Department of Chemistry and Biochemistry, The University of Arizona, P.O. Box 210041, Tucson, AZ 85721-0041, USA.
Chem Biodivers. 2012 Sep;9(9):1739-55. doi: 10.1002/cbdv.201100449.
The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli, the wild-type gene of the mature protein retains the methionine-0, which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli (R. E. Berry, T. K. Shokhireva, I. Filippov, M. N. Shokhirev, H. Zhang, F. A. Walker, Biochemistry 2007, 46, 6830). Native N-terminus nitrophorin 2 ((ΔM0)NP2) has been prepared by employing periplasmic expression of NP2 in E. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen). This paper details the similarities and differences between the three different N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (ΔM0)NP2. It is found that the NMR spectra of high- and low-spin (ΔM0)NP2 are essentially identical to those of NP2(D1A), but the rate and equilibrium constants for histamine and NO dissociation/association of the two are different.
成熟天然硝酸酯结合蛋白 2 的第一个氨基酸是天冬氨酸，当在大肠杆菌中表达时，成熟蛋白的野生型基因保留了翻译起始密码子时产生的蛋氨酸-0。这种形式的 NP2（M0）NP2，与它的 D1A 突变体具有不同的性质，大肠杆菌的蛋氨酸氨肽酶可以将 M0 切割（R. E. Berry、T. K. Shokhireva、I. Filippov、M. N. Shokhirev、H. Zhang、F. A. Walker，Biochemistry 2007，46，6830）。通过使用来自欧文氏菌的 pelB 启动子序列在大肠杆菌中进行 NP2 的周质表达，已经制备了天然 N 端硝酸酯结合蛋白 2（（ΔM0）NP2），该启动子存在于 pET-26b 表达质粒（Novagen）中。本文详细介绍了硝酸酯结合蛋白 2 的三种不同 N 端形式（M0）NP2、NP2（D1A）和（ΔM0）NP2 的异同。结果发现，高自旋和低自旋（ΔM0）NP2 的 NMR 谱与 NP2（D1A）的基本相同，但两者的组氨酸和 NO 解离/结合的速率和平衡常数不同。
