Bam N B, Cleland J L, Randolph T W
Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520, USA.
Biotechnol Prog. 1996 Nov-Dec;12(6):801-9. doi: 10.1021/bp960068b.
We demonstrate that a surfactant-stabilized molten globule intermediate exists for recombinant human growth hormone (rhGH), is very hydrophobic, and tends to form aggregates. Characterization of this intermediate included equilibrium denaturation measured by electron paramagnetic resonance (EPR) and CD spectroscopy, assessment of aggregation during refolding, and fluorescence studies of its binding to the hydrophobic probe, 1-anilinonapthalene-8-sulfonate (1,8-ANS). We have found that at 4.5 M guanidinium hydrochloride (GuHCl), a molten globule intermediate of rhGH is stabilized and results in significant aggregation upon refolding. This intermediate is populated by the addition of the nonionic surfactant, Tween. This surfactant also reduces the extent of aggregation during refolding of rhGH from 4.5 M GuHCl. Overall, our studies reveal that rhGH forms a molten globule-like intermediate during folding and this intermediate self-associates. This self-association is reduced upon formation of a Tween-rhGH complex. Tween also binds to the native protein. Thus, nonionic surfactants such as Tween may act like molecular chaperones in facilitating protein folding while not altering the native conformation.
我们证明重组人生长激素（rhGH）存在一种由表面活性剂稳定的熔融球状中间体，它具有很强的疏水性，且易于形成聚集体。对该中间体的表征包括通过电子顺磁共振（EPR）和圆二色光谱（CD）测量平衡变性、评估复性过程中的聚集情况以及对其与疏水探针1-苯胺基萘-8-磺酸盐（1,8-ANS）结合的荧光研究。我们发现，在4.5 M盐酸胍（GuHCl）条件下，rhGH的熔融球状中间体得以稳定，并且在复性时会导致显著的聚集。通过添加非离子表面活性剂吐温可形成这种中间体。该表面活性剂还降低了rhGH从4.5 M GuHCl复性过程中的聚集程度。总体而言，我们的研究表明rhGH在折叠过程中形成了类似熔融球状的中间体，且该中间体发生了自缔合。形成吐温-rhGH复合物后，这种自缔合作用减弱。吐温还能与天然蛋白质结合。因此，诸如吐温之类的非离子表面活性剂可能起到分子伴侣的作用，促进蛋白质折叠，同时又不改变其天然构象。
