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Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany
Reprint requests to: Arne Skerra, Lehrstuhl für Biologische Chemie, Technische Universität München, D-85350 Freising-Weihenstephan, Germany; e-mail: skerra{at}wzw.tum.de; fax: +49-8161-714352.
(RECEIVED August 15, 2005; FINAL REVISION September 30, 2005; ACCEPTED October 3, 2005)
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Abstract |
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= 90°), thus demonstrating its conformationally homogeneous behavior and providing a basis for the future X-ray structural analysis of this functionally still puzzling protein. Keywords: E. coli expression; lipocalin; protein engineering; gel permeation chromatography; protein structure/folding; stability and mutagenesis; lipoproteins; isolation; characterization
Abbreviations: ApoD, apolipoprotein D • BBP, bilin-binding protein • GPC, gel permeation chromatography • HDL, high-density lipoprotein • IMAC, immobilized metal affinity chromatography • KD, dissociation constant • OmpA, outer membrane protein A
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051775606.
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Introduction |
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Besides progesterone, a series of potential low molecular weight ligands was described for ApoD in different studies (see Vogt and Skerra 2001). However, thorough investigation with the recombinant protein in our own laboratory revealed measurable affinities—in the low micromolar range—merely for one further ligand, arachidonic acid (Vogt and Skerra 2001). Indeed, ApoD could mediate the transport of arachidonate—and possibly of chemically related fatty acids—during neuronal cell growth.
Based on the detectable sequence homology with the structurally well-characterized insect bilin-binding protein (BBP) (Huber et al. 1987), human ApoD was assigned as a member of the lipocalin family (Peitsch and Boguski 1990). Lipocalins are compact globular proteins that serve for the transport or storage of vitamins, hormones, and secondary metabolites in many organisms. Their tertiary structure comprises a circularly closed eight-stranded anti-parallel -sheet. This -barrel supports four loops at one of its ends, which form the entrance to the ligand pocket.
Despite low mutual sequence identity, the -barrel is structurally well conserved among the lipocalins (Skerra 2000). Yet, the four loops at its open end exhibit large conformational differences and size variation between individual lipocalins, thus giving rise to the diverse natural ligand specificities. In principle, the architecture of lipocalins resembles that of the immunoglobulin variable region, where altogether six hypervariable loops are supported on a rigid framework. Consequently, it was possible to employ ApoD as molecular scaffold to reshape its binding site for the recognition of a prescribed ligand (Vogt and Skerra 2004).
The glycosylation of natural human ApoD varies depending on the tissue where it is synthesized (Rassart et al. 2000). When co-isolated with lecithin: cholesterol acyl-transferase from plasma, ApoD was shown to be modified at both of its potential N-glycosylation sites, Asn45 and Asn78 (Schindler et al. 1995), whereas ApoD shows a different glycosylation pattern when isolated from apocrine gland secretions (Zeng et al. 1996). However, ApoD can also be produced as a soluble protein without glycosylation, e.g., via secretion into the periplasm of E. coli, where its two intrachain disulfide bonds are efficiently formed (Vogt and Skerra 2001).
Although ApoD exhibits specific ligand-binding activity when isolated from E. coli, the recombinant protein reveals a pronounced tendency to adsorb to glass surfaces and to form aggregates upon storage (Vogt and Skerra 2001). Remarkably, attempts to purify this protein by gel permeation chromatography (GPC) failed because ApoD apparently adsorbed to the column matrix and could be eluted only under denaturing conditions. It seems unlikely that this peculiar behavior of recombinant ApoD is attributable to the missing glycosylation alone; rather, this lipocalin may exhibit hydrophobic surface properties that also enable it to interact with HDL particles and with cell membranes in the physiological environment.
Nevertheless, there is a need for monodisperse and highly soluble protein preparations in order to permit biophysical studies and to obtain crystals for X-ray structural analysis. Herein, we report the systematic substitution of hydrophobic surface residues of ApoD without loss of its ligand-binding function, and we describe the successful crystallization of one of the resulting mutants.
