Kortt A A, Guthrie R E, Hinds M G, Power B E, Ivancic N, Caldwell J B, Gruen L C, Norton R S, Hudson P J
CSIRO, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
J Protein Chem. 1995 Apr;14(3):167-78. doi: 10.1007/BF01980329.
The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG), has been expressed in high yield (15-27 mg/L) in Escherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of approximately 4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20 degrees C and concentrations of 5-10 mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its 1H NMR spectrum. Reagents such as CHAPS, n-ocytylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.
抗流感神经氨酸酶抗体NC41的VH结构域，带有和不带有C末端亲水性标记肽（FLAG），已在大肠杆菌中高产表达（15 - 27 mg/L）。两种形式均分泌到周质中，在那里形成不溶性聚集体，用2 M盐酸胍定量溶解，并通过离子交换色谱纯化至同质。VH - FLAG由三种异构体组成（pI值约为4.6、4.9和5.3），VH分子由两种pI值为5.1和6.7的异构体组成；VH异构体之间的差异表明是由于pI 5.1异构体中N末端谷氨酰胺残基的环化。在20℃和5 - 10 mg/ml的浓度下，VH结构域在溶液中发生二聚化，然后部分沉淀，导致其1H NMR谱中共振峰变宽。诸如CHAPS、正辛基葡萄糖苷和乙二醇等试剂，可能掩盖了VH分子暴露的疏水界面，阻止了VH的二聚化，并使得能够获得同位素标记蛋白质的高质量NMR谱。
