Ko te tikanga whakakore tika he take nui mo te angitu o nga whakamatautau peptide. Ko te whakakore tika ka ngaro nga peptides me te kore o te whakamatautau. Heoi ano, Ko te kimi i te tikanga wetewete pai i etahi wa he tino uaua. Ko enei e whai ake nei nga tikanga whakakore i whakaarohia e nga roopu hangarau e rua o nga Huringa MOL. tikanga
MOL Huri Hangarau Aratohu Whakarewa Rōpū
Ko te raruraru nui i roto i te whakakorenga peptide ko te hanganga o te hanganga tuarua.
Ahakoa ko te hanganga o nga hanganga tuarua o nga mekameka peptide hydrophobic he tino kitea, engari mo nga mekameka peptide poto rawa atu, puta tenei āhuatanga i roto i tata katoa mekameka peptide, ahakoa te polarity. No reira, Ko te maataapono tuatahi mo te whakakore i nga polypeptides ko te whakamahi i te wai parakore, te wai Deionized ranei (he wai kore hāora ranei mena ka whakaaetia e nga ahuatanga).
Ko te otinga peptide ka pa ki te whakahekenga huakita. Hei karo i tenei ahuatanga, kia rewa te peptide i roto i te wai maturu horomata, ka tātarihia ki te kiriuhi tātari me te rahi pore o 0.45 ranei 0.2 μm mo te whakahoromata. Polypeptides kei roto Cys, I tutaki, me te Trp he tino whakaraerae ki te waikura, no reira me rewa ki roto i te wai kore-hauora. Ka taea te whiwhi wai kore-hauora ma te whakaheke hau i raro i te pehanga iti ma te wero i nga hau koretake (hauota, helium, me te argon).
Ki te he insoluble te peptide i roto i te wai parakore, Ka taea e te maimoatanga ultrasonic te awhina i te pakaru i nga matūriki me te whakanui ake i te whakarewatanga. Tuhipoka: Ko te maimoatanga Ultrasonic ka whakamahana i te otinga me te paheketanga o te peptide.
Mena he maha nga waikawa amino taketake kei roto i te peptide, whakamahi 1~10% wairewa wai waikawa acetic; mo nga peptides tino hydrophobic, whakamahi 50% waikawa ahetiki.
Mena kei roto i te polypeptide te nui o nga waikawa amino waikawa, ka taea te rewa i roto i te a 1 ki 10% he wairewa haukini, he parepare kawakore ranei penei i te ethyl morphine acetate, bicarbonate ranei. Me whakatika te uara pH i mua i te chromatography.
Mena ka utaina te peptide ki runga i te pou, me iti rawa te nui o te whakarewa pararopi, ki te kore ka tino pa te wa pupuri.
Mena he tino hydrophobic te polypeptide na te mea kei roto i nga mekameka taha waiwaro kakara penei i a Val, Leu, I tutaki, Phe, Tiro, Ala, etc., he peptide kūpapa rānei, ka taea e te whakamahi i nga denaturants membrane penei i te DMF me te DMSO te awhina ki te whakakore i te polypeptide:
a. Ka awhina nga denaturants membrane teitei ki te whakakore i te polypeptide ma te whakangaro i tana hanganga tuarua.
b. Ko nga denaturants membrane e tika ana mo te whakarite i nga otinga tātaritanga peptide, engari ka raru pea te rangahau mo o raatau mahi koiora.
c. Ko DMF te denaturant pai (te kukū teitei ka taea 30%), a ka tapirihia kia maturuturu kia rewa te peptide.
d. I te wa whakamuri-wahanga chromatography, Ka rere tahi te DMF me te tihi o mua o te eluent. I runga i te rahinga werohanga, ka nui pea te uara teitei. Ka taea e te nuinga o nga peptides te whakaheke i roto i etahi meneti i muri i te nui o te DMF elutes. Mēnā he iti te mekameka peptide, ā, moata rawa ka puta, ka heke te nui o te peptide.
