thelabile a5para lylresiclues bっ gluし 曲 yl iesidues prevellmd deamWaLjon effe砒 廴 ve 取 WeゆDn5trUu 陀 @thequl ロ 加 pl 已 utant CE .皿」 t has no de 直da 廿 Dn gi加. The overa] 100nfi 〕rmatiQn o @thlsmutan し wa δ eqUlvulent to 止 a し of 、粗d 騨e 血 the nativa state . Thc mD -t inte estingP ヒvpert } 「 。 「 CML molten gl。bde fomllation w・ xg alsu }。 hserv' erl as we ] ] as "1i しン . pe、 Stab セ li7a し 重o 匸 1 Df Uie C : 乢agalts し deamidation hnP es the } ield of pure Teo binantp 漁 in and thgrepmducib 皿ty

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... ntS . in addtion , mbm 皿 匸 CN 乢 expv sed inE .co ]i bas an eitra N ・ しerm 捌methio ne residue 魚. let 』 he pvesence げ Mε t O ln ⊂ 脳 . 口 Ld reas 騷 thc thermal stabrU1 . M. solublity . and perhaps metal b 血 dhlg a畄血 隊Wn tninoClr by eva ]し 服t 血 呂 tlle therlll り dynall 血 s 鳳 b丗 prt , iSely wi 匸 hdifferAnrinl scannlng caTol ・ e婚 ・ ΦSC ) 、 The pair ・f・ the・muta 七 i ・n sites t @be displaced b}・ cyste 血were deロerrnitied using the following し hT [te criteria:ωthe atio 【 1 sites were gely記 P 曲 曲 ng 曲 e a o .acid sequenee,伽ie slde で hai Qf the lnu 馳 tlon鵬 were es 血 na 匸 edto be n巳 ar enonghbo rnake a disulfide } x ) nd in thon 釦v @three .dimensional5 c , 皿 d (3 )也e side chain of 匸 he 5 」陀 3 were eyms め しh w 且 ven 匸 、 皿 Ld 圃 yAl1C 脚 C. mutant ・ was seleabEd as the most hopeft 匸 1 血 血 加 . 「 11he m 曲 t gen 曲 ytwu ・ step PCR 輔 th 山 e" 訌 dt } pe ten 叩 late con 匸洫 血 コg HLg. 匸 agwas hユt u d m E .coli hoc . t .BL21 D 〕Piodirce 出 e mutMlt . Tho cxpresgsed Plvtoins Viere on dennttiTed , p囲且ed byNl ・MAcoh ,曲 1dedb 魍 畑舳 m 曲 . sdep diilution ofthe rl aturant , mid finnllypr e5sedb } . TEV pmtease tQ remσ vethe 且 is . 巴g . Ftum the C @m 匸 Llsし 肥ment , 七 he mu 匸 was found 加 have 出e SHIIIe t 』 dnllensianal st Iculle a @that the、 祀d type. Thetransitiontemperature evalua 匸 edbyDSC at pH3 . 5wash均 r 亡 han 血 a 亡of he 輌 d甲 pe mDTe than 20K . 2PO70 on anial } asiS . A且 though i匸has beenthgg し unedthat th治 phenots1 , e is caし 罷 ed by stmctur ins 亡 ab 趾貯derived 丘 τ , m mutation , itsde 1 a 皿 迥 } asiS su 〔 in as a therma ⊥ n8i 恥 n analy3is ofpur 血 ed mutant proteins, has ne 、 erbean 面 ne. Wb 〔 rmi { 三 d ⊂ 〕 Llt such ana ]yslg . FlhAC ,血e C船 rm血温 domain of a proteincomponen しoftle flmgellur prub 曲 n巳卿江 apPar 肌 s. Sa]mone ]la fulgell し 翻al comp19x Bdi 廟 ded into 3 parts :the basa ユ bod } 」 廿 1e hua C and the 田 e11 しAU the fiagelLar aXiaT 騨脱 h職 mヨ tr3n51 嘸 樋 d 励D the centr d dof 宙 efL 昭 e 且 し皿 for . lelf・assembly 趾 , te 血 kcat1Dn 給medLatrri by the fiagellar pro し e export apparatus , whi ¢h捻heheved D∂bel a 陀don 匸he cyヒ oplas 【 /side of the basa1 body. is ex1 ))rt appara 亡 −' and plvaTmaeeutiaal hldし 一 t . ries 、 We show thati s・cy he 幽1血e e踊 vdy pre 鴨 n陪出ε鯏 c 伽 a 面11 nf ribonuclekge A 〔 RNase ム) andhenegglysozym D At neutral p 且〔 〈 8 ),heaレ 血duc 巳 diattivati エ m 【 fRNaseA dl } mzymg wa 南 mp じ . oved by the ad〔htiou of trans ・1.2 一 酬 ohexane し五a 血 e (12eHDA ) better than trans . 1, 4 ℃ ydohe ャ皿ed 蝕 m血e工 14CHDA ) in su [LLtiorL wh丑e aじ al hne pH 〔> 8 ), the immiviition of RNaseA an @lysoz }me was improved mo ハ∋effective且 y by 14CHDAth 日 皿 12CI {DA . Thc resu [ts lndicate atthe aggregation ot 」 RNaseAandlysozyme was a 且」 bortedby the e ]ev匸 rostutic repu 踊 由n the presenre of 廿ans . cyclohexanediamlne mo ] eculeE 、 Moreov 鱗hydroph面Lc a 皿o aeid @LoUld be diss ⊂ , lved betber in 12CHDAsoLution than hl14CHDAsロ且ution . 「出e re5ult5 di じate口1at 12C.II )AsLrongl }・interaets with hydrophobicre 虹 dues ,1eading 匸 op皿・ entag 即g リon ot ' lltNaseAand ] y5Dz } . τ ne at . neuLral pHs . IricreaEe 血the repUltlive 飼rce b 巳 tsl ・