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`SIS 10-2016-0108260
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`AAAS 22] ofF] eA AMS 72 ABS TAT BMeEpol =r} ASLA +O] 2} (epidermal growth factor, EGF)¢l
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`SISA 10-2016-0108260
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`TA] Cc);
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`(2008).The skin:an indispensable barrier. Exp Dermatol. 17(12):1063-72].
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`

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`SISA 10-2016-0108260
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`tr},
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`ct al. Expcrimental Neurology
`188(€2004)
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`
`al., Neuroscience letters 421(2007) 110-114 @]. 4, AZ=EW°) Se] s4] Sl BAA] BAyAassenel
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`2 22 ASWS 22] oe ASS AAG ASS TAT PeEbol=E7, AAA e914epidermal growth
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`
`ASHS 29] HMA ASS 74 HSS TAT WEE} EEA BACThymosinB4, TB4)ol et
`of BE ASS TAT MEE SSH BABA BAAS WSs}.
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`ea ne,Node or
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`#2 oye
`hGH)¢] $23 AS 34°98 SE ASS TAT PESOS SS HGH BMAS Asa.
`
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`
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`factor, EGF] @eslo] Baad AS AT Mepo]= Se AGF GAS BesH= 7S Sdeowe se age
`
`ANSE ASAT. OM, 87) oe “29Se UG dS, WE Se AAS SE BAP ws} YAS SE BP
`AVS] AW - Meo. EGFE BE Se 7A, WA ws Ye), BW Age] Bah} Me zAewe VaA 37]
`W-°]C}+ (Fallon JH, Seroogy KB, Loughlin SE, Morrison RS, Bradshaw RA, Knaver DJ, Cunningham DD (June
`
`

