Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags
Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags
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DOI：
10.1016/j.bbapap.2017.09.012
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发表时间：
2018-02-01
影响因子：
3.2
通讯作者：
Kuroda, Yutaka
中科院分区：
文献类型：
--
作者：
Kabir, Md. Golam;Islam, Mohammad Monirul;Kuroda, Yutaka
仔细分析显微镜下可见的无定形聚集体，其中蛋白质在天然或变性状态下非共价结合而不形成特定的四级结构，可以深入了解蛋白质聚集和溶解性的机制。在这里，我们报告了我们的模型蛋白，牛胰胰蛋白酶抑制剂变体（BPTI-19 A），其寡聚化的控制通过附加溶解度控制肽标签（SCP标签）到其C末端，这是由一个单一类型的氨基酸组成的短肽，调节蛋白质溶解度的生物物理和生化分析。在25 ℃下的动态光散射和静态光散射表明，15个SCP标签中的11个仅影响我们的参考变体BPTI-19 A和BPTI-C2 G的流体动力学半径和光散射强度。另一方面，由5个Ile（C5 I）或5个Leu（C5 L）组成的疏水性SCP标签结合成亚可见聚集体。圆二色性表明，所有标记的BPTI变体在25 ° C下具有与参考BPTI-19 A相同的二级结构含量，表明BPTI-C5 I和C5 L在缔合后保持其天然结构。此外，所有BPTI变体的热变性是完全可逆的，并且是天然折叠的小球状蛋白的典型热变性，如通过CD在222 nm处监测的。然而，疏水残基标记的BPTI-19 A的热稳定性随着蛋白质浓度和标记物疏水性的增加而降低，并且BPTI-C5 I和C5 L在37 ℃下部分变性。通过胃蛋白酶的有限蛋白水解评估的生化稳定性与变体聚集的程度相关，并且由BPTI-C5 I和C5 L形成的大的亚可见聚集体显著增加了它们对胃蛋白酶蛋白水解的抗性。总而言之，这些观察结果表明，疏水性SCP标签导致天然样蛋白质可逆地结合成耐胃蛋白酶消化的亚可见可溶性无定形聚集体。
Careful analysis of sub-visible amorphous aggregates, where proteins associate non-covalently in either native or denatured states without forming a specific quaternary structure, may shed insight into the mechanisms of protein aggregation and solubility. Here we report a biophysical and biochemical analysis of our model protein, a bovine pancreatic trypsin inhibitor variant (BPTI-19A), whose oligomerization were controlled by attaching solubility controlling peptide tags (SCP tags) to its C terminus, which are short peptides composed of a single type of amino acid that modulate protein solubility. The dynamic light scattering and static light scattering at 25 degrees C indicated that 11 out of 15 SCP tags merely affected the hydrodynamic radius and light scattering intensity of our reference variants BPTI-19A and BPTI-C2G. On the other hand, hydrophobic SCP tags composed of 5 Ile (C5I) or 5 Leu (C5L) were associated into sub-visible aggregates. Circular dichroism indicated that all tagged BPTI variants had the same secondary structure contents as the reference BPTI-19A at 25 degrees C, suggesting that BPTI-C5I and C5L kept their native structure upon association. Furthermore, the thermal denaturation of all of the BPTI variants was fully reversible and typical of natively folded small globular proteins, as monitored by CD at 222 nm. However, the thermal stability of BPTI-19A tagged with hydrophobic residues decreased with increasing protein concentration and tag's hydrophobicity, and BPTI-C5I and C5L were partially denatured at 37 degrees C. Biochemical stability assessed by limited proteolysis with pepsin correlated with the extent of the variants' aggregation, and the large sub-visible aggregates formed by BPTI-C5I and C5L significantly increased their resistance to pepsin proteolysis. Altogether, these observations indicated that hydrophobic SCP tags led to the reversible association of native-like proteins into sub-visible soluble amorphous aggregates resistant to pepsin digestion.