i n t u r n s o f t h e p r o t e i na n d i n h y d r o p h o b i c p a t c h e s w h i c h l e d t o d e c r e a s e d inv i t r o a n d i n v i v o a g g r e g a t i o n o f r e c o m b i n a n t a n t i b o d yf r a g m e n t s . A s e c o n d s t r a t e g y i n v o lv e s t h e u s e o f a n t i b o d i e sw h i c h p r e f e r e n t i a l l y b i n d h y d r o p h o b i c p a t c h e s a w a y f r o mt h e a c t i v e s i t e t o p r o t e c t t h e p r o t e i n f r o m i n t e r m o l e c u l a ra s s o c ia t io n s l e a d in g to a g g re g a t io n . T h i s s t r a t e g y w a st e s t e d b y K a t z a v - G o z a n s k y e t a l . [3 4 ] w h o s h o w e d th a tc a r b o x y p e p t i d a s e A a g g r e g a t i o n c a n b e p r e v e n t e d u s i n gs p e c i f i c m o n o c lo n a l a n t ib o d ie s . In t e re s t in g ly , P l t i c k th u n ' sg ro u p [32 ,3 3 ] m u t a t e d a m in o a c id s l i k e ly to b e o n th e s u r -f a c e o f t h e n a t i v e p r o t e i n , w h i l e S o l o m o n a n d c o - w o r k e r s

[3 4 " ] r a i s e d th e i r a n t ib o d ie s u s in g n a t iv e a n t ig e n t s . T h e s er e s u lt s s e e m t o in d i c a t e t h a t i n t e r m e d i a t e s r e s p o n s i b l e f o ra g g r e g a t i o n m a y h a v e m o r e n a t i v e - l i k e s t r u c t u r a l f e a t u r e st h a n c u r r e n t l y s p e c u l a t e d .I m p r o v i n g r e n a t u r a t i o n y i e l d sA s i m p l e r s t r a t e g y t o p r e v e n t a g g r e g a t i o n b y i n t e r f e r i n gw i t h i n t e r m o l e c u l a r h y d r o p h o b i c i n t e r a c t i o n s i s t o u s ea d d i t iv e s , s m a l l m o le c u le s th a t a re r e l a t iv e ly in e x p e n s iv ea n d e a s y t o r e m o v e o n c e r e f o l d i n g g o e s t o c o m p l e t i o n .A v a r i e ty o f a d d i ti v e s h a v e b e e n t e s t e d f o r t h e i r a b i li t yt o p r e v e n t a g g r e g a t i o n . T h e y m a y a c t b y s t a b i l i z i n g t h en a t iv e s t a t e , b y p re fe re n t i a l ly d e s t a b i l i z in g in c o r re c t lyfo ld e d m o le c u le s , b y in c re a s in g th e s o lu b i l i t y o f fo ld in gin te rm e d ia t e s , o r b y in c re a s in g th e s o lu b i l i t y o f t h eu n fo ld e d s t a t e . In g e n e ra l , t h e s e a d d i t iv e s d o n o t s e e mto a c c e le ra t e th e r a t e o f fo ld in g , b u t t h e y d o in h ib i tt h e u n w a n t e d a g g r e g a t i o n r e a c t i o n . A d d i t i v e s t h a t h a v eb e e n s h o w n t o p r o m o t e h i g h e r r e f o l d i n g y i e l d s a r e l i s t e di n T a b l e 1 .A s T a b l e 1 i n d i ca t e s, s u r f a c t an t s a n d d e t e r g e n t s h a v ep r o v e n t o b e v e r y e f f i c i e n t f o l d i n g a i d s , a n d h a v e b e e ns h o w n to w o rk w i th a v a r i e ty o f p ro te in s , i n p a r t i c u la rm u l t i m e r i c d i s u l p h i d e b o n d e d o n e s . C o r r e c t d i s u l p h i d eb o n d f o r m a t i o n b y t h i o l / d i s u l p h i d e e x c h a n g e u s i n g o x i d os h u f f l i n g s y s t e m s a n d a i r o x i d a t i o n h a v e b e e n s h o w n t ob e p ro m o te d in th e p r e s e n c e o f d e t e r g e n t s [7, 8" ,3 5 "] .O n e d r a w b a c k o f t h e u s e o f s u r f ac t a n t s a n d d e t e r g e n t si s t h a t t h e y a re d i f f i c u l t t o r e m o v e , a d i r e c t r e s u l t o fth e i r a b i l i t y to b in d to p ro te in s a n d to fo rm m ic e l l e s .M u c h e a s i e r t o r e m o v e , l o w d e n a t u r a n t c o n c e n t r a t i o n sa n d L - a r g i n i n e h a v e s h o w n e x c e l l e n t f o l d i n g e n h a n c i n gc a p a b i l i t i e s (T a b le 1 ) ; h o w e v e r , b e c a u s e th e y a re u s e d int h e m o l a r c o n c e n t r a t i o n r a n g e , t h e y m a y i n t e r f er e w i t h t h ea s s e m b l y o f o l i g o m c r i c p r o t e i n s .A s i n v i v o f o l d i n g a n d a g g r e g a t i o n p r o c e s s e s a r e m o d u l a t e db y t h e p r e s e n c e o f c h a p e r o n e s a n d f o l d a s e s i n t h e c e ll u l a re n v i ro n m e n t , i t i s n o t s u rp r i s in g th a t s u c h p ro te in s c a n a l s oi m p a c t t h e c o m p e t i t i o n b e t w e e n f o l d i n g a n d a g g r e g a t i o nin i n v i t r o p ro te in fo ld in g [3 6 " ] . C h a p e ro n e s a n d fo ld a s e s ,h o w e v e r , a r e p r o t e i n s t h a t n e e d t o b e r e m o v e d f r o m t h ere n a tu ra t io n s o lu t io n a t t h e e n d o f t h e r e fo ld in g p ro c e s s ,a n d m a y b e c o s t l y t o p r o d u c e u n l e ss a r e c o v e r y - r e u s es c h e m e c a n b e i m p l e m e n t e d [ 3 7 ] . A p r a c t i c a l s o l u t i o nt o t h is p r o b l e m w a s p r o p o s e d a n d i m p l e m e n t e d b yA l t a m i r a n o e t a l . [ 3 8 " ] w h o u s e d i m m o b i l i z e d m i n i - c h a p -e r o n e s t o p r o m o t e p r o p e r f o l d i n g o f s e v er a l p r o t ei n sw h i c h p r o v e d d i f f i c u l t t o r e f o l d b y o t h e r m e a n s . T h ei m m o b i l i z e d m i n i - c h a p e r o n e s c o n s i s t ed o f f r a g m e n t s o fG r o E L a t t a c h e d t o c h r o m a t o g r a p h i c r e s i n s . T h e t e c h n i q u ei s o n l y a p p l i c a b l e t o G r o E L s u b s t r a t e s a n d h a s n o t b e e nt e s t e d u n d e r o x i d a t i v e r e n a t u r a t i o n c o n d i t i o n s .I n a n a t t e m p t t o m i m i c t h e G r o E L - G r o E S c h a p e r o n i na c t io n , R o z e m a a n d G e l lm a n [3 5 ", 39 ] d e v e lo p e d a fo ld in gs t r a t e g y in w h ic h th e d e n a tu re d p ro te in i s f i r st e x p o s e d

