Samal B, Boone T, Karan B, Chen K, Sachdev R, Arakawa T
Amgen Inc., Thousand Oaks, California 91320, USA.
Adv Exp Med Biol. 1996;379:95-104. doi: 10.1007/978-1-4613-0319-0_10.
We have isolated the cDNA and the genomic clones encoding a novel serine proteinase, named proteinase T, from the fungus Tritirachium album Limber. The coding region of the gene is interrupted by two introns. The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 56% identical to that of proteinase K. Four cysteines are present in the mature proteinase, probably in the form of disulfide bonds. We have also purified the native proteinase from Tritirachium album Limber grown in the presence of 2% skim milk. Proteinase T is extremely stable at 50 degrees C. The thermal stability is not affected in the presence of 1% SDS either at pH 8.0 or 10.0. We have expressed the cDNA of proteinase T in Escherichia coli. The authenticity of the proteinase has been characterized by Western blotting and amino terminal analysis of the recombinant product. High level expression of proteinase T in E. coli as well as the refolding process to generate active proteinase will be discussed in detail.
我们从真菌白僵菌中分离出了编码一种新型丝氨酸蛋白酶（命名为蛋白酶T）的cDNA和基因组克隆。该基因的编码区被两个内含子打断。从核苷酸序列推导的蛋白酶T的氨基酸序列与蛋白酶K的氨基酸序列约56%相同。成熟蛋白酶中有四个半胱氨酸，可能以二硫键的形式存在。我们还从在2%脱脂牛奶存在下生长的白僵菌中纯化了天然蛋白酶。蛋白酶T在50℃时极其稳定。在pH 8.0或10.0时，1% SDS的存在也不会影响其热稳定性。我们已在大肠杆菌中表达了蛋白酶T的cDNA。通过蛋白质印迹法和重组产物的氨基末端分析对蛋白酶的真实性进行了表征。将详细讨论蛋白酶T在大肠杆菌中的高水平表达以及产生活性蛋白酶的重折叠过程。
