Agarwal A, Tan J, Eren M, Tevelev A, Lui S M, Cowan J A
Evans Laboratory of Chemistry, Ohio State University, Columbus 43210.
Biochem Biophys Res Commun. 1993 Dec 30;197(3):1357-62. doi: 10.1006/bbrc.1993.2626.
A synthetic gene encoding the peptide sequence for the low molecular weight (M(r) approximately 9600 Da) high-potential iron protein (HiPIP) from the photosynthetic bacterium Chromatium vinosum has been constructed by shotgun ligation of twelve complimentary oligonucleotides varying in size from 42-mers to 48-mers. After cloning the gene into a pET-21d(+) vector, expression of holoprotein in yields of 35 mg/liter of culture was obtained following induction with isopropyl-beta-D-thiogalactoside (IPTG). The recombinant protein was characterized by electronic absorption, 1H NMR, electrochemistry, N-terminal sequencing and amino acid analysis. This is the first example of the expression of a high potential ferredoxin containing a fully constituted [Fe4S4] cluster.
通过对12条长度从42聚体到48聚体不等的互补寡核苷酸进行鸟枪法连接，构建了一个合成基因，该基因编码来自光合细菌嗜酒色杆菌的低分子量（相对分子质量约9600道尔顿）高电位铁蛋白（HiPIP）的肽序列。将该基因克隆到pET-21d(+)载体中后，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导，全蛋白表达量可达35毫克/升培养物。通过电子吸收、1H核磁共振、电化学、N端测序和氨基酸分析对重组蛋白进行了表征。这是含有完全构成的[Fe4S4]簇的高电位铁氧还蛋白表达的首个实例。