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R e f o l d in g o f r e c o m b i n a n t p r o t e i n s D e B e r n a r d e z C l a r k 1 6 1

Table 1In vitro folding aids.Add itive Protein ReferenceNon-denaturing concentrat ions ofchaotropic agentsG d m C I

Urea

L-arginine

P. fluorescens ipase [10]Hen egg-wh i te lysozyme [22"]Carbonic anhydrase I I [41]Interferon-~-polypeptides [P1Porcine growth hormone [4]Hen egg-whi te ysozyme [42]IGF-I [P2"]Interferon-13-polypeptides [P1P. fluorescens ipase [10lFab fragments [14]Hen egg-whi te lysozyme [22*]t-gluocosidase [20

SaltsAmmonium sulphate Hen egg-wh i te lysozyme [42 ]SugarsGlycerol P. fluorescens ipase [10]Hen egg-wh i te lysozyme [42 ]IGF-I [P2o]Sucro se IGF-I [P2]Glucose Hen egg-wh i te ysozyme [42"]N-acety l glucosarnine Hen egg-wh i te ysozyme [42 ]Seresine Hen egg-wh i te ysozyme [42]Detergents and surfactantsCha ps TGF-~,-l ike protein s [P3]Carbon ic anhydrase I I [41]Tween Human growth hormone [44]SDS Interferon-~-polypep tides [P1Sarkosyl RNA polymerase q factor [6]Sodium lauorsylsarcosine Single chain Fv fragment [7]Dodecy l mal ts ide Class I I MHC {8]Tri ton X-100 Carbon ic anhydrase I I [41]Polyethylene glycol Carbon ic anhydrase I I [41]Octaethylene glycol Carbon ic anhydrase I I [41]monolaurylPhosphol ipids Hen egg-wh i te lysozyme [g' ]TGF-~-I ike proteins [P3]Sulphobetaines Hen egg-wh i te lysozyme [431I~-D-galactosidase [431Short chain alcoholsn-pentanol Carbon ic anhydrase I I [41 ]n-hexanol Carbon ic anhydrase I I [41]cyclohexanol Carbon ic anhydrase I I [41]

t o a d e t e r g e n t - c o n t a i n i n g s o l u t i o n t o p r e v e n t a g g r e g a t i o n ,f o l l o w e d b y s t r ip p i n g o f t h e d e t e r g e n t w i t h c y c l o d e x t r i n t op r o m o t e f o l d i n g . T h e t e c h n i q u e h a s b e e n n a m e d ' a r t i f i c i a lc h a p e r o n e - a s s i s t e d r e f o l d i n g ' a n d h a s b e e n a p p l i e d t oth e r e fo ld in g o f c a rb o n ic a n h y d ra s e B [39 ], a n d th eo x i d a t i v e r e n a t u r a t i o n o f l y s o z y m e [ 3 5 ]. T h i s p r o c e d u r eh a s al so b e e n s h o w n t o w o r k i n t h e r e f o l d i n g M M - c r e a t i n ek inase [40] .C o n c l u s i o n sI n c l u s i o n b o d y p r o t e i n r e f o l d i n g u s e d t o b e c o n s i d e r e da d i f f i c u l t t a s k . A p ro to c o l t h a t w o rk e d fo r o n e p ro te ind id n o t w o rk fo r o th e r s . F in d in g th e r ig h t c o n d i t io n s tofo ld a g iv e n p ro te in w a s a t r i a l a n d e r ro r p ro c e s s in w h ic he x i s t i n g m e t h o d s w e r e t r i e d u n t i l a s u c c e s s f u l o n e w a sf o u n d . T h i s w a s i n p a r t d u e t o o u r l a ck o f u n d e r s t a n d i n go f t h e c o m p e t i t i o n b e t w e e n f o l d i n g a n d a g g r e g a t i o n inin vitro p r o t e i n f o l d i ng . D e s p i t e t h is l a c k o f k n o w l e d g e ,

