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鏈霉親和素可以提高融合率的原因
2019-11-15 05:13
鏈霉親和素(streptavidin下稱(chēng)SA)是與親和素(svidin下稱(chēng)AV)有相似生物學(xué)特性的一種蛋白質(zhì)。
鏈霉親和素分子由4條相同的肽鏈組成，其氨基酸組成中，甘氨酸和丙氨酸的含量較大，而且結(jié)合生物素的活性基團(tuán)也是肽鏈中的色氨酸殘基；鏈霉親和素是一種稍偏酸性（pH6.0）的蛋白質(zhì)，并且不帶任何糖基。在蛋白水解酶作用下，鏈霉親和素可在N端10～12和C端19-21間斷裂，形成的核心鏈霉親和素仍然保持完整的結(jié)合生物素的能力，所以可以提高融合率。
鏈霉親和素(SA)是streptomyces avidinii菌的分泌物，其分子量及結(jié)臺(tái)生物素的能力與雞蛋清中的親和素相似，等電點(diǎn)6.0，非特異性結(jié)合遠(yuǎn)比親和素低。
一 、來(lái)源：AV 存在于雞蛋清中，主要通過(guò)離子交換和分子篩方法提純，從1L蛋清中可提取純AV15~25mg。SA 是Streptomyces avicllrdi菌培養(yǎng)過(guò)程中的分泌產(chǎn)物，主要通過(guò)2一亞氨基生物索親和層析法提純，1L培養(yǎng)液中含蛋白1 0~60mg 。
二、分子量 ：一條完整的SA肽鏈中有159個(gè)氨基酸殘基，分子量為16450，而AV 的一條肽鏈其含128個(gè)氨基酸殘基，但分子量也達(dá)15600，兩者十分接近，這是因?yàn)镾A 的氨基酸中，甘、丙氨基酸含量較大，且又不含任何糖基，而AV 的每條肽鏈含4個(gè)甘露糖，3個(gè)氨基葡糖，含糖量達(dá)lO ，故分子量相對(duì)較大。
三、比消光系數(shù)：消光系數(shù)與蛋白中Tyr含量有關(guān)， 由于SA 中Tyr的含量比AV 多，所以SA的比消光系數(shù)也較AV 為高，兩者分別為E 1mg/ml=3．40和E1mg/ml=1.57。在282nm 上這兩種蛋白結(jié)合生物素后并不改變其消光系數(shù)，而在233nm上，它們結(jié)合生物素后郡便其消光系數(shù)值增高，現(xiàn)在一般都是利用蛋白的這種吸光特點(diǎn)來(lái)測(cè)定它們的活性。
Streptavidin, or SA, is a protein with similar biological properties to Avidin Svidin, or AV. The Streptavidin molecule is composed of four identical peptide chains, in which glycine and Alanine are more abundant in the amino acid composition, and the active group of biotin-binding is also the tryptophan residue in the peptide chain; Streptavidin is a slightly acidic (Ph 6.0) protein with no glycosyl groups. Under the action of proteolytic enzymes, Streptavidin can break between N Terminal 10 ~ 12 and C Terminal 19 ~ 21, and the formed core Streptavidin still retains the intact biotin binding capacity, so the fusion rate can be improved. Streptavidin is a secretory product of streptomyces AVIDINII, and its molecular weight and binding biotin ability are similar to those of Avidin in egg white. Its isoelectric point is 6.0, and its nonspecific binding is much lower than Avidin. Source: Av is present in egg white and purified by ion exchange and molecular sieve. AV15 ~ 25mg can be extracted from 1L egg white. Sa was isolated from Streptomyces Avicllrdi and purified by 2-imino affinity chromatography. The protein content of SA was 10 ~ 60 MG IN 1L culture medium. 2. Molecular Weight: A Complete SA peptide chain has 159 amino acid residues with a molecular weight of 16450, whereas a AV peptide chain has 128 amino acid residues with a molecular weight of 15600, which is very similar because SA Amino Acids The content of glycine and Alanine is high, and there is no sugar group. The molecular weight of Av is relatively high because it contains 4 mannose and 3 glucosamine. 3. Specific Extinction Coefficient: the Extinction Coefficient is related to the content of Tyr in protein. Because the content of Tyr in SA is more than that in Av, the specific extinction coefficient of Sa is also higher than that of Av, the two are e 1 mg / ml 3.40 and e 1 mg / ml 1.57 respectively. At 282 nm, the two proteins did not change their extinction coefficient when they combined with Biotin, but at 233 nm, their extinction coefficient increased It is now generally used to determine the protein activity of this light-absorbing characteristics.
Streptavidin, or SA, is a protein with similar biological properties to Avidin Svidin, or AV. The Streptavidin molecule is composed of four identical peptide chains, in which glycine and Alanine are more abundant in the amino acid composition, and the active group of biotin-binding is also the tryptophan residue in the peptide chain; Streptavidin is a slightly acidic (Ph 6.0) protein with no glycosyl groups. Under the action of proteolytic enzymes, Streptavidin can break between N Terminal 10 ~ 12 and C Terminal 19 ~ 21, and the formed core Streptavidin still retains the intact biotin binding capacity, so the fusion rate can be improved. Streptavidin is a secretory product of streptomyces AVIDINII, and its molecular weight and binding biotin ability are similar to those of Avidin in egg white. Its isoelectric point is 6.0, and its nonspecific binding is much lower than Avidin. Source: Av is present in egg white and purified by ion exchange and molecular sieve. AV15 ~ 25mg can be extracted from 1L egg white. Sa was isolated from Streptomyces Avicllrdi and purified by 2-imino affinity chromatography. The protein content of SA was 10 ~ 60 MG IN 1L culture medium. 2. Molecular Weight: A Complete SA peptide chain has 159 amino acid residues with a molecular weight of 16450, whereas a AV peptide chain has 128 amino acid residues with a molecular weight of 15600, which is very similar because SA Amino Acids The content of glycine and Alanine is high, and there is no sugar group. The molecular weight of Av is relatively high because it contains 4 mannose and 3 glucosamine. 3. Specific Extinction Coefficient: the Extinction Coefficient is related to the content of Tyr in protein. Because the content of Tyr in SA is more than that in Av, the specific extinction coefficient of Sa is also higher than that of Av, the two are e 1 mg / ml 3.40 and e 1 mg / ml 1.57 respectively. At 282 nm, the two proteins did not change their extinction coefficient when they combined with Biotin, but at 233 nm, their extinction coefficient increased It is now generally used to determine the protein activity of this light-absorbing characteristics.
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