1m ammonium sulfate the new solution was subjected to hplc using phenyl 5 pw tosoh corp equilibrated with 10 mm phosphate buffer ph 6 8 containing 1m ammonium sulfate followed by linear concentration gradient elution with 1m ammonium sulfate to 0m ammonium sulfate a fraction containing the desired protease inhibitor activity was thus recovered the fraction was dialyzed against 20 mm tris hydrochloride buffer ph 8 containing 0 05 chaps and then subjected to hplc using deae equilibrated with 20 mm tris hydrochloride buffer ph 8 containing 0 05 chaps followed by linear concentration gradient elution with 0m to 500 mm nacl whereby a fraction showing the desired protease inhibitor activity was recovered the fraction was dialyzed against 5 mm phosphate buffer ph 6 8 containing 0 05 chaps and then subjected to hplc using a hca a 4007 column product of mitsui toatsu chemicals equilibrated with 5 mm phosphate buffer ph 6 8 containing 0 05 chaps and the non adsorbed fraction was recovered the fraction was submitted to gs 520 equilibrated with pbs containing 0 05 chaps and an active fraction fraction of about 40 to 20 kda was recovered for eliminating minor bands the fraction was applied to a ymc pack c4 column obtained from ymc linear concentration gradient elution was carried out over 40 minutes using acetonitrile isopropyl alcohol 3 7 containing 0 1 tfa and varying the concentration thereof from 10 to 50 and the active fraction was neutralized with 1m tris hydrochloride buffer ph 8 and then dried under reduced pressure after drying the solid obtained was dissolved in pbs containing 0 05 chaps.