Reznik Gabriel O, Yu Yong, Tarr George E, Cantor Charles R
Center for Advanced Biotechnology, Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.
J Proteome Res. 2003 May-Jun;2(3):243-8. doi: 10.1021/pr0255809.
A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.
一种名为RBP-S的人血浆视黄醇结合蛋白（RBP）突变体已被设计并生产出来，其中参与形成三个二硫键的六个天然半胱氨酸残基已被丝氨酸取代。还在RBP的C末端添加了一个六组氨酸标签，以便于纯化。二硫键的去除导致RBP对全反式视黄醇的亲和力下降。数据表明，在约20摄氏度时，全反式视黄醇与RBP和RBP-S结合的解离常数（Kd）分别为4×10⁻⁸ M和1×10⁻⁷ M。与天然RBP相比，RBP-S在pH 8.0以下和室温下稳定性降低。远紫外圆二色性表明，去除二硫键后RBP结构发生了松弛。近紫外圆二色性表明，在没有二硫键的情况下，RBP在310 - 330 nm处的光学活性高于在280 - 290 nm范围内的光学活性。这项工作表明，三个天然二硫键有助于RBP的折叠，但对于产生可溶性活性蛋白并非必不可少。
