Vlahos C J, Wilhelm O G, Hassell T, Jaskunas S R, Bang N U
Lilly Research Laboratories, Indianapolis, Indiana 46285.
J Biol Chem. 1991 Jun 5;266(16):10070-2.
The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.R., Vlahos, C.J., and Bang, N.U. (1990) J. Biol. Chem. 265, 14606-14611), was internally radiolabeled using [35S]methionine-cysteine. Following refolding and isolation, the labeled polypeptide was further purified by reverse-phase high performance liquid chromatography. The purified kringle-2 domain was digested with thermolysin, and the resulting peptides were purified by high performance liquid chromatography. Five major peptides containing 35S were obtained. Amino acid sequence analysis showed that these peptides represented various cleavage products containing one or more of the following disulfides: Cys180-Cys261, Cys201-Cys243, Cys232-Cys256 (sequence numbering based on Pennica et al. (Pennica, D., Holmes, W.E., Kohr, W.J., Hakins, R.N., Vehar, G. A., Ward, C.A., Bennett, W.F., Yelverton E., Seeburg, P.H., Heynecker, H.L., Goeddel, E.V., and Collen, D. (1983) Nature 301, 214-221)). These results confirm that the refolding methodology used produced kringle-2 with the predicted disulfide linkage and, thus, yielded material suitable for structural and functional studies.
组织型纤溶酶原激活剂的kringle-2结构域在大肠杆菌中克隆并表达（威廉姆，O.G.，贾斯库纳斯，S.R.，弗拉霍斯，C.J.，和班，N.U.（1990年）《生物化学杂志》265，14606 - 14611），使用[35S]甲硫氨酸 - 半胱氨酸进行内部放射性标记。复性和分离后，标记的多肽通过反相高效液相色谱进一步纯化。纯化的kringle-2结构域用嗜热菌蛋白酶消化，所得肽段通过高效液相色谱纯化。获得了五个含35S的主要肽段。氨基酸序列分析表明，这些肽段代表了包含以下一种或多种二硫键的各种裂解产物：Cys180 - Cys261、Cys201 - Cys243、Cys232 - Cys256（序列编号基于彭尼卡等人（彭尼卡，D.，霍姆斯，W.E.，科尔，W.J.，哈金斯，R.N.，韦哈尔，G.A.，沃德，C.A.，贝内特，W.F.，耶尔弗顿，E.，西伯尔格，P.H.，海内克，H.L.，戈德尔，E.V.，和科伦，D.（1983年）《自然》301，214 - 221））。这些结果证实，所使用的复性方法产生了具有预测二硫键连接的kringle-2，因此，得到了适合进行结构和功能研究的材料。
