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1、技术与方法 T B k Z E *刘福英null 宋淑霞null 郑null 龙null 张焕铃null 尤红煜null 王俊霞* *( D S v L null F E null 050017)null null * 8 S / O * * Y T null Te:l 0311null86266147, Enullmai:l wangjunx62 163null comg : 2005null10null28, : 2006null01null18K 1 : T B ak , Z E ! A v s , T E V ) Lnull183 ! A B T ( SA ), k , SA l q
2、75% 85%bk V , 1 SA s 0 74null5kD, s 0 SAV 3null2 3 s 0 , 11null2U /mg, pI 7null4b1 SA 3 D M b1 o M : T , B k , T m s | : Q814null null D S M : Anull null c I | : 0253null2654 ( 2006) 05null0112null05Study on Purification and Identification of Streptavidin*LIU FunullYingnull SONG ShunullXianull ZHENG
3、 Longnull ZHANGHuannullLingYOU HongnullYunull WANG JunnullXia* *(D epartment of Laboratory Animal, H ebeiM edical University, Shijiazhuang 050017)Abstract: Theaim of this research is to refine the protocolofpurification ofSA and identify the character ofSAnullBy utilizing the coldnulldenaturingmetho
4、d, most ofother kinds of protein were screened out and SA was purifiedfrom the fermentation broth ofLnull183 byusing the refined affinity chromatographymethodnull The rateofrecollectionwaschecked to be75% 85% null By identification, it is indicated that themolecularweightof selfnullmade SA was74null
5、5kD, the biotinnullcombiningnumber 3null2, the activity11null2u /mg, thepIaround 7null4null So, the essentialcharacnullters ofSA are same asdescribed by documentsnullKey words: Streptavidin, Purification and identification, Affinity chromatography T ( Streptavidin, SA ) ) ( Streptomyces avid inii) A
6、TCC27419 3 V s B b SA c # 3 T ,y N SAX null V F E u r s B SAs ) , Lnull1831b SA 3 k # s B Z E b 3 ak SA $ b1null Z E1null1null ) : ) Lnull183 ( i i ), SA ( i 1 ), S T 3 null112null 3 Y null null null null null null null null null null null null 2006M 33 ( 5)(Promega ), 2null 3 j 4B ( Sigma ), 3 V (
7、S 3 )b1null2null Z E1null2null1null SA s B : ? ! : v ) Z E 2b? , | ! A4 , 5 000g 15m in, l bb ! A ) : | 1L b, _ F ; 80% , 4 2h , 4 ,10 000g 15min, b, l b_ F 100mL , s ( , null20 b48h | , i , 4 , 10 000g 15min, l b, 6mol/L_ ) pH 11bl , bT : P pH 11 0null05mol/L ) A | 2null 3 j 4BT , g , | B 8 ) b , 3
8、7 5m inbu 7 g , 5 0null05mol/L ) A ! , ! A OD280 0null05 / , pH 4 0null1mol/LZ ) A , l ! A b i : | l ! A X ) S 6 000 8 000 i , i F Y = ? 6000i 8 1/10, i 24h, W 5 6Q , V _ 3b1null2null2null SA k : i : SAE282nm1cm = 3null4L/g4, | 20nullL1 SA A d 3mL, 752, ; E s ; ; 9 282nm A , 9 SAi bB k : T ! A l w L
9、 SDSnulld bs 0 : SDSnulld b M k : K 5, 6, H q b : OD233nm, ; E , _ 1 F 1mg/mL1 SA 2null5mL, null , , Y , null D S v , 2001, 22 (5): 257 260null 2 ) , g + , b , null D S v , 2002, 23 (1): 4 6null 3 I V , _ , null D v , 1995, 26 (1): 113 115null 4 I V , null r , _ null D v , 1995, 26 (4): 436 438null
10、5 Bayer E A, BennullHurH, W ilchek Mnull M ethods in Emzymologg, 1990, 184: 8 89null 6 Bayer E A, BennullHurH, H illerY, et alnull J Biochem, 1989, 259: 369 376null 7 ChaietL, W olfF Jnull Arch Biochem Biophy, 1964, 106: 1 5null 8 o , null , P , null Z f , 1993, 13 (5): 303 306null 9 Diamandis E P, ChristopoulosT Knull C lin Chem, 1991, 37: 625 636null 10 Bayer E A,
