生成、纯化和结晶原核生物中SLC26同源蛋白用于结构研究

Yung-Ning Chang; Farooque  R. Shaik; Yvonne Neldner; Eric R. Geertsma

doi:10.21769/BioProtoc.2116

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Peer-reviewed

Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies

生成、纯化和结晶原核生物中SLC26同源蛋白用于结构研究

Yung-Ning Chang

Farooque R. Shaik

Yvonne Neldner

Eric R. Geertsma email

发布: 2017年02月05日第7卷第3期 DOI: 10.21769/BioProtoc.2116 浏览次数: 9551

评审: Arsalan DaudiChing Yao YangAnonymous reviewer(s)

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Cover of Nature Structural & Molecular Biology, featuring study using the protocol.

Oct 2015

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Abstract

The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, termed SLC26Dg, from Deinococcus geothermalis. Following these instructions we obtained well-folded and homogenous material of the membrane protein SLC26Dg and the nanobody Nb5776 that enabled us to crystallize the complex and determine its structure (Geertsma et al., 2015). The procedure may be adapted to purify and crystallize other membrane protein complexes.

Keywords: Membrane transport protein (膜转运蛋白)

Nanobody (纳米抗体)

Crystallization chaperone (结晶伴侣)

Solute carrier (溶质载体)

SLC26 (SLC26)

Background

With few exceptions, structural characterization of membrane proteins involves challenges at the level of protein production, stabilization in the detergent-solubilized state, and crystallization. The strategy we have followed to overcome these hurdles relied on the efficient selection of SLC26 homologs with superior biochemical properties and the use of antibodies as crystallization chaperones (Geertsma et al., 2015). The procedures described here do not greatly deviate from those of colleagues, but on a few points we do follow alternative approaches. For example, for protein production we make use of the araBAD promoter (Guzman et al., 1995) and not the popular T7 promoter (Studier et al., 1990). In contrast to the T7 promoter, the P_BAD promoter allows direct tuning of the protein production levels and its adjustment to the capacity of the downstream folding machinery, thereby reducing the formation of inclusion bodies (Geertsma et al., 2008). Furthermore, we prefer nanobodies, the variable domain of camelid heavy chain only antibodies (Pardon et al., 2014), as crystallization chaperones over the more commonly used Fabs. In our hands, the generation, selection, and production of nanobodies is far more robust and straightforward. Though we are aware that alternative protein production strategies (Henderson et al., 2000; Kunji et al., 2003; Miroux and Walker, 1996; Studier, 2005; Wagner et al., 2008) and crystallization chaperones (Koide, 2009; Seeger et al., 2013) exist, we did not explore these as the presented procedures proved very robust and successful.

Materials and Reagents

Dialysis tube
MWCO 8 kDa (Carl Roth, catalog number: 1924.1 )
MWCO 3.5 kDa (Carl Roth, catalog number: E860.1 )
Concentrators
MWCO 50 kDa (EMD Millipore, catalog number: UFC905024 )
MWCO 3 kDa (EMD Millipore, catalog number: UFC800324 )
Crystallization plates (HAMPTON RESEARCH, catalog number: HR3-158 )
Potter tube and piston (VWR, catalog numbers: 432-0205 and 432-0211 )
50 ml tube
E. coli MC1061 (Coli Genetic Stock Center, catalog number: 6649 )
Plasmid pBXNPHM3-Nb5776 (Figure 1A)
Note: This plasmid holds the gene coding for the nanobody under control of the araBAD promoter and results in the production of the nanobody fused to an N-terminal pelB leader sequence followed by decaHis-tag, MBP, and a HRV 3C protease site. It contains a beta-lactam antibiotic marker. Plasmid available for non-commercial use upon request.
Plasmid pBXC3GH-SLC26Dg (Figure 1B)
Note: This plasmid holds the gene coding for SLC26Dg under control of the araBAD promoter and results in the production of SLC26Dg fused to a C-terminal HRV 3C protease site, GFP, and a decaHis-tag. It contains a beta-lactam antibiotic marker. Plasmid available for non-commercial use upon request.

