Obukowicz M G, Gustafson M E, Junger K D, Leimgruber R M, Wittwer A J, Wun T C, Warren T G, Bishop B F, Mathis K J, McPherson D T
Corporate Research and Development Staff, Monsanto Company, St. Louis, Missouri 63198.
Biochemistry. 1990 Oct 16;29(41):9737-45. doi: 10.1021/bi00493a033.
Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized. MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds. MB1004 without a pro region was secreted into the periplasm of E. coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter. Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E. coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography. Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence. The specific activity of purified MB1004 from E. coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N). Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities. Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays. These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered. By utilization of secretion in E. coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies.
活性人组织纤溶酶原激活物变体kringle-2-丝氨酸蛋白酶（K2 + SP结构域；称为MB1004）在大肠杆菌中作为分泌蛋白合成、分离并进行了表征。MB1004是一种相对较大且复杂的蛋白质，大小约为38 kDa，含有9个二硫键。通过将该蛋白与从tac启动子表达的PhoA前导肽融合，将没有前体区域的MB1004分泌到大肠杆菌的周质中。通过赖氨酸和刺桐抑制剂亲和层析相结合，从大肠杆菌匀浆中纯化出约1%（20微克/升肉汤）的分泌型MB1004，作为一种可溶性活性酶。纯化后的MB1004为单体单链，其N端与预测的氨基酸序列一致。将从大肠杆菌中纯化的MB1004的比活性与从哺乳动物细胞中纯化的等效重组材料进行比较，后者天然糖基化（MB1004G）或用N-聚糖酶处理后去糖基化（MB1004N）。四种不同体外试验的结果表明，MB1004和MB1004N具有相似的活性。在纤溶酶原激活试验中，两者的比活性均比MB1004G高4至12倍。这些结果表明，如果不考虑糖基化的影响，可能会得出不准确的比活性情况。通过利用大肠杆菌中的分泌作用，未进行体外重折叠就纯化出了非糖基化的MB1004，并且表明其适用于结构-功能研究。
