α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates
α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates
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α1 → 3-半乳糖基转移酶：使用重组酶合成 α-半乳糖基化糖复合物
DOI：
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
J. H. Shaper
中科院分区：
文献类型：
--
作者：
D. Joziasse;N. Shaper;L. S. Salyer;D. H. Eijnden;A. C. Spoel;J. H. Shaper
我们已经报道了一个牛cDNA克隆的分离和鉴定，该克隆含有UDP-Gal：Galβ1 4GlcNAc α 3-半乳糖基转移酶的完整编码序列[Joziasse，D. H、Shaper，J.H.，货车登艾因登D. H、货车图嫩J. & Shaper，N. L.（1989）J.Biol.Chem.264，14290-14297]。将该cDNA克隆插入苜蓿银纹夜蛾核型多角体病毒（Autographa californica nuclear polyhedrosis virus，AcNPV）基因组，感染草地贪夜蛾（Spodoptera frugiperda，Sf 9）昆虫细胞，获得了具有酶活性的α1 - 3-半乳糖基转移酶的高效表达。表达的酶约占细胞蛋白的2%;相应的比酶活性比小牛胸腺中观察到的高1000倍，小牛胸腺是迄今报道的比酶活性最高的组织。重组α1 - 3-半乳糖基转移酶易于洗涤剂溶解，随后通过UDP-己醇胺-Sepharose亲和层析纯化。重组α1 β 3-半乳糖基转移酶显示出对受体底物N-乙酰乳糖胺（Galβ1 4GlcNAc）的预期偏好，并证明了与先前报道的亲和纯化小牛胸腺α1 β 3-半乳糖基转移酶相同的酶动力学[Blanken，W. M. &货车den Eijnden，D. H.（1985）J.Biol.Chem.260，12927-12934]。
在初步研究中，检测了重组酶合成α 1 - 3-半乳糖基化寡糖、糖脂和糖蛋白的能力。通过结合1H-NMR、甲基化分析、HPLC。和外切糖苷酶消化，确定了对于每种模型化合物，半乳糖转移产物具有预期的末端结构，Galα1 3Galβ1 4-R。我们的研究结果表明，重组α1 - 3-半乳糖基转移酶的催化作用可用于获得制备量的各种α1 - 3-半乳糖基化糖缀合物。因此，使用重组酶的酶促合成是这些生物学相关化合物的化学合成的有效替代方案。
We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP-Gal:Galβ1 4GlcNAc α 3-galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J. & Shaper, N. L. (1989) J. Biol. Chem. 264, 14290–14297]. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera frugiperda (Sf9) insect cells with recombinant virus, resulted in high-level expression of enzymatically active α1 3-galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000-fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant α1 3-galactosyltransferase could be readily detergent-solubilized and subsequently purified by affinity chromatography on UDP-hexanolamine -Sepharose. The recombinant α1 3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (Galβ1 4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus α1 3-galactosyltransferase [Blanken, W. M. & Van den Eijnden, D. H. (1985) J. Biol. Chem. 260, 12927–12934].
In pilot studies, the recombinant enzyme was examined for the ability to synthesize αl 3-galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of 1H-NMR, methylation analysis, HPLC. and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Galα1 3Galβ1 4-R. Our results demonstrate that catalysis by recombinant α1 3-galactosyltransferase can be used to obtain preparative quantities of various α1 3-galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.
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DOI：
10.4049/jimmunol.1202204
发表时间：
2013-02-15
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Olaru F;Wang XP;Luo W;Ge L;Miner JH;Kleinau S;Geiger XJ;Wasiluk A;Heidet L;Kitching AR;Borza DB
通讯作者：
Borza DB
DOI：
10.1073/pnas.84.5.1369
发表时间：
1987-03-01
影响因子：
11.1
作者：
GALILI, U;CLARK, MR;MACHER, BA
通讯作者：
MACHER, BA
天冬酰胺连接的寡糖加工的调节。
DOI：
--
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hsieh,P;Robbins,PW
通讯作者：
Robbins,PW
牛 α 1----3-半乳糖基转移酶：cDNA 克隆的分离和表征。
DOI：
--
发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Joziasse,DH;Shaper,JH;VandenEijnden,DH;VanTunen,AJ;Shaper,NL
通讯作者：
Shaper,NL
鼠 β-1,4-半乳糖基转移酶全长 cDNA 的表征。
DOI：
--
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Shaper,NL;Hollis,GF;Douglas,JG;Kirsch,IR;Shaper,JH
通讯作者：
Shaper,JH