MOL Huringa Hangarau Roopu II Aratohu Whakarewa
Me pehea te wehewehe mena he koretake te waikawa amino, waikawa, kawakore ranei, me te tatau i te tikanga whakakore:
1. Whakaritea ia waikawa amino waikawa i roto i te raupapa polypeptide tae atu ki te waikawa aspartic Asp (D), waikawa glutamic Glu (E) me te C-terminal carboxyl-COOH rite -1; ia waikawa amino taketake tae atu ki te arginine Arg ( R), raihine Lys (K), histidine Tona (H) me te N-terminal amino-NH2 kua tohua he uara o +1; he waikawa amino kūpapa 0. I runga i tenei, ka tatauhia te uara o te polypeptide katoa, a he pai te kaute Ko nga Peptides me te kaute kino ka kiia ko nga peptides taketake, ko te hunga he tohu kino ka kiia ko te peptides waikawa, a ko te hunga whai kaute kore ka kiia he peptides kore.
2. Mena he peptide kawakore, ngana ki te memeha ki te wai i te tuatahi; ki te kore e taea te rewa, ngana ki te whakamahi 10% – 30% waikawa ahetiki; ki te kore tonu e memeha te peptide, ngana ki te whakamahi i te waikawa acetic parakore me te waikawa trifluoroacetic TFA (<50 μl) ki te memeha, katahi ka whakahiatotia te otinga peptide ki te kukū e hiahiatia ana.
3. Mena he peptide waikawa, ngana ki te memeha ki te wai i te tuatahi; ki te kore e taea te rewa ki te wai, ngana ki te whakamahi 13% wai haukini (v/v) ki te memeha, me te waimeha i te otinga peptide ki te kukū e hiahiatia ana. Mena kei roto i te raupapa peptide he cysteine Cys (C), e kore e taea te rewa ki te wairewa kawakore. Tena koa ngana nga tikanga i raro nei.
4. Ko nga peptides kupapa kei te nuinga o te waa ka memeha i roto i nga whakarewa pararopi. Tuatahi, ngana ki te whakamahi acetonitrile (acetonitrile), mewaro ranei (mewaro) isopropanol ranei (isopropanol) ki te memeha; mo nga peptides tino hydrophobic, tuatahi memeha ki te iti o te dimethyl sulfoxide (DMSO) me te waimeha ki te wai ki te kukū e hiahiatia ana; mehemea kei roto i te raupapa peptide he cysteine Cys (C), whakamahia te dimethylformamide (DMF) N-methylpyrrolidone ranei (NMP) mo te whakakorenga. Mo nga peptides e kaha ana ki te whakahiato, tuatahi tāpiri 6M guanidine·HCl 8M Urea rānei (Urea) ki te whakakore i a ratou, a ka waimeha ki te kukū e hiahiatia ana.
whakaahua:
1. Kia pai ai te rokiroki me te whakamahi i muri mai, e tūtohu ana ki te tareka i te peptide ki te kukū e pā ana ki 1-2 mg/ml.
2. Ka taea te rongoa i te paura maroke peptide noa ki te -20°C neke atu i te kotahi tau. Mo nga peptides memeha, e tūtohu ana ki te aliquot me te rokiroki i -20°C; mehemea kei roto i te raupapa peptide te methionine Met (M), Cys Acid Cystine (C) ranei tyrosine Tyr (Y) e taunaki ana kia penapena ki roto i te taiao kore-hauora hei aukati i te waikura.
Rarangi waikawa amino kua taapiri:
1.waikawa amino kūpapa: glycine, aranina, riihi, isoleucine, valine, hauteine, methionine, toronine, serine, phenylalanine, tirohina, te waikawa tryptophan, proline, asparagine me te glutamine
Fnga kai: Ko tenei momo ngota ngota waikawa amino kotahi anake te roopu amino me tetahi roopu carboxyl
2.waikawa amino waikawa: waikawa glutamic, waikawa aspartic
Ngā āhuatanga: Ko tenei momo ngota ngota waikawa amino he roopu amino kotahi me nga roopu carboxyl e rua
3.Nga waikawa amino taketake: raihine, arginine, histidine
Ngā āhuatanga: Kei roto i tenei momo waikawa amino he roopu diaminocarboxyl kei roto i tana ngota; He mowhiti hauota te histidine, he ngoikore te kawakore, he waikawa amino taketake ano hoki.
