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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 November 1; 64(Pt 11): 1060–1062.
Published online 2008 October 31. doi:  10.1107/S1744309108032454
PMCID: PMC2581705

Expression, purification, crystallization and preliminary X-ray diffraction analysis of Sagittaria sagittifolia arrowhead protease inhibitor API-A in complex with bovine trypsin

Chunhui Jiang,a, Rui Bao,a, and Yuxing Chena,b,*

Abstract

Protease inhibitors play key roles in physiological processes. Arrowhead protease inhibitor A (API-A), a member of the serine protease inhibitor family, can inhibit two trypsin molecules simultaneously. In the present work, API-A from Sagittaria sagittifolia has been cloned, expressed, purified and crystallized in complex with bovine trypsin. The crystals were obtained by the sitting-drop method. A data set was collected to 2.48 Å resolution from a single crystal. The crystal belonged to space group C2221, with unit-cell parameters a = 76.63, b = 110.86, c = 152.99 Å, α = β = γ = 90°.

Keywords: arrowhead protease inhibitors, serine protease inhibitors, Sagittaria sagittifolia

1. Introduction

Protease inhibitors are ubiquitously present in a variety of species that includes plants and animals as well as microorganisms (Las­kowski & Kato, 1980 [triangle]). They play vital roles in many physiological processes such as protein digestion, cell signal transmission, inflammation, apoptosis, blood clotting, embryogenesis etc. (Leung et al., 2000 [triangle]). As proteases are involved in many diseases, such as cancer (Kobayashi et al., 2004 [triangle]), nephrotoxicity (Umeki et al., 1989 [triangle]) and AIDS (Ng et al., 2003 [triangle]), their inhibitors have been extensively studied over the last few decades as starting points for potential therapeutic agents. One of the most studied specific protease-inhibitor families, the Kunitz-type superfamily from serine protease inhibitors, has been well characterized (Laskowski & Kato, 1980 [triangle]; Nielsen et al., 2004 [triangle]; Onesti et al., 1991 [triangle]; Schmidt et al., 2005 [triangle]; Song & Suh, 1998 [triangle]; Sweet et al., 1974 [triangle]). Members of the family usually contain 170–200 residues and one or two intrachain disulfide bonds. They have a strong affinity for their target proteases and form stable complexes that are difficult to separate into the constituent proteases and inhibitors (Baillargeon et al., 1980 [triangle]).

A few members of this family have two active sites and can thus act on two molecules of protease simultaneously; these are the so-called ‘double-headed inhibitors’ (Yang et al., 1992 [triangle]). The arrowhead protease inhibitors A and B (API-A and API-B) were first purified from the tubers of Sagittaria sagittifolia L. and characterized by Chang et al. (1979 [triangle]). API-A and API-B share 91% sequence homology, but their inhibitory activities are distinctly different from each other. API-A inhibits equimolar amounts of trypsin and chymotrypsin simultaneously, whereas API-B inhibits two molecules of trypsin at the same time. Both of them can inhibit pig tissue kallikrein in a 1:1 ratio (Xie et al., 1997 [triangle]). There has been some controversy about the exact locations of the active sites (Li et al., 2002 [triangle]; Xie et al., 1997 [triangle]). Therefore, high-resolution crystallographic structures are necessary to answer these and other questions.

In the present work, we report the crystallization and preliminary X-ray diffraction analysis of API-A in complex with two trypsin molecules. The structure will enable us to confirm the locations of the active sites and to better understand the characteristics of API-A and its structure–function relationship.

2. Materials and methods

2.1. Cloning and purification

Total RNA was isolated from 100 mg fresh arrowhead (S. sagittifolia L.) buds using Trizol reagent (Invitrogen) according to the protocol of Chomczynski & Mackey (1995 [triangle]). The first chain of cDNA was reverse-transcribed from the total RNA using a specific primer 5′-­CGCCGCGGCCGCTTAGAGTGCGTCGRACTTMTG-3′ with a NotI site (where R represents G/A and M is A/C) and Superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s protocols. The coding sequence of API-A was PCR-amplified from cDNA using the forward primer 5′-CTCGCATATGGATCCCGTC­GTCGACAGC-3′ containing an NdeI site. The PCR product was cloned into a pET28a-derived expression vector, which gives a pro­tein with a hexahistidine tag just after the start codon. The resulting construct was then co-transformed with PKY206 (a plasmid con­taining the groESL genes of Escherichia coli, resulting in the synthesis of chaperones GroEL and GroES; Mizobata et al., 1992 [triangle]) into the E. coli BL21 (DE3) strain and the transformed cells were grown at 310 K in LB containing 10 µg ml−1 kanamycin until the OD600 reached about 0.6; the cells were then induced with 0.2 mM IPTG at 291 K overnight.

