Frayser M, Sato A K, Xu L, Stern L J
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts, 02139, USA.
Protein Expr Purif. 1999 Feb;15(1):105-14. doi: 10.1006/prep.1998.0987.
The human class II major histocompatibility complex protein HLA-DR1 has been expressed in Escherichia coli as denatured alpha and beta subunits and folded in vitro to form the native structure. DR1 folding yields are 30-50% in the presence or absence of tight-binding antigenic peptides. The protein produced in this manner is soluble and monomeric with the expected apparent molecular weight. It reacts with conformation-sensitive anti-DR antibodies and exhibits peptide-dependent resistance to SDS-induced chain dissociation and to proteolysis as does the native protein. The observed peptide specificity and dissociation kinetics are similar to those of native DR produced in B-cells and finally the protein exhibits circular dichroism spectra and cooperative thermal denaturation as expected for a folded protein. We conclude that the recombinant DR1 has adopted the native fold. We have folded DR1 in the absence of peptide and isolated a soluble, peptide-free alphabeta-heterodimer. The empty DR1 can bind antigenic peptide but exhibits altered far UV-circular dichroism and thermal denaturation relative to the peptide-bound form.
人类II类主要组织相容性复合体蛋白HLA - DR1已在大肠杆菌中作为变性的α和β亚基表达，并在体外折叠形成天然结构。无论是否存在紧密结合的抗原肽，DR1的折叠产率为30 - 50%。以这种方式产生的蛋白质是可溶的单体，具有预期的表观分子量。它与构象敏感的抗DR抗体反应，并表现出与天然蛋白质一样的对SDS诱导的链解离和蛋白水解的肽依赖性抗性。观察到的肽特异性和解离动力学与在B细胞中产生的天然DR相似，最后该蛋白质表现出折叠蛋白预期的圆二色光谱和协同热变性。我们得出结论，重组DR1已采用天然折叠。我们在没有肽的情况下折叠了DR1，并分离出一种可溶的、无肽的αβ异二聚体。空的DR1可以结合抗原肽，但相对于肽结合形式，其远紫外圆二色性和热变性发生了改变。