`

`BIS 10-2016-0108260
`
`immunorcactive matcrial
`"Epidermal growth factor
`1984).
`development". Science 224 (4653): 11079).
`
`in the central nervous system:
`
`location and
`
`2 WS AUUE 29) obese MAS elt WB TAT ete] S7} ENALB ACThymosinBa, TBABe
` mi
`SJ) B4Q AVY TT eps Se YRABs PMAS BPE AS SAOR Fe IE AYLS ay
`ach. oli, 27] OR SASS, A AS, a ns} W]g BE spt Was Be waded ad
`2 UT. YMAWP RB BI, WP PY, BMYQH Ao] Asp} Ye Rom Tey gly] wee]ch
`(Crockford D, Turjman N, Allan C, Angel
`J
`(April 2010).
`"Thymosin beta4:
`structure,
`function,
`and
`biological properties supporting current
`and future clinical applications". Ann. N. Y. Acad. Sci.
`1194: 17989).
`
`Howe ASRS 22] oe] e4 ASS WAS NES TAT PEolE7} 2121-9422 ES (human growth hormon,
`hGHSo} SYS FY AT MES Sey hGH WYAS MAME AS Soe Be as ayes
`BAY, old, Q7 BQe AYBS,
`UY oS, HL FS AAR BE HA Vs YS BE ge Wage
`AQ - Qt}. hGH= WA ee 7A, BY Ss} 2], WBAad B77} Me AO VA By] Waro|t+
`(Hyun-Joo Cho etc (2007),
`"Analysis of
`the Effects of
`hGH Using Living Skin Equivalents". Tissue
`
`Engineering and Regenerative Medicine, Vol. 4, No. 3, pp 406-410)
`
`ofMpesk MAS Mae MPS TAT MEpo|L7} AYP
`# UGS AAUR 29]
`(epidermal growth
`
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`factor, EGF)@asjo] Aaa ARYA TAT Be}o]= Le AGF GYAS dsss Haas
`ASH MS ABE Gal (a); Avis] ASI Mes Use Edsel yawe Padagesa As
`Fe dges ARIS Ga (b): Ae) ABW GATS ws H, PRY WaT Ba
`BE a gosye
`ABS TAT Wepole Set RGF GAS Bese BA (c)) S VHS AS Soe se WHS TAT We}
`JE GH AF PA] Yap Vs asa. Pe wr PSs SHAA VS,
`chases car yeas
`roa
`ys1g WoO YT + Bee] sy] B wes] AMS Fay gely
`SHA 7S BP Ble TAT § 3} EGF Ey
`ole.
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`ai“
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`Bowes yaa 2 Aas 22] oe e424 AAS 74S ASS TAT MepolE7} AS41901] 4} Cepiderma
`growth factor, EGF)] @82459 S493 +5 4a Bowes TAT Mepole @S EGF GBUNAS Tespse 7A
`AS Age] Hs Aza @OQS AZS BA (a); A719] ABA AOS Vato] EMs
`
`AAA VLE AAW YAts AASE GA Oh): B27] ASH T4aws "Wea |S
`
`EE 2 YeoSsSy yA 2 ASS TAT PO= 8S EG PIAS BASS BA (e); 7-7] 1] 4
`
`a]et +e) WS TAT Webole BE EGF GASES HAAS MASE G4 D'S BPE A
`om,
`She Wes TAT BEbol= BS Lar GDMss] YsAyyes asac. HyA_ase S4rwas] ws
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`TAGS Aa] aasz So Uh AaaArS
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`itin Specific Protease, USP)O] Bay AGO AA.
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`32,
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`% 3S a1, to
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`ey-xoe & a& ae
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`BASS AT MS Se eA PARYY HAUS AAS GA (Hit BWAg Hom 3
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`i: APA UT Mepl=S SF TBa PAA] APHIS ABM. Hygade Bqugag wae osu
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`(a); A719) AG WHYS dato] EVsdats BG4AassA2eHenm Aes Ws
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`-o]] 4) 3 TAT 4} hGH UMASS you Ws — Yeo) s7) HB Bee] ABS Sa Slsaect.
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`2 292 +A 2 ASS 29)eA ASS AAS MSS TAT BEPOE7} WesS=E (human
`growth hormon, hGH)¢] so 24a+a 2 ASS TAT Bebol= Ss HGH BAAS csbshse FA
`Ae HWE ASsho] ALY WAYS aAsoh= WA) (a); A719] ALS AHS W4arta Edad dats @
`AAA Zoey ABW Wats ARSE BA (hb): BW?) ABB DAS joes SF, PRB Aaaay
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`(co); BIA #
`UTS 2 WySFH OSILE, H-HSEL WAS TAT BEFO|S $s} HGH MAS Bess Bal
`let uaeS TAT BEfOlS BS hGH UPYASSEE THABS AAS GA (dS ZHse AS
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`SQls se WES TAT MeE=S BH bhGY Al] AIGA! Aeacy. HyAae S4awae] Pas
`SHEE PAPE 244 ge aMadd, # Bs Sas HES] HH Aes + We Aol. B
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`Qh
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`Boe) =BZeobk] WUbiquitin Specific Protease, USP] Bay] 2459 aA.
`
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`= BgolAs} Zo], ASS ATS SBSHA714, cf ae] TATS BSA 7le Ael Fla] EGFCepidermal growth
`factor), E]J@4lB4(ThymosinB4, TB4),
`hGHChuman growth hormon, hGH)S Woes WArse |= gic}. tes,
`Buoys So qe Aste 7S TAT SS} EGF, E]RAB4, hGHE 345 Go] Ar7tso] ASS S| ach.
`
`m1 64a 6S] Gal £.coli AN LA ALS Bel Axrpolcy.
`
`[0027]
`
`(0028)
`
`[0030]
`
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`EB 2 Sada 25 Na +74 ae | BSA ASS Sele Axwpol-} (SL jar fermenter scale).
`
`= 3& H6Ub-TAT-TB4 <8 ae] SP FF QA] Axo] c},
`
`& 4% H6Ub-TAL"-1B4 GNA S)
`
`Ei 542 IIGUb-TAT-hGll
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`Ni FF B SP FF Qa) Aypojry,
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`SP FF | Azole},
`
`% 6& H6Ub-TAT'-hGH Ni FF 4a] Axz}o)c}
`
`
`
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`% 10 USAl(Cinclusion body) Ae BAP BBY AGUb-TAT'-EGFS] ea (Refolding) =, SP FF B Ni
`
`
`
`% 72: H6Ub-TAT-hGH SP FF 44] Axpolc} (1°)
`
`& 8 HGUb-TAL"-hGH SP FF 4a) 2 s}o]5} (2)
`
`T2942 JIGUb-TAT'-hGIl
`
`SP FF 4a Azpolc} (3°)
`
`
`
`
`
`
`
`