m a n y e f f i c i e n t r e f o l d i n g p r o c e s s h a v e b e e n d e v e l o p e di n w h i c h a g g r e g a t i o n i s r e d u c e d b y t h e u s e o f a d d it i v e st h a t i n t e r f e r e w i t h i n t e r m o l e c u l a r i n t e r a c t i o n s r e s p o n s i b l efo r a g g re g a t io n . A s m o re a n d m o re a d d i t iv e s a re a d d e dto th e l i s t , t h e re i s a p re s s in g n e e d to c h a ra c t e r i z e th ea g g re g a t io n p ro c e s s a t t h e m o le c u la r l e v e l i n o rd e r t os e l e c t t h e r i g h t a d d i t i v e . A d v a n c e s i n o u r u n d e r s t a n d i n go f t h e a g g r e ga t io n p a t h w a y c o m b i n e d w i t h k n o w l e d g e o nt h e r o l e t h a t c h a p e r o n e s p l a y i n in vivo p ro te in fo ld in gp ro v id e th e to o l s t h a t w i l l a l l o w u s to d e v e lo p e f f i c i e n tr e f o l d i n g p r o c e s s e s . A m o n g t h e s e d e v e l o p m e n t s , f i n d i n gs i t e s o n t h e p r o t e i n m o l e c u l e t h a t i n t e r a c t w i t h m o l e c u l a rc h a p e r o n e s , a n d i d e n t i f y i n g p r o t e i n r e g i o n s i n v o l v e d i nin t e rm o le c u la r i n t e ra c t io n s w i l l p ro v id e a r a t io n a l b a s i sfo r f in d in g s p e c i f ic m u ta t io n s a n d d e s ig n in g s m a l l b in d in gm o l e c u l e s t h a t p r e v e n t a g g r e g a t i o n .

R e f e re n c e s a n d r e c o m m e n d e d r e a d in gPapers o f par t i cu la r i n te res t , pub l i shed w i th in the annua l per iod o f rev iew ,h a v e b e e n h i g h l i g h t e d a s :

o f s p e c i a l i n t e r e s t* * o f o u t s t a n d i n g in t e r e s t1 . C l a r k e A R , W a l t h o J P : Protein fo ld ing pathw ays andin termediates. Cu r t O p i n B i o t e ch n o 1 1 9 9 7 , 8 : 4 0 0 - 4 0 5 .2 . Herman R : Protein Folding. M u n i c h : E u r o p e a n P a t e n t O f f i c e ;1 9 9 3 . [ E P O A p p l i e d T e c h n o l o g y S e r i e s v o l 1 2 .]3 . Rud o lph R , B~hm G, L i li e H , Jaen icke R : Folding proteins, In Protein Funct ion. A Pract ical Approach, e d n 2 . E d i t e d b yC r e i g h t o n T E . N e w Y o r k : I R L P r e s s ; 1 9 9 7 : 5 7 - 9 9 .T h i s c h a p t e r c o v e r s r e l e v a n t t o p i c s a n d t e c h n i q u e s i n p r o t e i n u n f o l d -i ng / fo ld ing . I t con ta ins p ro toco ls fo r ce l l l ys i s , i so la t i on o f i nc lus ion bod ies ,and renatura ti on o f so lub i l i zed p ro te ins w i th sp ec ia l emp has is on the renat -u r a t i o n o f d i s u l p h i d e b o n d e d p r o t e i n s . I t a l s o c o v e r s m o n i t o r i n g o f p r o t e i nf o l d i n g , k i n e t i c s a n d e q u i l i b r i u m c o n s i d e r a t i o n s , f o l d i n g a n d a s s o c i a t i o n o fo l i gomer i c p ro te ins , the e f fec ts o f l i gands and the e f fec t o f aux i l i a ry p ro te insass i s t i ng p ro te in fo ld ing .4 . C a r d a m o n e M , P u r l N K , B r a n d o n M R : C o m p ar i n g th e r e fo l di n gan d r eo x i da t io n o f r eco m b i n an t p o r ci n e g r o wth h o r m o n e f r o ma u r ea d en a tu r ed s ta te an d f r o m E s c h e r i c h ie c o i l inclusionbodies. Biochemistry 1 9 9 5 , 3 4 : 5 7 7 3 - 5 7 9 4 ,5 . C o w l e y DJ , M a c k i n R B : E x p r e s s i o n , p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f r e c o m b i n a n t h u m a n p r o i n s u l i n . FE B S L e tt1 9 9 7 , 4 0 2 : 1 2 4 - 1 3 0 .E. coi l d e r i v e d r e c o m b i n a n t h u m a n p r o i n s u l i n p r o d u c e d i n in c lu s io n b o d i e sw a s s o l u b i l iz e d w i t h 7 0 % f o r m i c a c i d a n d c l e a v e d w i t h c y a n o g e n b r o m i d e .A f te r so l ven t evapo ra t i on and f reeze-dry ing , the l yoph i l i zed p ro te in wa s d i s -s o l v e d i n 7 M u r e a a n d s u b j e c t e d t o o x i d a t i v e s u lp h i t o ly s i s , T h e s u l p h o n a t e dm a t e ri a l w a s p u r i fi e d b y a n i o n e x c h a n g e , a n d r e f o l d e d a t p H 1 0 . 5 i n t h epres enc e o f a i r and l~ -mercaptoe thano l . The re fo lde d pro insu l i n con ta ine dt h e c o r r e c t d i s u l p h i d e b o n d p a t te r n .6 . B u r g e s s R R : P u r i f i c a t i o n o f o ver p r o d u ced E s c h e r i c h ie c o i l R N A

