Morag E, Lapidot A, Govorko D, Lamed R, Wilchek M, Bayer E A, Shoham Y
Department of Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Appl Environ Microbiol. 1995 May;61(5):1980-6. doi: 10.1128/aem.61.5.1980-1986.1995.
The major cellulose-binding domain (CBD) from the cellulosome of Clostridium thermocellum YS was cloned and overexpressed in Escherichia coli. The expressed protein was purified efficiently by a modification of a novel procedure termed affinity digestion. The properties of the purified polypeptide were compared with those of a related CBD derived from a cellulosome-like complex of a similar (but mesophilic) clostridial species, Clostridium cellulovorans. The binding properties of the two proteins with their common substrate were found to be very similar. Despite the similarity in the amino acid sequences of the two CBDs, polyclonal antibodies raised against the CBD from C. thermocellum failed to interact with the protein from C. cellulovorans. Chemical modification of the single cysteine of the CBD had little effect on the binding to cellulose. Biotinylation of this cysteine allowed the efficient binding of avidin to cellulose, and the resultant matrix is appropriate for use as a universal affinity system.
克隆了来自嗜热栖热梭菌YS纤维小体的主要纤维素结合结构域（CBD），并在大肠杆菌中进行了过量表达。通过对一种称为亲和消化的新方法进行改进，高效纯化了表达的蛋白。将纯化多肽的性质与源自相似（但嗜温）梭菌属纤维素分解梭菌的类纤维小体复合物的相关CBD的性质进行了比较。发现这两种蛋白质与它们共同底物的结合性质非常相似。尽管两种CBD的氨基酸序列相似，但针对嗜热栖热梭菌的CBD产生的多克隆抗体未能与纤维素分解梭菌的蛋白相互作用。CBD单个半胱氨酸的化学修饰对与纤维素的结合影响很小。该半胱氨酸的生物素化使得抗生物素蛋白能够有效结合到纤维素上，所得基质适合用作通用亲和系统。
