Porcelli M, Fusco S, Inizio T, Zappia V, Cacciapuoti G
Istituto di Biochimica delle Macromolecole, Facoltà di Medicina e Chirurgia, Seconda Università degli Studi di Napoli, Via Costantinopoli 16, Naples, 80138, Italy.
Protein Expr Purif. 2000 Feb;18(1):27-35. doi: 10.1006/prep.1999.1161.
S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.
通过将含有编码S-腺苷同型半胱氨酸水解酶基因的基因组片段插入到pTrc99A表达载体的异丙基-β-D-硫代半乳糖苷诱导型启动子下游，在大肠杆菌中表达来自嗜热栖热菌的S-腺苷同型半胱氨酸水解酶。将位于该基因上游25 bp且与终止密码子读码框一致的ATG用作起始密码子。该构建体用于转化大肠杆菌RB791和大肠杆菌JM105菌株。通过快速高效的两步法纯化的重组蛋白（每克细胞产生0.4毫克酶），由于存在一种对应于缺少前24个氨基酸残基的“截短”S-腺苷同型半胱氨酸水解酶亚基的蛋白质污染物，在SDS-PAGE上看起来不均匀。重组酶显示出与从嗜热栖热菌分离的S-腺苷同型半胱氨酸水解酶相同的分子量、最适温度和动力学特征，但热稳定性较差。为了构建一个在核糖体结合位点和S-腺苷同型半胱氨酸水解酶基因起始密码子之间具有正确距离的载体，使用定点诱变在翻译起始密码子处创建了一个NcoI位点。高水平实现了均一突变体S-腺苷同型半胱氨酸水解酶的表达（每克细胞1.7毫克突变蛋白）。突变体S-腺苷同型半胱氨酸水解酶和天然酶在所有物理化学和动力学性质（包括热稳定性）上无法区分，表明涉及该蛋白质NH2末端序列的相互作用在嗜热栖热菌S-腺苷同型半胱氨酸水解酶的热稳定性中起作用。