p o l ym er ase a f ac to r s b y s o l u b i l i z i n g inclusion bod ies andrefo ld ing from sarkosyl . M e t h o d s E n z y m o l 1 9 9 6 , 2 7 3 : 1 4 5 - 1 4 9 .7 . K u r u cz I , T i t u s J A , J o s t C A , S e g a l D M : C o r r e c t d i s u l p h i d epair ing an d efficient refolding of detergen t-solubil ized single-chain Fv pro teins from bacter ia l inclusion bod ies. M o / I m m u n o l1 9 9 5 , 1 2 : 1 4 4 3 - 1 4 5 2 .8 . S t ~ c k e l J , D 6 r i n g K , M a l o t k a J , J ~ h n ig F , D o r n m a i r K : P a t h w a y o f d e te r g en t -m e d i a ted an d p ep t i d e l i g an d -m ed i a ted r e fo l d in g o fh e te r o d i m e r c lass I I m a j o r h i s to co m p at ib i li ty co m p l ex (M H C )m o l e c u l e s . E u r J B i o c h e m 1 9 9 7 , 2 4 8 : 6 8 4 - 6 9 1 .A n i n d e p t h d i s c u s s i o n o f t h e m e c h a n i s m o f d e t e r g e n t - m e d i a t e d p r o t e i n f o l d -i n g o f a h e t e r o d i m e r i c d i s u lp h i d e b o n d e d p r o t e i n w i t h f o u r d o m a i ns . T h e c o n -t r i bu t i ons o f de te rgen t head grou p and a l i pha t i c ta i l to the s tab il iza t ion o f fo ld -i n g i n te r m e d i a t e s a r e d i s s e c t e d . O p t i m a l d e t e r g e n t c o n c e n t r a t i o n d e c r e a s e sw i t h i n c r e a s i n g c r i t i c a l m i c e l l e c o n c e n t r a t i o n . F o r m a t i o n o f s e c o n d a r y s t r u c-t u r e o c c u r s e a r l y i n t h e f o l d in g p a t h w a y w h e n t h e d e n a t u r i n g d e t e r g e n t S D Si s r e p l a c e d b y a m i ld d e t e r g e n t . T e r t ia r y s t r u c t u r e f o r m a t i o n a n d h e t e r e d i m e r

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1 6 2 B i o c h e m i c a l e n g i n e e r i n g

a s s o c i a t i o n o c c u r s l a t e r in t h e f o l d i n g p a t h w a y c o n c o m i t a n t l y w i t h d i s u l p h i d ebond fo rmat ion .9 . M a a c h u p a l l i - R e d d y J , K e l l e y B D , D e B e r n a r d e z C l a r k E : E f f e c t o f inclusion bo dy contaminants on the oxidat ive r e n a t u r a t i o n o fh e n e g g w h i te l y s o z y m e . B io techno /P rog 1 9 9 7 , 1 3 : 1 4 4 - 1 5 0 .T h i s p a p e r s h o w s t h a t n o n - p r o t e i n a c e o u s c o n t am i n a nt s , s u c h a s p l a s m i dD N A , r i b o s o m a l R N A , a n d l i p o p o l y s a c c h a r i d e s , h a v e l i tt l e e f fe c t o n p r o t e i nr e n at u ra t io n . P h o s p h o l i p i d s i m p r o v e f o l d i n g y i e l d s b y a b o u t 1 5 % . P r o te i na -c e o u s c o n ta m i n a nt s , o n t h e o t h e r h a n d , c a n h a v e a s i g n i fi c a n t d e t r i m e n t a le f f e c t o n f o l d i n g y i e l d s b y c a u s i n g c o - a g g r e g a t i o n o f t h e p r o t e i n o f i n t e r -e s t . T h e p a p e r a l s o s h o w s t h a t c o n t a m i n a n t s a f f e c t t h e o v e r a l l r a t e o f t h eaggregat ion reac t i on w i thou t a f fec t i ng the fo ld ing ra te .10 . Ahn JH , Lee YP , Rhee JS : Investigation of refolding conditionfo r Pseudomonas fluorescens l ipase by response sur facemethodo logy. J Biotechno/ 1 9 9 7 , 5 4 : 1 5 1 - 1 6 0 .11 . S imm ons T, Ne who use YM, A rno ld KS , Innerari t y TL ,W e i s g r a b e r K H : Hum an low densi ty l ipopro tein receptorf r a g m e n t . S u c c e s s f u l r e f o l d i n g o f a functionally active l igand-b i nd i n g d o m a i n p r o d u c e d i n Escherichia col l . J B/o/ Chem1 9 97 , 2 7 2 : 2 5 5 3 1 - 2 5 5 3 6 .1 2 . N e g r o A, O n i s t o M , G r a s s a t o L , C a e n a z z o C , G a r b i s a S :R e c o m b i n a n t h u m a n T I M P - 3 f r o m Escherichia coi l : synthesis ,refo ld ing , physico -chemical and funct ional insigh ts. Protein Eng1 9 9 7 , 1 0 : 5 9 3 - 5 9 9 .1 3 . K i m S , B a u m J , A n d e r s o n S : P r o d u c t i o n o f correctly folded

recombinant [13C,15N] enr iched gu ine a p ig [Val9 0] -~ -lacta lbumin . Protein Eng 1 9 9 7 , 1 0 : 4 5 5 - 4 6 2 .1 4 . B u c h n e r J , R u d o l p h R : R e n a t u r a t i o n , p u r i f i c a t i o n a n dcharacter izat ion o f recombinant Fab fragm ents p r o d u c e d i n

