Efficient preparation of natural and synthetic galactosides with a recombinant β-1,4-galactosyltransferase-/UDP-4′-gal epimerase fusion protein
Efficient preparation of natural and synthetic galactosides with a recombinant β-1,4-galactosyltransferase-/UDP-4′-gal epimerase fusion protein
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DOI：
10.1021/jo0057809
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发表时间：
2001-04-06
影响因子：
3.6
通讯作者：
Paulson, JC
中科院分区：
文献类型：
--
作者：
Blixt, O;Brown, J;Paulson, JC
基于LacNAc的寡糖的众多生物学作用导致了对这些结构在生物学研究中的需求增加。本文介绍了一种利用细菌β-4-半乳糖基转移酶/-UDP-4‘-半乳糖基-差向异构酶融合蛋白合成β-半乳糖苷的有效途径。将脑膜炎奈瑟菌的LGTB基因与嗜热链球菌的Gale基因融合，克隆到表达载体PCW中。融合蛋白将半乳糖转移到各种不同的含葡萄糖和氨基葡萄糖的受体上，并利用UDP-半乳糖或UDP-葡萄糖作为供体底物。表达融合蛋白的大肠杆菌粗裂解产物被证明足以从廉价的UDP-葡萄糖中有效地制备多克规模的半乳糖化寡糖。含有融合蛋白的裂解产物也被发现用于在偶联反应混合物中生产更复杂的寡糖，例如在从N-乙酰氨基葡萄糖制备唾液酸苷的过程中，因此，细菌表达的融合蛋白非常适合于简便和经济地制备天然低聚糖和基于乳糖胺核心的合成衍生物。
The numerous biological roles of LacNAc-based oligosaccharides have led to an increased demand for these structures for biological studies. In this report, an efficient route for the synthesis of beta -galactosides using a bacterial beta -4-galactosyltransferase/-UDP-4 ' -gal-epimerase fusion protein is described. The lgtB gene from Neisseria meningitidis and the galE gene from Streptococcus thermophilus were fused and cloned into an expression vector pCW. The fusion protein transfers galactose to a variety of different glucose- and glucosamine-containing accepters, and utilizes either UDP-galactose or UDP-glucose as donor substrates. A crude lysate from Escherichia coli expressing the fusion protein is demonstrated to be sufficient for the efficient preparation of galactosylated oligosaccharides from inexpensive UDP-glucose in a multigram scale. Lysates containing the fusion protein are also found to be useful in the production of more complex oligosaccharides in coupled reaction mixtures, e.g., in the preparation of sialosides from N-acetylglucosamine, Thus, bacterially expressed fusion protein is well suited for the facile and economic preparation of natural oligosaccharides and synthetic derivatives based on the lactosamine core.