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Results |
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The recombinant "wild-type" ApoD (wtApoD), encoded on the corresponding vector pApo D10 (Vogt and Skerra 2004), already carried four side-chain substitutions at surface positions. Cys116, the residue that forms the disulfide bridge to Cys6 of human apolipoprotein A-II, had been replaced by Ser (Vogt and Skerra 2001). Furthermore, the mutations Leu23Pro, Pro13Val, and Asn134Ala served to introduce two BstXI restriction sites for simplified cloning and mutagenesis of the central coding region. These amino acid replacements were previously shown not to influence the gross protein behavior or ligand-binding activity of recombinant ApoD (Vogt and Skerra 2004).
Using the three-dimensional model based on the sequence homology with the bilin-binding protein (Peitsch and Boguski 1990), exposed hydrophobic residues of ApoD that were likely to cause its sticky protein character were identified (Fig. 1
). To investigate the influence of these hydrophobic side chains on the solubility of the bacterially produced protein, we prepared several mutants of ApoD via site-directed mutagenesis (Table 1). These mutants were tested for the yield of soluble production in the bacterial periplasm and the behavior in gel filtration experiments as detailed below. Based on the experimental observations that were made with the initially constructed ApoD mutants, some promising combinations of side-chain substitutions were prepared later on.
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-barrel in the vicinity of Asn78, i.e., one of the two glycosylation sites. In fact, its side chain forms a hydrophobic cluster together with Leu101 and Val13. Our hypothesis was that this region might be more exposed to solvent in the unglycosylated recombinant protein, and, therefore, hydrophilic side-chain substitutions were introduced at these positions. Trp99 was again replaced by a His side chain, either alone or in combination with the substitution of Leu101 by Gln or Val13 by Thr, respectively. Finally, loop 4 seems to exhibit a pair of large aliphatic side chains at its tip, Ile118 and Leu120, both in the vicinity of Cys116, which serves for covalent attachment to HDL in human ApoD. Together with Phe3 and Leu5, close to its N terminus, these side chains might give rise to another cluster of exposed hydrophobic residues, at least when assuming that the N-terminal segment packs against loop 4 in a similar manner as in the underlying crystal structure of BBP (Huber et al. 1987). Due to their mutually neighboring arrangement in the primary structure of ApoD, these four residues were exchanged in a pairwise manner: Phe3/Leu5 by Asn/Gln, and Ile118/Leu120 by Ser/Ser, respectively.
Following expression in the E. coli K-12 strain JM83, all mutants could be recovered from the bacterial periplasm by mild osmotic shock and efficiently purified via the Strep-tag II. Their purity was judged to be >95% as estimated from Coomassie-stained SDS-PAGE (Fig. 2). The purified wtApoD and all mutants investigated here appeared as single homogeneous bands with an apparent molecular size of 24 kDa. An increased electrophoretic mobility under nonreducing conditions indicated the presence of two properly formed disulfide bonds in each case (not shown). Notably, compared with the recombinant wtApoD, the yields of the mutants ApoD (W99H) and ApoD(W99H/I118S/L120S) were significantly improved by a factor between two and three (Table 1
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Thus, gel filtration was employed as a sensitive indicator of improved solubility of ApoD mutants, and the impact of side-chain substitutions on its chromatographic behavior was studied. The large exposed side chains of Trp91 and Trp99 appeared to be the first promising candidates for replacement and were separately substituted by His residues, resulting in the mutants ApoD(W91H) and ApoD(W99H). In analytical GPC experiments, using a calibrated Superdex 75 HR 10/30 column (resolution range of 3000–70,000 molecular mass) and 20 mM Tris/HCl (pH 8.0) buffer, the mutant ApoD(W91H) exhibited the same behavior as wtApoD and was not eluted. In contrast, ApoD (W99H) showed elution in one prominent peak, yet with an unexpectedly large retention volume (Fig. 3).
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GPC of both ApoD(W99H/F3N/L5Q) and ApoD (W99H/V13T)— performed under the optimized conditions—revealed just very low peaks, and none of them contained detectable amounts of protein (data not shown). Hence, these mutants exhibited essentially the same behavior as wtApoD, and the entire protein adsorbed to the column. On the contrary, the mutant ApoD(W99H/L101Q) predominantly eluted in the exclusion volume of the column, indicating the formation of microdispersed aggregates in this case, even though accompanied by a minor fraction that corresponded to monomeric protein.