Escherichia co i l . B io techno/1991, 9 : 1 5 7 - 1 6 2 .15 . Ham aker KH, L iu J , See ly RJ , L a d i s c h C M , L a d i s c h M R :C h r o m a to g r a p h y fo r r a p id b u f fe r e x c h a n g e a n d r e fo l d in g o fsecretory leukocyte p ro tease i n h i b i t o r . Biotechnol Prog 1 9 9 6 ,1 2 : 1 8 4 - 1 8 9 .1 6 . B a t a s B , J o n e s H R , C h a u d h u r i J B : S t u d i e s o f t h e hydrodynamicv o l u m e c h a n g e s t h a t o c c u r d u r i n g r e f o l d in g o f l y s o z y m e u s i n gs i z e - e x cl u s io n c h r o m a t o g r a p h y . J Ohromatog A 1 9 9 7 , 7 6 6 : 1 0 9 -119.17 . Ba tas B , Cha udhu r i JB : Protein refolding at high concentrationusing s ize-exclusion chrom atogra phy. B io techno /B ioeng 1 9 9 6 ,5 0 :1 6 -2 3 .1 8 . Q i u H , W e n D , B e l a n g e r A , B u n n H F : Over -expression andrefo ld ing o f b io log ical ly act ive hum an e r y t h r o p o i e t i n f r o mE. co i l Pro te in Eng 1 9 9 7 , 1 0 ( s u p p l ) : 3 3 .1 9 . S t e m p f e r G , R u d o l p h R : Improved refo ld ing o f a m a t r i x - b o u n dfusion protein. Ann N Y Acad Sc i 1 9 9 6 , 7 8 2 : 5 0 6 - 5 1 2 .2 0 . S t e m p f e r G , H S I I - N e u g e b a u e r B , R u d o l p h R : Improved refo ld ing o f a n i m m o b i l i z e d f u s i o n p r o t e in . Nat B io techno l 1 9 9 6 , 1 4 : 3 2 9 -3 3 4 .T h e a u t h o r s d e m o n s t r a t e a s t r a t e g y t o p r e v e n t a g g r e g a t i o n u p o n f o l d i n g b yphys i ca l l y i so la t i ng p ro te in mo lecu les f rom each o ther th rough immobi l i z i ngo n t o a s o l i d s u p p o r t . I n t h i s t e c h n i q u e , t h e p r o t e i n o f i n t e r e s t i s p r o d u c e das a fus ion p ro te in w i th e i ther an amino- o r a carboxy - te rmina l hexa-arg in inep e p t i d e . T h e d e n a t u r e d p r o t e i n i s a t t a c h e d t o a s o l i d s u p p o r t c o n t a i n i n gp o l y a n i o n i c g r o u p s a n d r e n a tu r a ti o n o n t h e i m m o b i l iz e d p r o t e i n i s p e r f o r m e dby remova l o f the denaturan t. The e f fec t o f i on i c s t reng th , add i t i on o f e thy leneg l y c o l a n d s u p p o r t m a t er ia l a r e t h o r o u g h l y i n v e s t i g a t e d .21 . Rud o lph R , L i ll e H : In vi tro fo ld ing o f inclusion bod y pro teins,An FASEB J 1 9 9 6 , 1 0 : 4 9 - 5 6 .

e x c e l l e n t r e v i e w w i th 8 0 r e f e r e n c e s d e t a i l i n g k e y a s p e c t s o f e x p r e s s i o nand re fo ld ing o f inc lus ion bod y pro te ins .2 2 . H s v e h a n D , D e B e rn a r d e z C l a rk E : O x i d a t i v e r e n a t u r a t io n o f l y s o z y m e a t h igh con centrat ions. Biotechnol Bioeng 1997,5 4 : 2 2 1 - 2 3 0 .T h i s p a p e r s h o w s t h a t i t i s p o s s i b l e t o r e f o l d a d i s u l p h i d e - b o n d e d p r o t e i nw i t h v e r y h i g h y i e l d s a t h i g h p r o t e i n c o n c e n t r a t i o n s ( u p t o 5 m g / m l ) b y a d d i -t i o n o f n o n - d e n a t u r i n g c o n c e n t r a t i o n s o f t h e c h a o t r o p i c a g e n t g u a n i d i n i u mc h l o r i d e ( G d m C I ) . I n c r e a s i n g G d m C I c o n c e n t r a t i o n s d e c r e a s e d b o t h t h er a t e o f t h e f o l d i n g a n d a g g r e g a t i o n r e a c t i o n s , w i t h a s t r o n g e r e f f e c t o n a g -g r e g a t i o n . T h e p a p e r a ls o s h o w s t h a t G d m C I i s a s e f f ic i e n t a s L - a rg i ni ne i ndec re as ing agg rega t ion wh i l e resu lt ing i n fas te r fo ld ing ra tes .2 3 . X i e Y , W e t l a u f e r D B : Contro l o f aggregat ion in p ro tein fo ld ing :th e t e m p e r a tu r e - l e a p t a c t i c . Protein Sci 1 9 9 6 , 5 : 5 1 7 - 5 2 3 .2 4 . M a e d a Y , U e d a T , I m o t o T : Effect ive renaturat ion o f den ature da n d r e d u c e d i m m u n o g l o b u l i n G in vi tro w i t h o u t a s s i s t a n c e o fchaperone. Protein Eng 1 9 9 6 , 9 : 9 5 - 1 0 0 .