`

`SIHSA 10-2016-0108260
`
`FF Qa) Axpolrp.
`
`= 12 W=AlCinclusion body) BAS Yay BSW HOU_-TAT’-EGE BIAS] 2 pH SAMNAS] BSQ B
`7 zi] spo] Ly,
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`
`% 12] W¥AlCinclusion body) @4Yz 2AM 284) TAT-EGFS] AA) HAS BYE SSE].
`
`4% 132 WXAl(inclusion body) BAe PAS BAY TAT-EGFS] Q EFS OBS Qa Axpoyyy.
`
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`A) 22] TAT 4-214) ALB 2S SAB PCR S= (MJ MiniTM from Bio-RAD, USA)S co] Bay Bes)acy.
`
`EGF€ Bell GI, Fong NM, Stempien MM, Wormsted MA, Caput D, Ku LL, Urdea MS, Rall LB,Sanchez-Pescador R.
`Human epidermal growth factor precursor:
`cDNA sequence,expression in vitro and gene organization.
`
`1986 Nov11;14(21):8427-46.), TB4(Yang SP, Lee HJ, Su Y. Molecular cloning and
`Nucleic Acids Res.
`tructural
`characterization
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`functional
`human
`thymosin
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`Biochem.
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`umangrowth hormone structural gene. Nucleic Acids Res. 1979 Sep 25;7(2):305-20.)= #AHS Xs] SB
`a rast AAS ASA, G4 S4U4ae Tarte SSS Hes] AS SYTIEVH HOUb AYA
`S ARS SBr}. HU GPYAAL BYARQG 67 H6)7+ HAA Ubiqitin) P44] SH5¢ VE aq
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`(Ubiquitin Specific Protease, USP)Seo] SUSAAeic}. 2 AZo] Ab
`25] EGF, TB4, hGHS) ofe]+4t AAS ASMHS 3, 4, 50] ZZ} vlASstc. SSH, BB AAA Apert
`ejay 2: Aa]4) 2° CSaccharomyces cerevisiae) tr tH] US AASSRE, oS ASS Cl 7) AWsArt
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`BF
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`NS TATE TAT HS +R’ BAlSsbrl = ate}.
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`15849.9 13|H6Ub-TAT-hGH (+ R protein)
`
`|Name Dal ton)
`[0041]
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`l1_|TAT-RGF(4+_R_ protein) 7920.0
`12s|HOUb-TAT-TB4 (+ R protein)
`33057. 4
`7763.8
`TAT-EGE
`15693.7
`H6Ub-TAT-T
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`16|H6Ub—TAT-hGH 32901.3 8
`H6Ub-TAT-EGF(+R_ protein)
`17150, 45
`4
`8.91
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`SIS 10-2016-0108260
`
`737) 13} BH Haz MAS Ae DNA SAS FE 2 FIA “ebo]AS co] Bs 2144 PCRS Sal GU,
`©]= pAPT (APTech's expression vector)o] @@s4 PSHyS assAry.
`
`
`
`product
`
`Primer
`F-TAT-EGF : CGT AAA AAA CGT CGT CAG CGT CGT CGT AAT AGT GAC TCT GAA
`
`[0043]
`
`[0044]
`
`R-BamHI-EGF : CAG CCG GAT CCT CAG CGC AGT TCC CAC CAC
`(+ R protein)
`
`H6Ub-TAT-TB49(+]F : AGA TAT ACA TAT GCA TCA TCA TCA TCA TCA TCA GAT TIT CGT CAA
`R protein)
`:
`ACG ACG CTG ACG ACG ACG TIT TIT ACG
`: CGT CAG OGT CGT CGT TCT GAC AAA CCC GAT
`:_ TCA GCC GGA TCC TTA CGA TTC GCC TGC TTG
`: AGA TAT ACA TAT GCA TCA TCA TCA TCA TCA TCA GAT TIT CGT CAA
`ACG ACG CTG ACG ACG ACG TIT TIT ACG
`> CGT CAG CGT CGT CGT TTT CCA ACC ATT CCA
`: GIT AGC AGC CGG ATC CTT AAA AAC CAC AAG AAC CTT CAA CAG AAC G
`: AGA TAT ACA TAT GTA CGG TCG TAA AAA ACG
`F-TAT-NdeI
`R-BamHI-EGF = CAG CCG GAT CCT CAG CGC AGT TCC CAC CAC
`
`H6Ub-TAT-hGH (+ RJF
`protein)
`
`:
`
`
`
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`TAT-EGF
`
`H6Ub-TAT-TB 4
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`H6Ub-TAT—-hGH
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`H6Ub-TAT-EGF (+R
`protein)
`
`;
`
`: AGA TAT ACA TAT GCA TCA TCA TCA TCA TCA TCA GAT TIT CCT CAA
`F-H6Ub-NdeI
`: ACG ACG ACG CTG ACG ACG TIT TTT ACG ACC GTA
`TATCori)
`TAT-TB4 : CGT CAG CGT CGT CGT TCT GAC AAA CCC GAT
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`+ TCA GCC GGA TCC TTA CGA TTC GCC TGC TTG
`: AGA TAT ACA TAT GCA TCA TCA TCA TCA TCA TCA GAT TTT CGT CAA
`> ACG ACG ACG CTG ACG ACG TIT TIT ACG ACC GTA
`> CGT CAG CGT CGT CGT TIT CCA ACC ATT CCA
`: GIT AGC AGC OGG ATC CTT AAA AAC CAC AAG AAC CTT CAA CAG AAC G
`AOG ACG CTG ACG ACG ACG TIT TTT ACG
`:
`TAT-EGF : CGT AAA AAA CGT CGT CAG CGT CGT CGT AAT AGT GAC TCT GAA
`F-H6Ub-Ndel: AGA TAT ACA TAT GCA TCA TCA TCA TCA TCA TCA GAT TIT CGT CAA
`R-BamHI-EGF =: CAG CCG GAT CCT CAG CGC AGT TCC CAC CAC
`
`[0046]
`
`7 YA Ae BAAS F.coli MC1061(P-A (ara-leu)7697 [araD1391B/r A(codB-lacI)3 galK16 galE15 A
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`Sole =, Faz] S7IASS BASU} (cosmogenetech, Korea).
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`s7]) ARIS + S29] SAASS AWAY E.coli Tol
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`wee] Hs Yet w244== LB vA)
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`from Duksan, Korea)=S S|] 23GOA, MAUASZS Sadao) 74+ek] 410100 ve/ml)S ARS SSAC.
`Hae GDMAS BAS7] Hspeo] 4~12% TUYc|AE A (gradient gel, KOMA Biotech, Korea)°]-26}Sic}.
`
`AQAayy = le] epics.
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`-Q-
`
`