In contrast, combination of the substitution Trp99His with the mutations Ile118Ser and Leu120Ser led to the proper elution of the recombinant protein from the gel filtration column (Fig. 3). ApoD(W99H/I118S/L120S) was obtained mainly as a monomeric protein fraction with the expected retention volume, almost identical to that of the structurally related BBP (Schlehuber et al. 2000), which was used as a positive control (not shown). Even after repeated chromatography, this mutant eluted as a stable monomeric protein with unchanged elution volume and without indication of aggregate formation (Fig. 3), demonstrating that the solubility properties of ApoD had been favorably improved.
Biochemical characterization of the engineered ApoD
The preparative bacterial production and purification of ApoD(W99H/I118S/L120S) via streptavidin affinity chromatography and subsequent gel filtration was analyzed by SDS-PAGE (Fig. 4). Gel electrophoresis under reducing conditions revealed a single homogeneous band with an apparent size of ~24 kDa. Under nonreducing conditions the electrophoretic mobility of the purified protein was markedly increased, thus indicating proper formation of the two intramolecular disulfide bridges. The circular dichroism (CD) spectrum of the engineered ApoD in the far-UV region revealed characteristic features of the predominant -sheet secondary structure (Brahms and Brahms 1980; Sreerama and Woody 2000), with a slightly blue-shifted negative band at < 205 nm and a positive band <200 nm (Fig. 5). In particular, the CD spectrum was virtually identical with that of the recombinant wtApoD, thus indicating conservation of the -barrel fold.
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), a typical saturation curve was observed, revealing a dissociation constant of 0.549 ± 0.032 µM. This value was essentially indistinguishable from the dissociation constant of 0.568 ± 0.095 µM measured for the recombinant wtApoD in an accompanying experiment under the same conditions.
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In conclusion, the three side-chain replacements that were introduced in order to raise the solubility of the recombinant Apo D did not affect its binding property for the steroid ligand progesterone. A comparable result was obtained with arachidonic acid, indicating that the ligand-binding profile of the engineered protein was fully retained (data not shown).
Optimized bacterial production and protein crystallization
Initial screens with ApoD(W99H/I118S/L120S) carrying the Strep-tag II at its C terminus, purified by streptavidin affinity chromatography and subsequent gel filtration, did not give rise to crystals suitable for X-ray diffraction. Based on the general observation that affinity tags may influence the crystallization behavior of recombinant proteins, the Strep-tag II was replaced by a His6 tag (Skerra 1994a).
ApoD(W99H/I118S/L120S)-His6 was successfully produced both in E. coli K-12 JM83 (Yanisch-Perron et al. 1985) in the shake flask and in the K-12 strain KS272 (Strauch and Beckwith 1988) in an 8-L bench top fermenter. During these expression experiments the influence of cotransformation with pTUM4, a generic vector for the periplasmic overexpression of the disulfide isomerases DsbA and DsbC as well as the chaperone-like proline isomerases SurA and FkpA (M. Schlapschy, S. Grimm, and A. Skerra, in prep.), was investigated. In the presence of pTUM4, the production of the soluble engineered ApoD was significantly improved, leading to a four times higher yield of purified protein (i.e., 0.4 mg/L vs. 0.1 mg/L for the shake flask).
After fermenter production, ApoD(W99H/I118S/L120S)-His6 was purified from the periplasmic cell fraction via immobilized metal affinity chromatography (Skerra 1994a), followed by gel filtration on a preparative column. The majority of the engineered ApoD eluted in a single fraction at a volume expected for the monomeric protein, and subsequent SDS-PAGE analysis revealed a homogeneous preparation, whereby the disulfide bonds were quantitatively formed. Fluorescence titration with progesterone revealed a dissociation constant similar to the one previously measured for the engineered protein carrying the Strep-tag II.
Screening for crystallization conditions was performed using the vapor diffusion technique (McPherson 1999) according to conventional procedures. Crystals with dimensions of ~200 x 200 x 50 µm3 were obtained after 1 d in the presence of 1.1–1.5 M Li2SO4, 73–100 mM Hepes/NaOH (pH 7.5), 5% (v/v) glycerol (Fig. 7). A complete X-ray diffraction data set with a maximal resolution of 3.5 Å was collected using a rotating anode source (CuK) and successfully indexed as belonging to the tetragonal space group I4 with unit cell parameters a = 75.1 Å, b = 75.1 Å, c = 166.0 Å, = = = 90°. The data with 5818 unique reflections were 99.8% complete from 30.00 to 3.50 Å (100% completeness in the 3.69–3.50 Å shell) with an Rmerge of 24.8% (42.1% in the last shell) and a multiplicity of 8.2.
