Bianchi E, Venturini S, Pessi A, Tramontano A, Sollazzo M
Department of Biochemistry, Istituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia, Italy.
J Mol Biol. 1994 Feb 18;236(2):649-59. doi: 10.1006/jmbi.1994.1174.
We recently described the design and chemical synthesis of the minibody, a 61-residue metal binding beta-protein with a novel fold. Characterization of the polypeptide by circular dichroism spectroscopy, size exclusion chromatography, and metal binding studies showed the molecule to be folded, monomeric, globular and able to bind metals. The main obstacle which prevented a more detailed characterization was the very low solubility of the protein in water (about 10 microM). To address this problem, we used two independent approaches: (1) mutagenesis of the beta-sheet framework residues and (2) addition of a solubilizing motif, made of three lysine residues, at the N or C termini. Engineering and production of mutants was facilitated by the achievement of high level expression of the protein in Escherichia coli. Both approaches led to minibody variants with a solubility ranging from tenfold higher up to millimolar levels. For the best-characterized variant obtained so far, the thermodynamic stability calculated from denaturant-induced transition is identical to that of the parent, poorly soluble, molecule.
我们最近描述了微型抗体的设计与化学合成，它是一种具有新型折叠结构的含61个残基的金属结合β蛋白。通过圆二色光谱、尺寸排阻色谱和金属结合研究对该多肽进行表征，结果表明该分子呈折叠状、单体球状且能够结合金属。妨碍进行更详细表征的主要障碍是该蛋白质在水中的溶解度极低（约10微摩尔）。为解决这个问题，我们采用了两种独立的方法：（1）对β折叠框架残基进行诱变；（2）在N端或C端添加由三个赖氨酸残基组成的增溶基序。大肠杆菌中该蛋白质的高水平表达促进了突变体的工程改造和生产。两种方法都产生了溶解度提高了10倍至毫摩尔水平的微型抗体变体。对于目前得到的表征最充分的变体，由变性剂诱导的转变计算出的热力学稳定性与亲本的、难溶的分子相同。
