Cicortas Gunnarsson L, Nordberg Karlsson E, Albrekt A-S, Andersson M, Holst O, Ohlin M
Department of Immunotechnology, Lund University, PO Box 7031, S-220 07 Lund, Sweden.
Protein Eng Des Sel. 2004 Mar;17(3):213-21. doi: 10.1093/protein/gzh026. Epub 2004 Apr 13.
The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
不断发展的生物技术领域一直需要具有新特性的结合蛋白。对于大规模应用而言，不仅结合特异性和亲和力，而且结构稳定性和产量都是重要的特性。为了找到这样的分子，通过使天然存在的结合蛋白多样化来创建文库，在这些情况下，天然结合蛋白充当支架。在本研究中，我们研究了使用来自嗜热栖热放线菌中发现的木聚糖酶的热稳定碳水化合物结合模块CBM4-2作为携带多样性的支架。通过在CBM4-2的碳水化合物结合位点的12个位置引入有限的变异来创建组合文库。尽管文库规模较小（1.6×10⁶个克隆），但使用噬菌体展示方法成功筛选出了对不同碳水化合物聚合物（桦木木聚糖、微晶纤维素和象牙果甘露聚糖）以及糖蛋白（人IgG4）具有特异性的变体。当在大肠杆菌中产生时，所研究的克隆显示出高产量（平均每摇瓶培养69毫克纯化蛋白），并且它们都是稳定的分子，具有高熔点转变温度（75.7±5.3℃）。我们所有的结果表明，CBM4-2分子是创建可用于不同生物技术应用的变体的合适支架。