`

`10-2016-0108260
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`SISA 10-2016-0108260
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`H6Ub-TAT'-T84, H6Ub-TAT'-hGH Y4b2 5L jar BAZ) eo] BSAA, HGUb-TAT'-TB4 AMALOD. coom 60 U2]
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`(1) H6Ub-TAT'-TB4 UYAe] Be] BZ Aal
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`Av] 2l@]& all H6Ub-TAT'-TB4 2 H6Ub-TAT’-hGH7} =24 Sqe Baws Bele |= glock, saad eg
`2) base 2494 ade Pea + Wy) qed eade vs 2S SadM= versa, AS y ye =
`244 GYaePhe Besta AQalaor Se see Geese HASZSo] Ac. BAITS lS Besa
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`Ay7] AAlal 214) 773) webs BSS] HOUb-TAT’-TB4 GAAS) Be], Aas Mote]
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`30mM Sodium phosphate, 0.5% Trilon X-100,
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`12g 2] wWalc(wet
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`
`2-9 AM Saedel Beas Braga. Brg 4
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`Seto] 21) 7]°] ] (SONOSMASHER from UL SSO HI“TECH, Korea) Aeo4 was sale =, AAs
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`taf
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`'B buffer'S Al-Ss}9 Bes #, SNS-PAGER BASS
`
`<AlA] a>
`
`- Column : SP Sepharose FF, 10ml
`
`(HiTrap)
`
`- Loading sample: 4 3}4]-= -d-sel, 60ml
`
`- A Buffer
`
`B Buffer
`
`:
`
`:
`
`30mM NaPi, pH 7.6
`
`30mM NaPi,
`
`1M NaCl, pH 7.6
`
`- Grandient Elution : 0~60%B, 6CV
`
`— Elution Fraction : F4, 40ml
`
`(Conductivity 52ms/cm)
`
`-11-
`
`

