Ghosh S, Pawelczyk T, Lowenstein J M
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
Protein Expr Purif. 1997 Mar;9(2):262-78. doi: 10.1006/prep.1996.0682.
Phospholipase C isoforms delta 1 and delta 3 (PLC delta 1 and delta 3) were expressed in Escherichia coli using the cDNA sequences from human fibroblasts. The enzymes were also expressed with the sequence Met-Gly-His6-Ser-Gly-Leu-Phe-Lys-Arg, a hexahistidine sequence followed by a Kex2 protease cleavage site, denoted as "-H6K2," attached to their amino termini. PLC delta 1, PLC delta 1-H6K2, PLC delta 3, PLC delta 3-H6K2 all expressed in highly active form. The H6K2-bearing isoforms were each purified to homogeneity in a single step, with yields of 90-100%, using agarose-iminodiacetic acid-Ni columns and imidazole buffer as eluting agent. Yields in terms of activity increased as the temperature of expression was decreased. Expression at 16 degrees C for 72 h yielded 33 mg of pure PLC delta 1-H6K2 and 13 mg of pure PLC delta 3-H6K2 per liter of culture. Removal of H6K2 from both isoforms with Kex2 protease resulted in little or no loss of activity. Expression of PLC isoforms bearing -H6K2 at the amino terminus resulted in about 12 times more activity than expression of the isoforms lacking -H6K2. PLC delta 3 is much less stable than PLC delta1. Successful purification and storage of PLC delta 3 depends on a suitable stabilizing medium. Both isoforms require 0.3 microM calcium ion for half-maximum activity. The specific activities of the isoforms expressed with and without -H6K2 are the same, as are their calcium saturation curves.
利用来自人成纤维细胞的cDNA序列，在大肠杆菌中表达了磷脂酶C同工型δ1和δ3（PLCδ1和PLCδ3）。这些酶还与序列Met-Gly-His6-Ser-Gly-Leu-Phe-Lys-Arg一起表达，该序列是一个六组氨酸序列，后面跟着一个Kex2蛋白酶切割位点，在其氨基末端标记为“-H6K2”。PLCδ1、PLCδ1-H6K2、PLCδ3、PLCδ3-H6K2均以高活性形式表达。使用琼脂糖-亚氨基二乙酸-Ni柱和咪唑缓冲液作为洗脱剂，带有H6K2的同工型均在一步中纯化至同质，产率为90-100%。随着表达温度的降低，活性产率增加。在16℃下表达72小时，每升培养物产生33mg纯PLCδ1-H6K2和13mg纯PLCδ3-H6K2。用Kex2蛋白酶从两种同工型中去除H6K2导致活性几乎没有损失或没有损失。在氨基末端带有-H6K2的PLC同工型的表达产生的活性比缺乏-H6K2的同工型的表达高约12倍。PLCδ3比PLCδ1稳定性差得多。PLCδ3的成功纯化和储存取决于合适的稳定介质。两种同工型都需要0.3μM钙离子才能达到最大活性的一半。带有和不带有-H6K2表达的同工型的比活性相同，它们的钙饱和曲线也是如此。