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Discussion |
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Residue Trp99 is probably located on the surface of the -barrel structure, in vicinity to the N-linked glycosylation site at position Asn78. Hence, its large aromatic side chain becomes exposed in the unglycosylated protein synthesized by E. coli. Indeed, exchange of Trp99 by His resulted in a first mutant of ApoD that not only gave rise to a higher yield of soluble periplasmic protein, but that could also be eluted from a gel permeation column under native conditions, albeit with an abnormally large retention volume. Nevertheless, the role of Trp99 in conferring hydrophobic properties to the unglycosylated ApoD seems to be a specific effect because a similar substitution of Trp91, which is probably even better exposed at the tip of loop 3, close to the open end of the -barrel, did not detectably improve the solubility properties.
Notably, the two additional side-chain substitutions, I118S and L120S, which finally resulted in a normal gel filtration behavior of ApoD, are located at the tip of loop 4 on the same face of the lipocalin fold as Trp99. These two residues are located close to Cys116, which is responsible for the covalent attachment of ApoD to human HDL via interchain disulfide bond formation with ApoA-II. During biogenesis this loop might mediate an initial hydrophobic interaction of ApoD with the HDL particle, by dipping into the lipid phase, before the covalent crosslink is formed. In other vertebrate species, such as rabbit, guinea pig, rat, and mouse, ApoD lacks the homologous Cys residue (Rassart et al. 2000). Therefore, the intermolecular disulfide bridge might be functionally dispensable, although not much is known about HDL association of ApoD in these animals.
Apart from the HDL-associated form that is abundant in plasma, human ApoD has been found as isolated protein in other body fluids such as apocrine secretion or organs, especially neuronal tissue. Under these circumstances, Cys116 is likely to be modified as a mixed disulfide with low molecular weight thiol compounds, e.g., glutathione. It is not clear to what extent ApoD exhibits hydrophobic association or adsorption behavior when prepared from those sources, but it seems likely that glycosylation of Asn78 together with post-translational modification of Cys116 with a hydrophilic substituent should alleviate this effect.
The correct fold of the engineered ApoD that results from the present investigation is supported by four experimental observations. First, the elution volume on a calibrated analytical gel filtration column was indistinguishable from the closely related BBP, whose three-dimensional structure already served for the construction of a model of ApoD (Peitsch and Boguski 1990). Second, its CD spectrum in the far-UV region revealed essentially the same secondary structure composition as for wtApoD. Third, ligand-binding studies with two well-characterized ligands of natural ApoD—progesterone and arachidonic acid—led to the same dissociation constant as for the recombinant wild-type protein (Vogt and Skerra 2001), thus demonstrating native biochemical activity. Finally, the engineered ApoD gave rise to crystals suitable for X-ray diffraction—even though at moderate resolution so far—thus demonstrating considerable molecular homogeneity of its protein preparation. In contrast, many attempts in our own and several other laboratories to crystallize either ApoD from human tissue or the bacterially produced recombinant protein have failed up to now.
The strategy of substituting exposed residues to influence the surface properties toward crystallizability of a recombinant protein has been applied before (Dale et al. 2003; Derewenda 2004). Previous examples mainly aimed at increasing the solubility of the protein versus nonspecific aggregation, for instance, by systematically mutating known surface residues of thymidylate synthase (McElroy et al. 1992) or with a fortuitously found Trp 
Glu mutation that allowed crystallization of human leptin (Zhang et al. 1997). Another example is the site-directed Phe Lys substitution in order to increase solubility and to successfully crystallize the catalytic domain of HIV-1 integrase (Jenkins et al. 1995).
While in this case the unmutated protein gave rise to oligomers and aggregates during gel filtration, a slightly retarded elution from the Superdex column was described for the solubilized mutant and could be abolished by adding a detergent. This behavior contrasts with the situation for the natural ApoD, which shows pronounced stickiness to the column and other surfaces, probably because of its physiological function as a lipid-associated protein, whereas our engineered ApoD does no longer adsorb to the chromatography matrix.