`

`[0076]
`
`Al@ Az}, 0.5~6M NaClel]A] HEUb-TAT'-TB4 UHlaap Be Bulge] RSG2 Selsar} (= 3).
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`[0078]
`
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`aa, 4 dds] ee AAS Met SP FF ABA, SBS fraction 48 UBPS 2+ %, Ni FFS o]-8-3}
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`o] TAT'-TB4 UWA ‘flow through’ WRBPsAce. ols, SP PPS ol Sse #74 AAS TBA
`
`SISA 10-2016-0108260
`
`[0079]
`
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`[0082]
`
`[0083]
`
`[0084]
`
`[0085]
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`OOR6}
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`
`<4 7] 2 a>
`
`- Column : SP Sepharose FF
`
`- Loading sample: Ni FF sample, 30ml + A buffer 30 ml
`
`- A Buffer
`
`B Buffer
`
`:
`
`:
`
`30mM NaPi, pH 7.6
`
`30mM NaPi,
`
`1M NaCl, pH 7.6
`
`- Wash : 0~60%B 12CV, and then 70%B buffer
`
`- Grandient Elution :
`
`100%B
`
`Elution Fraction : F3, 7.5ml, 0.3mg/ml: 2.3mg
`
`0088}
`
`AQaAy, ee Se TAT-TB4 GMAS Best += WMO, TAT-TB4 AMAL 0.2 mg / g (wet cell) 9
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`5SmM
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`'A buffer'S Sa#a, NaCl= &e+o4 SDS-PAGES HASACH.
`
`<Aal)>
`
`— Column : SP Sepharose FF, 10ml
`
`- Loading sample: # S}3]
`
`-F, 4heod
`
`- A Buffer
`
`B Buffer
`
`:
`
`:
`
`30mM NaPi, pll 7.6
`
`30mM NaPi,
`
`1M NaCl, pH 7.6
`
`- Grandient Elution : 0~50%B, 6CV
`
`Elution Fraction : F4, 25ml
`
`fO102]
`
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`
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`
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`
`-12-
`
`

`

`SISA 10-2016-0108260
`
`AAA] OM US Cimidazolc)= SS}a1, SDS-PAGES. 44S} Sit}.
`
`<Qal A>
`
`- Colum : Ni” SP Sepharose FF
`
`- Loading sample: SP FF elution F4 + UBP Img + A buffer 15ml
`
`— A Buffer
`
`B Buffer
`
`:
`
`:
`
`20mM Tris, pll 7.5
`
`20mM Tris, 0.5M Imidazole, pll 7.3
`
`- Grandient Elution : 0~50%B & 100%B
`
`[0105]
`
`[0106]
`
`[0107]
`
`[0108]
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`
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`
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`
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`
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`
`(0125)
`
`[0126]
`
`[0127]
`
`[0128]
`
`[0129]
`
`(0130)
`
`- Column : SP Sepharose FF, 10ml
`
`(HiTrap)
`
`- Loading sample: # 3}2]-@ -d-eo4
`
`- A Buffer
`
`B Buffer
`
`— Method
`
`:
`
`:
`
`30mM NaPi, pH 7.6
`
`30mM NaPi,
`
`1M NaCl, pH 7.6
`
`
`
`
`
`1. Column Equilibration CA buffer)
`
`2. wading Cloding sample 80ml1 )
`
`3. Column Wash (0~40%B)
`
`4.5 — ution : 55%B
`
`- Elution Fraction : F4, 46ml
`
`{0132}
`
`SDS-PAGE el As},
`
`55%B NaCloq II6Ub-TAT-hGll Yo] SSEAS SBels-Arp(% 8). olf,
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`1%} SP FF
`
`ol] 231 HOUb-TAT-hGHe] AGHA UPS Ase] geayds age Aayaqu, ad ayae sp
`
`-138-
`
`

`

`FF AWS ABse] Bales ASS.
`
`<A) zi] 42>
`
`SIHSA 10-2016-0108260
`
`- Column : SP Scpharose FF, 10ml
`
`(HiTrap)
`
`
`
`
`
`3. Column Wash (40%B)
`
`- Loading sample: LD SP FF elution + A buffer(pH6.1) + 0.6mg UBP
`
`- A Buffer
`
`B Buffer
`
`:
`
`:
`
`30mM NaPi, pll 7.6
`
`30mM NaPi,
`
`1M NaCl, pll 7.6
`
`— Method
`
`1. Column Equilibration (A buffer)
`
`2. Loading (loding sample 80m1)
`
`4. Elution : 40~80%B,
`
`100%B
`
`- Elution Fraction : F4, 10.5ml, 0.38g/L: 3.5mg
`
`[0133]
`
`[0134]
`
`[0135]
`
`[0136]
`
`(0137)
`
`(0138)
`
`f0139]
`
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`

`SIHS 10-2016-0108260
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`- 23 -
`
`

`

`SIS 10-2016-0108260
`
`Glu Ser
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`