Cloning, purification, crystallization and preliminary X-ray studies of human α₁-microglobulin

Here, the expression, purification, crystallization and preliminary crystallographic analysis of human α₁m are reported.

Abstract

α₁-Microglobulin (α₁m) is one of the phylogenetically most widespread lipocalins and is distributed in various organs and tissues, including liver, heart, eye, kidney, brain, lung, pancreas and skeletal muscle. α₁m has been found to exert multifarious functions, including interacting with IgA, albumin and prothrombin, binding strongly to haem and exhibiting reductase activity. Nevertheless, little structural information is available regarding these functions of α₁m. Since determination of three-dimensional structure is a powerful means of functional characterization, X-ray crystallography was used to accomplish this task. Here, the expression, purification, crystallization and preliminary crystallographic analysis of human α₁m are reported. The crystal belonged to space group P4₃, with unit-cell parameters a = b = 36.45, c = 112.68 Å, and diffracted to a resolution of 2.0 Å. The crystals are most likely to contain one molecule in the asymmetric unit, with a V _M value of 1.63 ų Da⁻¹.

1. Introduction  

The lipocalins, which are found in animals, plants and bacteria, are a superfamily of proteins consisting of 30–35 members (Flower, 1996 ▶; Åkerström et al., 2000 ▶). The family has a well conserved three-dimensional structure, folding into a barrel consisting of eight antiparallel β-strands with a closed end and an open end which provides a binding site for small hydrophobic ligands. Interestingly, lipocalins exert a surprisingly wide array of biological functions, but only a few members have been characterized, including the plant enzyme violaxanthin de-epoxygenase (Hieber et al., 2000 ▶), insect bilin-binding protein (Huber et al., 1987 ▶), and prostaglandin D-synthase (Urade & Hayaishi, 2000 ▶) and α₁-microglobulin (α₁m) from animals (Åkerström & Lögdberg, 2006 ▶).

α₁m, also called protein HC, is one of the phylogenetically most widespread lipocalins (Tejler & Grubb, 1976 ▶). To date, it has been found in mammals, birds, fish and amphibians (Åkerström & Lögdberg, 2006 ▶). α₁m is mainly synthesized in the liver, secreted into the blood and rapidly distributed to various organs and tissues, including liver, heart, eye, kidney, brain, lung, pancreas, skeletal muscle and blood. The lipocalin exits in a free form or as a component of protein complexes by binding to IgA, albumin and prothrombin (Berggård et al., 1998 ▶; Allhorn et al., 2002 ▶). The amino-acid sequence is highly conserved and the extent of its distribution indicates that α₁m plays an important physiological function. Previous research has found that α₁m can bind strongly to haem and that t-α₁m, a truncated variant α₁m that lacks the C-terminal tetrapeptide LIPR, can degrade haem with the help of Hb (Allhorn et al., 2002 ▶). In addition, the secreted protein also exerts reductase and antioxidant activities by serving as a radical scavenger (Åkerström et al., 2007 ▶). Nevertheless, little information is available about the active sites (or regions) and the mechanisms of the reductase and antioxidant activities of α₁m. In order to further investigate the biochemical and physiological functions of α₁m, we report the expression, purification, crystallization and preliminary X-ray crystallographic studies of human α₁m. Structure determination will be pursued using experimental phasing methods. Determination of the three-dimensional structure of α₁m will help in understanding how this widespread protein exerts its multitudinous functions.

2. Materials and methods  

2.1. Cloning and expression  

The gene encoding α₁m was amplified from human cDNA by PCR using primers (α₁mFwd and α₁mRev) that contained EcoRI and XhoI restriction sites, respectively. Both the PCR products and the plasmid pET28a were digested with EcoRI and XhoI restriction enzymes. The ligation mixture was transformed into chemically competent Escherichia coli DH5α cells and insertion was verified by PCR using T7 promoter and T7 terminator primers. The identity of the insert was further confirmed by DNA-sequence analysis. The final construct (pET28a-α₁m) encodes α₁m protein with an N-terminal His tag. The recombinant expression vector was transformed into chemically competent E. coli BL21 (DE3) cells for expression. Cultures of bacteria carrying pET28a-α₁m were grown overnight in 20 ml LB medium supplemented with 50 mg l⁻¹ kanamycin. The bacteria were then used to inoculate 2.0 l LB medium and cultured at 310 K for about 4 h. The protein was induced by adding isopropyl β-­d-1-thiogalactopyranoside (IPTG; 0.3 mM) when the OD_600 nm reached 0.4; the culture was allowed to grow at 293 K for a further 15 h before the cells were harvested by centrifugation.

