| Autor: |
Reznik, G. O., Yu, Y., Tarr, G. E., Cantor, C. R. |
| Zdroj: |
Journal of Proteome Research; June 2003, Vol. 2 Issue: 3 p243-248, 6p |
| Abstrakt: |
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 × 10-8 M and 1 × 10-7 M, respectively, at approximately 20 °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. Keywords: human plasma retinol-binding protein • all-trans-retinol • genetic engineering • disulfide bonds |
| Databáze: |
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