2 5 . P t it s yn O B , B y c h k o v a V E , U v e r s k y V N : Kinet ic and e q u i l i b r i u mfolding intermediates. Phi los Trans R Soc Lend B B io l Sci1 9 9 5 , 3 4 8 : 3 5 - 4 1 .2 6 . C r e i g h to n T E : H o w im p o r t a n t is t h e m o l t e n g lobu le fo r cor rect~n p r o t e i n f o l d i n g ? Trends B iochem Sci 1997, 22 :6 -10 .t h i s p a p e r , T E C r e i g h t o n a n a l y z e s e x p e r im e n t a l o b s e r v a t i o n s o f t h e o x i d a .t i ve re fo ld ing o f o~- Iac ta lbumin ob ta ined in h i s l abora to ry and by o ther i nves t i.g a t o r s . H e s h o w s t h a t , c o n t r a r y t o w h a t i s w i d e l y b e l i e v e d , t h e m o l t e n g l o b u l ehas l it t le na t i ve- li ke topo log y and i t cou ld be an o f f -pa thway , non prod uc t i vespec ies , ra ther than the key to rap id fo ld ing .2 7 . Y o n J M : T h e s p e c i f i c i t y o f protein aggregation. Nat Biotechnol1 9 9 6 , 1 4 : 1 2 3 1 .2 8 . G o l d b e r g M E , R u d o l p h R , J a e n i c k e R : A kinet ic study o f thecompet i t ion be twe en renaturat ion an d aggregat ion dur ingth e r e fo ld i n g o f d e n a tu r e d - r e d u c e d e g g w h i te l y s o z y m e .Biochemistry 1 9 9 1 , 3 0 : 2 7 9 0 - 2 7 9 72 9 . S p e e d M A , W a n g D I C , K i n g J : Speci f ic aggregat ion o f p a r t i a l l y fo lded po lypept ide chains: the molecu lar basis o f inclusionbody composi t ion . Nat Biotechnol 1 9 9 6 , 1 4 : 1 2 8 3 - 1 2 8 ? .T h e a u t h o r s r e f o l d e d a m i x t u r e o f P 2 2 t a i l s p i k e a n d c o a t p r o t e i n s u n d e rc o n d i t i o n s w h i c h p r o m o t e a g g r e g a t i o n o f b o t h p r o t e i n s . C o - a g g r e g a t i o n o ft h e p r o t e i n s w a s n o t o b s e r v e d i n d i c a t i n g t h a t a g g r e g a t i o n i s d u e t o s p e c i fi ci n te rac t ions . As on ly so lub le m u l t imer i c sp ec ies w ere ana lyzed, the poss ib il it yo f fo r m a ti o n o f h e t e r o g e n e o u s i n s o l u b l e a g g r e g a t e s c a n n o t b e e x c l u d e d .3 0 . D e B e r n a r d e z C l a r k E , H e v e h a n D , S z e l a S , M a a c h u p a l l i- R e d d y J :O x i d a t i v e r e n a t u r a t i o n o f hen egg-wh i te lysozyme. Fo ld ing vs.aggregat ion . Biotechnol Prog 1 9 9 8 , 1 4 : 4 7 - 5 4 .3 1 . S p e e d M A , K i n g J , W a n g D I C : P o l y m e r i z a t i o n m e c h a n i s m o fpo lypept ide chain aggregat ion . Biotechno l B ioeng 1 9 9 ? , 5 4 : 3 3 3 -3 4 3 .3 2 . K n a p p i k A , P l e ck t hu n A : E n g i n e e r e d t u r n s o f a recombinanta n t i b o d y i m p r o v e it s in vivo f o l d i n g . Protein Eng 1 9 9 5 , 8 : 8 1 - 8 9 .3 3 . N i e b a L , H o n e g g e r A , K r e b b e r C , P l (J c k th u n A : Disrupting th e h y d r o p h o b ic p a tc h e s a t th e a n t i b o dy v a r i a b l e / c o n s ta n tdomain in ter face: improved in vivo fo ld ing and physicalcharacter izat ion o f an eng inee red scFv fragm enL Protein Eng1 9 9 7 , 1 0 : 4 3 5 - 4 4 4 .A m i n o a c i d m u t a t i o n s a w a y f r o m t h e a n t i b o d y fr a g m e n t b i n d i n g s i te r e -s u l t e d i n d e c r e a s e d a g g r e g a t i o n r a t e s in vitro. M u t a t i o n s w e r e p e r f o r m e do n e x p o s e d h y d r o p h o b i c r e s i d u e s w h i c h a r e a d j a c e n t t o a n o t h e r e x p o s e dh y d r o p h o b i c r e s i d u e in o r d e r t o d