2.2. Purification  

The cells were resuspended in lysis buffer (20 mM Tris–HCl pH 8.0, 300 mM NaCl) and disrupted by sonication. The cell lysate was centrifuged at 15 000g for 30 min at 277 K and the cell debris was removed. The recombinant protein in the supernatant was applied onto a Ni²⁺–NTA (Qiagen) column pre-equilibrated with lysis buffer. Nonspecifically bound proteins were washed from the column with 100 ml lysis buffer containing 50 mM imidazole. The recombinant protein was then eluted from the column with 20 ml elution buffer (20 mM Tris–HCl pH 8.0, 200 mM imidazole). The elution buffer was then loaded onto a HiTrap Q column (GE Healthcare) pre-equilibrated with 20 mM Tris–HCl pH 8.0 and the target protein was eluted with a linear gradient of 0–1000 mM NaCl in 20 mM Tris–HCl pH 8.0. For crystallization, fractions containing α₁m were concentrated and buffer-exchanged into the final buffer (5 mM Tris–HCl pH 8.0, 50 mM NaCl) using a Millipore Amicon concentrator with a 10 kDa cutoff membrane. The concentration of the purified protein was 10 mg ml⁻¹ and its purity was determined to be about 95% by SDS–PAGE.

2.3. Crystallization  

Preliminary screening of crystallization conditions was performed by the hanging-drop vapour-diffusion method with Crystal Screen, Index and PEG/Ion kits (Hampton Research). The crystallization experiments consisted of 1.0 µl protein solution mixed with 1.0 µl reservoir solution and equilibrated against 400 µl reservoir solution at 293 K. After 6 d, small crystals of α₁m were observed using a reservoir condition consisting of 20%(w/v) PEG 3350, 0.2 M ammonium citrate tribasic pH 7.0. Further optimization of the conditions using PEGs of different molecular weights at various concentrations gave good diffraction-quality crystals using 25–35%(w/v) PEG 3350 in the presence of 0.1 M HEPES pH 6.6–7.5. Crystals were obtained after 5 d equilibration against the crystallization solution and grew to full size (0.05 × 0.05 × 0.1 mm) in 15 d (Fig. 1 ▶).

Figure 1.

A crystal of human α₁m as grown by the hanging-drop method. The average dimensions of the crystals were 0.1 × 0.05 × 0.05 mm.

2.4. Data collection and processing  

Crystals were transferred to a reservoir solution adjusted to 20%(v/v) glycerol and immediately placed in a 100 K nitrogen-gas stream. X-ray diffraction data were collected from a single crystal using a MAR CCD detector on beamline U17O at SSRF (Fig. 2 ▶). The images were processed using MOSFLM (Leslie, 1992 ▶) and SCALA from the CCP4 suite (Evans, 2006 ▶; Winn et al., 2011 ▶). The final data-collection and processing statistics are given in Table 1 ▶.

Figure 2.

X-ray diffraction image from a native human α₁m crystal recorded using a MAR CCD detector. The edge of the detector corresponds to a resolution of 2.0 Å.

Table 1. Data-collection statistics.

Values in parentheses are for the highest resolution shell.

Wavelength (Å)	1.0
Space group	P43
Unit-cell parameters (Å)	a = b = 36.45, c = 112.68
Resolution range (Å)	50–2.0 (2.07–2.00)
Total reflections	284280 (1946)
Unique reflections	9947
Completeness (%)	99.9 (98.9)
〈I/σ(I)〉	16.7 (8.5)
Multiplicity	7.1 (7.3)
Rmerge†	7.0 (35.8)

^† R _merge = , where I_i(hkl) is the intensity of the ith measurement of an equivalent reflection with indices hkl.

3. Analysis and discussion of preliminary X-ray diffraction results  

The overexpression method led to 20 mg pure protein being obtained from 3 l LB culture medium after purification by metal-affinity and ion-exchange chromatography. The purified α₁m was crystallized under PEG-containing conditions. Crystals suitable for X-ray diffraction were obtained after 15 d. A total of 284 280 measured reflections were merged into 9947 unique reflections with an R _merge of 7.0%. The merged data set was 99.9% complete to 2.0 Å resolution. The relevant data-collection statistics are given in Table 1 ▶. The α₁m crystal belonged to the tetragonal space group P4₃, with unit-cell parameters a = b = 36.45, c = 112.68 Å (Table 1 ▶). Based on the molecular weight of α₁m (about 23 kDa) and space group P4₃, solvent-content analysis indicated that one molecule could be accommodated per asymmetric unit, suggesting a V _M value (Matthews, 1968 ▶) of 1.63 ų Da⁻¹ and a solvent content of 24.4%. Molecular replacement using AMoRe (Navaza, 2001 ▶), Phaser (McCoy et al., 2005 ▶) and MOLREP (Vagin & Teplyakov, 2010 ▶) was carried out using the human prostaglandin D-synthase structure (PDB entry 3o19; Zhou et al., 2010 ▶) as a search model. In order to solve the structure of α₁m by multiwavelength anomalous dispersion methods, a selenomethionyl derivative of α₁m, which contains four methionine residues, is being prepared.