The cell pellets were resuspended in 40 ml cold lysis buffer (20 mM Tris–HCl pH 7.5, 100 mM NaCl) and lysed by sonification. The lysate was clarified by centrifugation at 16 000g for 30 min and the supernatant was filtered and loaded onto 2 ml nickel-agarose affinity resin (Qiagen) equilibrated with lysis buffer. After washing with ten volumes of lysis buffer plus 10 mM imidazole, His-tagged protein was then eluted with lysis buffer plus 200 mM imidazole. The fractions containing the target protein were further purified by size-exclusion chromatography on a Superdex 200 column (GE Healthcare; Fig. 1 [triangle] a). The purity of the protein was checked by SDS–PAGE (Fig. 1 [triangle] b).

Figure 1
(a) Gel filtration of API-A using size-exclusion chromatography on a Superdex 200 column. The flow rate was 1 ml min−1. The main peaks were collected as follows: peak I, 47.90 min; peak II, 78.21 min; peak III, ...

2.2. Complex formation

For complex formation, the fractions corresponding to the highest peak, which contained about 5 mg protein, were pooled and mixed with 12 mg β-trypsin (bovine trypsin, USP grade; Ameresco) and CaCl2 was then added to a final concentration of 20 mM. After in­cubation at 277 K for 2 h, the mixture was loaded onto a HiLoad 16/60 Superdex 200 (Amersham Biosciences) column equilibrated with buffer (20 mM Tris–HCl pH 7.5, 100 mM NaCl, 20 mM CaCl2; Fig. 2 [triangle]). The fractions containing the API-A–trypsin complex were collected and concentrated to 15 mg ml−1 by ultrafiltration using an Amicon Ultra 10 kDa cutoff concentrator (Millipore) and stored at 193 K.

Figure 2
Gel filtration of API-A complexed with trypsin using a HiLoad 16/60 Superdex 200 column. The flow rate was 1 ml min−1. The peak at 81.40 min corresponds to the complex.

2.3. Crystallization experiments

Crystallization trials were carried out by the hanging-drop vapour-diffusion method at 291 K using Crystal Screens I and II (Hampton Research) for initial screening. Drops were prepared by mixing 1 µl protein solution with 1 µl precipitant solution and were equilibrated against 500 µl reservoir solution. For optimization, the sitting-drop method at 291 K was used.

2.4. Data collection

X-ray diffraction data were collected at 100 K using an in-house Rigaku MM007 X-ray generator (λ = 1.54179 Å) with a MAR Research 345 detector at the School of Life Sciences, University of Science and Technology of China (USTC, Hefei, People’s Republic of China). The crystal was flash-frozen and maintained at 100 K using nitrogen gas during data collection. 20%(v/v) glycerol was added to the reservoir as a cryoprotectant. The cryoprotectant was introduced into the crystal by soaking.