i s r u p t h y d r o p h o b i c p a t c h e s . T h e p a p e r a l s oe x p l o r e s th e r o l e o f t h e s e m u t a t i o n s o n in vivo f o l d i n g a n d a g g r e g a t i o n o fthe resu lt ing scFv f ragmen ts .3 4 . K a t z a v - G o z a n s k y T , H a n a n E , S o l o m o n B : E f f e c t o f m o n o c l o n a l an t ibod ies in p revent ing carbo xyp ept idas e A aggregat ion .Biotechnol Appl Biochem 1 9 9 6 , 2 3 : 2 2 7 - 2 3 0 .T h is p a p e r s h o w s t h a t c e r t a i n m o n o c l o n a l a n t i b o d i e s t h a t b i n d t o t h e a n t i g e na t l o c a t i o n s r e m o v e d f r o m t h e a c t i v e s i t e , a r e c a p a b l e o f p r o m o t i n g p r o p e rf o l d i n g b y d e c r e a s i n g a g g r e g a t i o n . T h us , m o n o c l o n a l a n t i b o d i e s c a n b e u s e da s f o l d i n g a d d i t i v e s t o p r e v e n t a g g r e g a t i o n , a n d a s t o o l s t o i d e n t i f y s i t e sw h e r e p r o t e i n a g g r e g a t i o n m a y b e i n it ia t ed .3 5 . R o z e m a D , G e l l m a n S H : A r t i f i c ia l c h a p e r o n e -a s s i s te d r e fo l d in g o f d e n a tu r e d - r e d u c e d ly s o z y m e : m o d u l a t i o n o f th e c o m p e t it io nbetw een renaturat ion a nd aggregation . Biochemistry 1 9 9 6 ,3 5 : 1 5 7 6 0 - 1 5 7 7 1 .The ar t i f i c i a l chaperone s t ra tegy cons is ts o f re fo ld ing by d i l u t i on i n the p res -e n c e o f a d e t e r g e n t f o l lo w e d b y s t r i p p i n g o f t h e d e t e r g e n t u s i n g m e t h y l- B -cyc lodex t r i n . On ly i on i c de te rgents a re e f fec t i ve i n the ro le o f a r t i f i c i a l chap-erones . Lysozyme i s no t ac t i ve i n the p resence o f de te rgent , and ac t i v i t yi s r e c o v e r e d o n l y a f t e r t h e s t r i p p i n g s t e p . T h i o l / d i s u l p h i d e r e a g e n t s c a n b ea d d e d e i t h e r i n t h e d i l u t i o n s t e p ( i n t h e p r e s e n c e o f d e t e r g e n t ) o r i n t h es t r i p p i n g s t e p . H i g h e r y i e l d s a r e o b t a i n e d w h e n t h i o l / d i s u l p h i d e e x c h a n g eo c c u r s i n t h e p r e s e n c e o f d e t e r g e n t s .3 6 . T h o m a s J G , A y l in g A , B a n e y x F : Molecu lar chaperones, fo ld ing cata lysts , and the recovery o f act ive recom binant p ro teinsf r o m E. coil. To fold o r t o r e f o l d . App/Biochem Bio techno11997,6 6 : 1 9 7 - 2 3 8 .A d e t a i l e d r e v i e w o f t h e r o l e o f c h a p e r o n e s a n d f o l d a s e s o n in vivo a n d invitro pro te in fo ld ing . Inc ludes an ex tens i ve l i s t o f re levant re fe rences .3 7 . K i n g J : R e f o l d i n g w i t h a p i e c e o f t h e r in g . Nat Biotechnol 1997,1 5 : 5 1 4 - 5 1 5 .38 . A I tami rano M, Go lb i k R , Zahn R , Buc le AM, Fersh t AR: Refo ld ing c h r o m a t o g r a p h y w i t h i m m o b i l iz e d m i n i - ch a p e r o n e s . Proc Nat/Acad Sc i USA 1 9 97 , 9 4 : 3 5 7 6 - 3 5 7 8 .T h e a u t h o r s d e m o n s t r a t e t h e p r a c t i c a l u s e o f t h e G r o E L a p i c a l d o m a i n( r e s i d u e s 1 9 1 - 3 7 6 ) a n d t h e c o r e o f t h e a p i c a l d o m a i n ( r e s id u e s 1 9 1 - 3 4 5 )as fo ld ing enhancers i n the renatura t i on o f severa l d i f f i cu l t to re fo ld p ro -t e in s . T h e m i n i -c h a p e r o n e s a r e i m m o b i l iz e d o n c h r o m a t o g r a p h i c r e s in s a n d

