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Crystallization and crystallographic studies of kallistatin.

Lin F, Zhou A, Wei Z - Acta Crystallogr F Struct Biol Commun (2015)

Bottom Line: Kallistatin is a serine protease inhibitor (serpin) which specifically inhibits human tissue kallikrein; however, its inhibitory activity is inhibited by heparin.The crystals were found to belong to space group P61, with unit-cell parameters a = 113.51, b = 113.51, c = 76.17 Å.Initial analysis indicated that the crystallized kallistatin was in a relaxed conformation, with its reactive-centre loop inserted in the central β-sheet.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hongqiao International Institute of Medicine, Shanghai Tongren Hospital and Faculty of Basic Medicine, and Department of Pathophysiology, Shanghai Jiaotong University School of Medicine, (Room 1006, Building 2, No 280, South Chongqing Road), Shanghai 200025, People's Republic of China.

ABSTRACT
Kallistatin is a serine protease inhibitor (serpin) which specifically inhibits human tissue kallikrein; however, its inhibitory activity is inhibited by heparin. In order to elucidate the underlying mechanism, recombinant human kallistatin was prepared in Escherichia coli and the protein was crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.9 Å resolution. The crystals were found to belong to space group P61, with unit-cell parameters a = 113.51, b = 113.51, c = 76.17 Å. Initial analysis indicated that the crystallized kallistatin was in a relaxed conformation, with its reactive-centre loop inserted in the central β-sheet.

No MeSH data available.


Related in: MedlinePlus

Purification of recombinant kallistatin by ion-exchange chromatography. The Sumo3-kallistatin fusion protein was cleaved with SENP2 and the mixture (lane S) was loaded onto a HiTrap SP column. The protein was eluted with an NaCl gradient from 0 to 1 M, measuring the absorbance at a wavelength of 280 nm (a). Flowthrough (FT) and fractions from elution were analysed by SDS–PAGE (b). Fractions from peak I (13 and 14) and peak II (15–17) were pooled separately and subjected to crystallization trials. Lane M contains molecular-weight marker (labelled in kDa).
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fig1: Purification of recombinant kallistatin by ion-exchange chromatography. The Sumo3-kallistatin fusion protein was cleaved with SENP2 and the mixture (lane S) was loaded onto a HiTrap SP column. The protein was eluted with an NaCl gradient from 0 to 1 M, measuring the absorbance at a wavelength of 280 nm (a). Flowthrough (FT) and fractions from elution were analysed by SDS–PAGE (b). Fractions from peak I (13 and 14) and peak II (15–17) were pooled separately and subjected to crystallization trials. Lane M contains molecular-weight marker (labelled in kDa).

Mentions: In order to understand the molecular mechanism of kallistatin in regulating protease activity, we expressed recombinant human kallistatin as a fusion protein using an E. coli expression system. After Ni-Sepharose affinity-column purification, the fusion tag was cleaved with SENP2 protease and kallistatin was further purified on a cation-exchange column. As shown in Fig. 1 ▸, the Sumo3 tag eluted in fractions 9 and 10 and kallistatin eluted as two peaks (I and II). Proteins from both peaks were pooled and screened for crystallization. Stick-shaped crystals were obtained with protein from peak II after two weeks using 30% tert-butanol as precipitant (Fig. 2 ▸). These crystals readily diffracted to better than 2 Å resolution and were found to belong to space group P61, with unit-cell parameters a = 113.51, b = 113.51, c = 76.17 Å. Diffraction data statistics are shown in Table 3 ▸.


Crystallization and crystallographic studies of kallistatin.

Lin F, Zhou A, Wei Z - Acta Crystallogr F Struct Biol Commun (2015)

Purification of recombinant kallistatin by ion-exchange chromatography. The Sumo3-kallistatin fusion protein was cleaved with SENP2 and the mixture (lane S) was loaded onto a HiTrap SP column. The protein was eluted with an NaCl gradient from 0 to 1 M, measuring the absorbance at a wavelength of 280 nm (a). Flowthrough (FT) and fractions from elution were analysed by SDS–PAGE (b). Fractions from peak I (13 and 14) and peak II (15–17) were pooled separately and subjected to crystallization trials. Lane M contains molecular-weight marker (labelled in kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555919&req=5

fig1: Purification of recombinant kallistatin by ion-exchange chromatography. The Sumo3-kallistatin fusion protein was cleaved with SENP2 and the mixture (lane S) was loaded onto a HiTrap SP column. The protein was eluted with an NaCl gradient from 0 to 1 M, measuring the absorbance at a wavelength of 280 nm (a). Flowthrough (FT) and fractions from elution were analysed by SDS–PAGE (b). Fractions from peak I (13 and 14) and peak II (15–17) were pooled separately and subjected to crystallization trials. Lane M contains molecular-weight marker (labelled in kDa).
Mentions: In order to understand the molecular mechanism of kallistatin in regulating protease activity, we expressed recombinant human kallistatin as a fusion protein using an E. coli expression system. After Ni-Sepharose affinity-column purification, the fusion tag was cleaved with SENP2 protease and kallistatin was further purified on a cation-exchange column. As shown in Fig. 1 ▸, the Sumo3 tag eluted in fractions 9 and 10 and kallistatin eluted as two peaks (I and II). Proteins from both peaks were pooled and screened for crystallization. Stick-shaped crystals were obtained with protein from peak II after two weeks using 30% tert-butanol as precipitant (Fig. 2 ▸). These crystals readily diffracted to better than 2 Å resolution and were found to belong to space group P61, with unit-cell parameters a = 113.51, b = 113.51, c = 76.17 Å. Diffraction data statistics are shown in Table 3 ▸.

Bottom Line: Kallistatin is a serine protease inhibitor (serpin) which specifically inhibits human tissue kallikrein; however, its inhibitory activity is inhibited by heparin.The crystals were found to belong to space group P61, with unit-cell parameters a = 113.51, b = 113.51, c = 76.17 Å.Initial analysis indicated that the crystallized kallistatin was in a relaxed conformation, with its reactive-centre loop inserted in the central β-sheet.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hongqiao International Institute of Medicine, Shanghai Tongren Hospital and Faculty of Basic Medicine, and Department of Pathophysiology, Shanghai Jiaotong University School of Medicine, (Room 1006, Building 2, No 280, South Chongqing Road), Shanghai 200025, People's Republic of China.

ABSTRACT
Kallistatin is a serine protease inhibitor (serpin) which specifically inhibits human tissue kallikrein; however, its inhibitory activity is inhibited by heparin. In order to elucidate the underlying mechanism, recombinant human kallistatin was prepared in Escherichia coli and the protein was crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.9 Å resolution. The crystals were found to belong to space group P61, with unit-cell parameters a = 113.51, b = 113.51, c = 76.17 Å. Initial analysis indicated that the crystallized kallistatin was in a relaxed conformation, with its reactive-centre loop inserted in the central β-sheet.

No MeSH data available.


Related in: MedlinePlus