Ybe J A, Hecht M H
Department of Chemistry, Princeton University, New Jersey 08544-1009.
Protein Expr Purif. 1994 Aug;5(4):317-23. doi: 10.1006/prep.1994.1047.
Poplar plastocyanin has been expressed in E. coli from a synthetic gene cloned into the T7 expression system. Despite the absence of a signal sequence, large quantities of the recombinant protein were readily obtained by procedures typically used to isolate proteins from the bacterial periplasm. Several different fractionation methods were equally successful. The presence of plastocyanin in these fractions does not reflect wholesale leakage of intracellular proteins, since neither beta-galactosidase activity nor the bulk of Escherichia coli proteins were released by the fractionation. The identity of the overexpressed protein was unequivocally proven to be poplar plastocyanin by N-terminal amino acid sequence analysis and by spectroscopic characterization of the purified blue copper protein.
杨树质体蓝素已通过克隆到T7表达系统中的合成基因在大肠杆菌中表达。尽管没有信号序列，但通过通常用于从细菌周质中分离蛋白质的方法很容易获得大量重组蛋白。几种不同的分级分离方法同样成功。这些级分中质体蓝素的存在并不反映细胞内蛋白质的大量泄漏，因为分级分离既没有释放β-半乳糖苷酶活性，也没有释放大部分大肠杆菌蛋白质。通过N端氨基酸序列分析和纯化的蓝色铜蛋白的光谱表征，明确证明过表达的蛋白质是杨树质体蓝素。