3. Results and discussion

The API-A protein was co-expressed with the chaperones GroES and GroEL in a soluble form in E. coli BL21 (DE3) and was purified to homogeneity after size-exclusion chromatography. After incubation with excess trypsin for 2 h, the putative complex was applied onto a HiLoad 16/60 Superdex 200 column. The peak corresponding to the target complex appeared at 81.40 min (Fig. 2 [triangle]), which was earlier than API-A (90.87 min; Fig. 1 [triangle] a). The elution time and symmetry of the peak indicated that API-A had formed a stable complex with trypsin. Hampton Research Crystal Screens I and II were used for preliminary screening and gave crystals from seven conditions, three of which contained ammonium sulfate and the other four different precipitants. These initial hits gave needle-shaped crystals that were not suitable for X-ray diffraction. Optimization was carried out with the sitting-drop method. Eventually, crystals that were suitable for diffraction studies and had dimensions of 0.5 × 0.2 × 0.03 mm appeared in drops with reservoirs containing 0.1 M sodium cacodylate pH 6.5, 0.2 M ammonium sulfate, 20% PEG 8000 (Fig. 3 [triangle] a). These crystals diffracted to 2.48 Å resolution (Fig. 3 [triangle] b). The diffraction data were processed with the program AUTOMAR v.1.2 (Bartels & Klein, 2003 [triangle]). These crystals belonged to space group C2221, with unit-cell parameters a = 76.63, b = 110.86, c = 152.99 Å, α = β = γ = 90°. The data-collection statistics are listed in Table 1 [triangle]. Structure solution is in progress and we hope that this will enable us to characterize the binding of API-A to trypsin more precisely.

Figure 3
(a) Crystals of S. sagittifolia API-A complexed with trypsin obtained by the sitting-drop vapour-diffusion method. The average dimensions of these crystals were 0.5 × 0.2 × 0.03 mm. (b) X-ray diffraction pattern at a resolution ...
Table 1
Data-collection statistics

Acknowledgments

This work was supported by the Ministry of Education of China (Talents Project of New Century NCET-06-0374 and Program PRA B07-02) and the National Natural Science Foundation of China (Grant No. 30670461).

References

  • Baillargeon, M. W., Laskowski, M. Jr, Neves, D. E., Porubcan, M. A., Santini, R. E. & Markley, J. L. (1980). Biochemistry, 19, 5703–5710. [PubMed]
  • Bartels, K. & Klein, C. (2003). The AUTOMAR User’s Guide http://www.marresearch.com/automar/.
  • Chang, H. Y., Chi, C. W. & Lo, C. Q. (1979). Sci. Sin.22, 1443–1454.
  • Chomczynski, P. & Mackey, K. (1995). Biotechniques, 19, 942–945. [PubMed]
  • Kobayashi, H., Yagyu, T., Inagaki, K., Kondo, T., Suzuki, M., Kanayama, N. & Terao, T. (2004). Cancer, 100, 869–877. [PubMed]
  • Laskowski, M. Jr & Kato, I. (1980). Annu. Rev. Biochem.49, 593–626. [PubMed]
  • Leung, D., Abbenante, G. & Fairlie, D. P. (2000). J. Med. Chem.43, 305–341. [PubMed]
  • Li, J., Ruan, K. C. & Chi, C. W. (2002). Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 34, 662–666. [PubMed]
  • Mizobata, T., Akiyama, Y., Ito, K., Yumoto, N. & Kawata, Y. (1992). J. Biol. Chem.267, 17773–17779. [PubMed]
  • Ng, T. B., Lam, S. K. & Fong, W. P. (2003). Biol. Chem.384, 289–293. [PubMed]
  • Nielsen, P. K., Bonsager, B. C., Fukuda, K. & Svensson, B. (2004). Biochim. Biophys. Acta, 1696, 157–164. [PubMed]
  • Onesti, S., Brick, P. & Blow, D. M. (1991). J. Mol. Biol.217, 153–176. [PubMed]
  • Schmidt, A. E., Chand, H. S., Cascio, D., Kisiel, W. & Bajaj, S. P. (2005). J. Biol. Chem.280, 27832–27838. [PubMed]
  • Song, H. K. & Suh, S. W. (1998). J. Mol. Biol.275, 347–363. [PubMed]
  • Sweet, R. M., Wright, H. T., Janin, J., Chothia, C. H. & Blow, D. M. (1974). Biochemistry, 13, 4212–4228. [PubMed]
  • Umeki, S., Tsukiyama, K., Okimoto, N. & Soejima, R. (1989). Am. J. Med. Sci.298, 221–226. [PubMed]
  • Xie, Z. W., Luo, M. J., Xu, W. F. & Chi, C. W. (1997). Biochemistry, 36, 5846–5852. [PubMed]
  • Yang, H. L., Luo, R. S., Wang, L. X., Zhu, D. X. & Chi, C. W. (1992). J. Biochem.111, 537–545. [PubMed]

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