Crystallization and preliminary X-ray crystallographic studies on a Kunitz-type potato serine protease inhibitor

Abstract

Interest in protease inhibitors has been renewed because of their potent activity in preventing carcinogenesis in a wide variety of in vivo and in vitro model systems. Potato tubers contain a wide range of such protease inhibitors. In cv. Elkana potato tubers, protease inhibitors represent about 50% of the total amount of soluble protein. Potato serine protease inhibitor (PSPI), one of the isoforms of the most abundant group of protease inhibitors, is a dimeric double-headed Kunitz-type inhibitor. No high-resolution structural information on this type of inhibitor has so far been obtained, as all currently known structures are of the monomeric single-headed or monomeric double-headed types. Crystals were grown in 0.1 M HEPES pH 7.5, 10% PEG 8000 and 8% ethylene glycol complemented with 9 mM 1-s-octyl--d-thioglucoside or 0.1 M glycine. Data were collected from a single crystal under cryoconditions to 1.8 A Ê resolution. The protein crystallized in space group P2 1 , with unit-cell parameters a = 54.82, b = 93.92, c = 55.44 A Ê , = 100.7; the scaling R sym is 0.044 for 45 456 unique re¯ections.

Heterogeneous nucleation of three-dimensional protein nanocrystals

“…Our studies on heterogeneous crystallization were extended further to other proteins that are more difficult to crystallize. Of special interest to us was the potato protease inhibitor 6.1, as this protein is not easy to crystallize and its structure has still not been resolved by X-ray crystallography (Thomassen et al, 2004). The crystals are difficult to manipulate and all attempts to soak them with heavy metals in order to obtain phase information have so far been unsuccessful.…”

“…Antipolymeric Lewis X Fab fragment 54 was expressed by papain digestion of MAP 54-5C10-A followed by extensive purification using affinity and ion-exchange chromatography as described previously by van Roon et al (2004). Potato serine protease inhibitor 6.1 (the number represents the isolectric point) was expressed and purified following the procedure described by Thomassen et al (2004). All chemicals used in the crystallization experiments were purchased from Merck and solutions were further filtered with Millipore filters (0.22 mm) directly before use.…”

Structure of a post-translationally processed heterodimeric double-headed Kunitz-type serine protease inhibitor from potato

“…Protein purification, crystallization and data collection have been reported previously (Thomassen et al, 2004). The crystal diffracted to 1.60 Å resolution, but the data completeness at this resolution was very low owing to the use of a square detector set to 1.8 Å , with only 18.4% completeness in the highest resolution shell (1.69-1.60 Å ).…”