Figure 1. Plasmid maps. A. Plasmid map of pBXNPHM3-Nb5776; B. Plasmid map of pBXC3GH-SLC26Dg. Unique restriction sites and important features in the plasmids are indicated.
Ampicillin sodium salt (Carl Roth, catalog number: K029.2 )
L-arabinose (20% w/v in ddH₂O) (Carl Roth, catalog number: 5118.2 )
1 M KPi, pH 7.5 (see Recipes)
K₂HPO₄(AppliChem, catalog number: 121512 )
KH₂PO₄(AppliChem, catalog number: 131509 )
Sodium chloride (NaCl, 2.5 M, ddH₂O) (Carl Roth, catalog number: 3957.1 )
Lysozyme (100 mg/ml in ddH₂O) (AppliChem, catalog number: A3711 )
DNAse I (2 mg/ml in ddH₂O) (AppliChem, catalog number: A3778 )
Magnesium sulphate heptahydrate (MgSO₄, 1 M, ddH₂O) (Carl Roth, catalog number: P027.2 )
Phenylmethyl sulphonyl fluoride (PMSF, 200 mM in ethanol) (Carl Roth, catalog number: 6367.1 )
NiNTA (50% w/v slurry in 20% EtOH) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 88223 )
ddH₂O
Imidazole (2 M, pH 7.5, HCl, ddH₂O) (Carl Roth, catalog number: X998.4 )
HEPES (1 M, pH 7.5, NaOH, ddH₂O) (Carl Roth, catalog number: HN78.2 )
HRV 3C protease (homemade)
Polypropylene glycol 2000 (10% v/v in ddH₂O) (Sigma-Aldrich, catalog number: 81380-1L )
EDTA, disodium salt dihydrate (Carl Roth, catalog number: X986.1 )
Glycerol, 86% (w/v) (Carl Roth, catalog number: 4043.3 )
Liquid nitrogen
Decylmaltoside (10% w/v in ddH₂O) (Anatrace, catalog numbers: D322S and D322LA )
Ammonium formate (Carl Roth, catalog number: 5093.1 )
Sodium acetate (Na-acetate) (Carl Roth, catalog number: 6773.2 )
PEG400 (Sigma-Aldrich, catalog number: 91893 )
Tryptone (AppliChem, catalog number: A1553 )
Yeast extract (AppliChem, catalog number: A1552 )
Agar (Applichem, catalog number: A0917 )
LB agar (see Recipes)
TB medium (see Recipes)
Reservoir solution (see Recipes)

Equipment

5 L baffled flask
Incubator at 37 °C
Shaker with adjustable temperature (Infors, model: HT Multitron )
Centrifuge for pelleting cultures (Thermo Fisher Scientific, Thermo Scientific, model: Thermo Sorvall Evolution RC )
Homogenizer (IKA, model: ULTRA-TURRAX^® T25 )
High pressure cell disrupter (Avestin, model: Emulsiflex C3 )
Nanodrop (Thermo Fisher Scientific, Nanodrop^TM, model: 1000 , discontinued)
Magnet stirrer (Heidolph Instruments, model: Hei-Mix S )
SEC column Superdex 75 10/300 GL (GE Healthcare, catalog number: 17-5174-01 )
SEC column Superdex 200 10/300 GL (GE Healthcare, catalog number: 28-9909-44 )
Table-centrifuge (VWR, model: Micro Star 17 )
HPLC (GE Healthcare, model: ÄKTAprime plus )
Fermenter (BIOENGiNEERiNG, model: NLF 22, 30 L)
Spectrophotometer (Amersham Biosciences, model: Ultrospec 10 )
Note: This product has been discontinued.
Ultracentrifuge (Beckman Coulter, model: Optima XPN-100K Ultracentrifuge )

Procedure

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Chang, Y., Shaik, F. R., Neldner, Y. and Geertsma, E. R. (2017). Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies. Bio-protocol 7(3): e2116. DOI: 10.21769/BioProtoc.2116.