Efficient processing of procathepsin K to the mature form

•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.