کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2020525 | 1069186 | 2013 | 5 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Efficient processing of procathepsin K to the mature form Efficient processing of procathepsin K to the mature form](/preview/png/2020525.png)
• Procathepsin K is well expressed in Pichia pastoris.
• Processing to the mature active form has proven unpredictable.
• Efficient autoprocessing occurs under non-reducing conditions at low dilution.
• Mature cathepsin K is then reversibly inhibited using methyl methanethiosulfonate.
• Cation exchange chromatography followed by diafiltration yields active cathepsin K.
The proteolysis of collagen fibrils by cathepsin K is a hallmark of bone catabolism and tissue degeneration. The production of active recombinant cathepsin K is central for our ability to study the mechanisms by which these processes occur. Here we report an efficient processing method for the preparation of recombinant cathepsin K expressed in Pichia pastoris. Methanol precipitation of crude media and autoactivation in the absence of a reducing agent allows for the reversible inhibition of the enzyme prior to subsequent purification steps. The resultant purified enzyme is both resistant to autolysis and effective at cleaving collagen.
Journal: Protein Expression and Purification - Volume 91, Issue 1, September 2013, Pages 37–41