8/3/2019 Refolding of ant Proteins

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R e f o l d i n g o f r e c o m b i n a n t p r o t e i n s D e B e r n a r d e z C L a rk 1 6 3

c h r o m a t o g r a p h y c a n b e c o n d u c t e d i n b o t h e l u t i o n a n d b a t c h m o d e s . T h et e c h n i q u e o n l y w o r k s w i t h p r o t e i n s t h a t a r e k n o w n t o b e G r o E L s u b s t r a t e s .I t s app l i cab i l i t y in the ox ida t i ve renatura ti on o f p ro te ins i s no t dem ons t ra ted .3 9 . R o z e m a D , G e l l m a n S H : A r t i f i c i a l ch ap er o n e -as s i s ted r e fo l di n go f car b o n i c an h yd r ase B. J B io l Chem 1 9 9 6 , 2 7 1 : 3 4 7 8 - 3 4 8 ? .4 0 . C o u t h o n F , C l o t t e s E , V i al C : R e f o l d i n g o f S D S - a n d t h e r m a l l yden atured MM-creat ine k inase using c y c l o d e x t r i n s . Biochem

Biophys R es Commun 1 9 9 6 , 2 2 7 : 8 5 4 - 8 6 0 .4 1 . W e t l a u fe r D B , X i e Y : C o n t r o l o f a g g r e g a t i o n i n p r o t e i n r e f o l d in g :a v a r i e t y o f sur factants prom ote renaturat ion o f carbon ica n h y d r a s e IL Protein Sci 1 9 9 5 , 4 : 1 5 3 5 - 1 5 4 3 .4 2 . M a e d a Y , Y a m a d a H , U e d a T , I m o t o T : E f f e c t o f addi t ives on the r e n a t u r a t i o n o f r ed u ced l yso z ym e i n t h e p r e s e n c e o f 4 M urea.Protein Eng 1 9 9 6 , 9 : 4 6 1 - 4 6 5 .I n c r e a s i n g c o n c e n t r a t i o n s o f a d d i t i v e s s u c h a s s a r c o s i n e , g l y c e r o l , a m m o -n i u m s u l p h a t e , g l u c o s e a n d N - a c e t y l g l u c o s a m i n e r e s u l t e d i n i m p r o v e d r e -fo ld ing ra tes (exce pt fo r g l ycero l ) and y ie ld s in the ox ida t i ve renatura ti on o fl ysozyme. Sarcos ine was the mos t e f fec t i ve fo ld ing enhancer .4 3 . G o l d b e r g M E , E x p e r t - B e z a n l o n N , V u i ll ar d L, R a b i l l o u d T : N o n -dete rgent su lphobetaines: a new class o f molecu les t h a tf a c i l i t a t e in vi tro protein renaturation. Fo/d Design 1 9 9 6 ,1:21-27.4 4 . B a r n N B , C l e l a n d J L , R a n d o l p h T W : Molten g lobu le i n t e r m e d i a t e

o f r e c o m b i n a nt h u m a n g r o w t h h o r m o n e : s t a b i l i z a t io n w i t hs u r f a c t a n t s . Biotechnol Prog 1 9 9 6 , 1 2 : 8 0 1 - 8 0 9 .

Pa t e n t sP f . D o r in G , M c A l a r y P , W o n g K : Bacterial production o fh y d r o p h o b i c p o l y p e p t i d e s . World (WO) Patent 1 9 9 6 , 9 6 / 3 9 5 2 3 .P 2 , B u i l d e r S , H a r t R , L e s t e r P , R e i f s n y d e r D : R e f o l d i n g o f misfo ldedA i n s u l i n - li k e g r o w t h f a c t o r -I . US Patent 1 9 9 7 , 5 6 6 3 3 0 4 .me thod i s d i sc los ed in wh ich the ox ida t i ve renatura t i on o f insu l in - l ikeg r o w t h f a c t or - I ( I G F - I) i s c o n d u c t e d u s i n g o x y g e n a n d i n th e p r e s e n c e o f l o wc o p p e r o r m a n g a n e s e c o n c e n t r a t i o n s , a n a l c o h o l i c o r p o l a r a p r o t i c s o l v e n t(such as 20o/o e thano l) , an e f fec t i ve amou nt o f chao t rop i c ag ent (such as 2 Murea) , an e f fec t i ve amoun t o f an a l ka li ne ear th , a l ka li meta l , o r ammonium sa l t(such as 1 M NaC I ) , an op t i on a l osmo ly te (such as g l ycero l ) , and a reduc ingagen t (such as I mM DTT) . An ex ten s i ve fac to r i a l des ign ana lys is o f thee f fec ts o f IGF- I concent ra t i on , sa l t t ype and concent ra t i on , u rea , e thano l andg l y c e r o l c o n c e n t r a t i o n s o n f o l d i n g y i e l d i s a l so i n c l u d e d .P 3 . C e r l e t t i N , M c M a s t e r G K , C o x D , S c h m i t z A , M e y b a c k B : Process= for refo ld ing recombinant ly produ ced TGF-~ - I ike pro teins. U SPatent 1 9 9 7 , 5 6 5 0 4 9 4 .M e t h o d s t o i m p r o v e t h e o x i d a t i v e r e n a t u r a t i o n y i e l d s o f d i m e r i c f o r m sof t rans fo rming grow th fac to r # - li ke p ro te ins a re d i sc losed . The pro-cedure requ i res the use o f mi ld de te rgents , such as 3 - (3 -ch lo lami -d o p r o p y l ) d i m e t h y l a m m o n i o - l - p r o p a n e s u l p h o n a t e . S e v e r a l m e t h o d s f o rt h i o l / d is u l p h i d e e x c h a n g e a r e d i s c l o s e d . T h e s e i n c l u d e t h e u s e o f r e d u c e da n d o x i d i z e d g lu t a t h io n e , t h i o r e d o x i n , a n d f o l d i n g f r o m a S - s u l p h o n a t emonomer .P 4 . S e e l y R , L a d i s c h M : P r o c e s s f o r protein refo ld ing by mean s o fb u f f e r exch an g e u s i n g a co n t i n u o u s s ta t i o n ar y p h ase cap ab l e

o f separating proteins f r o m s a l t . World (WO) Patent 199'7,9 7 / 0 4 0 0 3 .