The diffraction quality of the presently described crystals of the "hydrophilized" ApoD should be sufficient to collect a data set with suitable resolution for structural elucidation at a synchroton X-ray beam line. An experimentally based tertiary structure of human ApoD, which will probably deviate from the existing theoretical model in several details, will not only shed new light on the physiological role of this abundant lipocalin but should also serve as a better starting point for functional protein engineering projects in the future.
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Site-directed mutagenesis was carried out with the single-stranded plasmid DNA according to published protocols (Geisselsoder et al. 1987) using the oligodeoxynucleotides 5'-TGCCGATGGCATAAAGTGGGAAAACTTAACTTC-3' to construct ApoD(W91H) and 5'-GGTGGCCAGGATGTGGTA CGGTGCCGA-3' to construct ApoD(W99H), thereby eliminating the BamHI restriction site. The vector encoding ApoD (W99H) was used as template for further mutagenesis with the primer 5'-AGGATTGGGGCACTTCCCTTGGTGGTTTGCTTGGGCCTGCGCTAC-3' to construct ApoD(W99H/F3N/L5Q), whereby a StyI restriction site was introduced; with the primer 5'-CGTCAAAATTCTCCTGCGTCGGAGGATTGG GGCA-3' to construct ApoD(W99H/V13T), whereby a BsaWI restriction site was eliminated; with the primer 5'-CATAGTCGGTGGCCTGGATGTGGTACGG-3' to construct ApoD(W99H/L101Q), whereby an MscI restriction site was eliminated; and with the primer 5'-CCAAGCAAAATCCAGGTGAAAAGATT GGGAGATGCTAGTACAGGAATACACG-3' to construct ApoD(W99H/I118S/L120S), whereby a SpeI restriction site was eliminated. Each mutagenesis was first checked by restriction analysis and then confirmed by double-stranded DNA sequencing (ABI-Prism 310 Genetic analyzer, Perkin-Elmer Applied Biosystems) using the BigDye terminator kit.
For production of ApoD(W99H/I118S/L120S) with a C-terminal His6 tag (-SAHHHHHH) instead of the Strep-tag II (-SAWSHPQFEK), the vector pApoD27 was constructed, which carries an ampicillin resistance gene instead of the chloramphenicol resistance gene. In this case the unique Eco47III restriction site upstream of the Strep-tag II permitted exchange with the His6 tag-encoding region from other compatible plasmids (Skerra 1994b; Fiedler et al. 2002).
Recombinant protein production and purification
E. coli K-12 strain JM83 (Yanisch-Perron et al. 1985) was used for the preparation of the recombinant ApoD and its mutants in 2-L shake-flask cultures as previously described (Vogt and Skerra 2001). For the fermenter scale production of ApoD(W99H/I118S/L120S)-His6, the E. coli K-12 strain KS272 (Strauch and Beckwith 1988) transformed with pApoD27 was employed. Bench-top fermentation was carried out at exponential growth in an 8-L bioreactor (Schütt Labortechnik) using a synthetic glucose minimal medium, similarly as described for the bacterial production of recombinant Fab fragments (Schiweck and Skerra 1995; Fiedler and Skerra 2001). Recombinant gene expression was induced with anhydrotetracycline (Skerra 1994b) as soon as the culture reached OD550 < 20, followed by an induction period of 2.5 h. Fermentation was also performed after cotransformation with the vector pTUM4 (M. Schlapschy, S. Grimm, and A. Skerra, in prep.), which leads to overexpression of the periplasmic protein-folding catalysts DsbA, DsbC, SurA, and FkpA, and carries the chloramphenicol resistance marker.
In each case the recombinant ApoD was extracted as a soluble protein from the periplasmic cell fraction. When using the vector pApoD10, purification was performed by means of the Strep-tag II via streptavidin affinity chromatography (Skerra and Schmidt 2000). When using pApoD27, the engineered protein was purified by means of the His6 tag via IMAC on Zn(II)-charged IDA Sepharose according to published protocols, either in the presence of betaine or without addition of a salt (Skerra 1994a; Schiweck and Skerra 1995).
Irrespective of the procedure, the recombinant ApoD had an apparent purity of 95% as judged by SDS-PAGE (Fling and Gregerson 1986). Protein concentration was determined according to the absorption at 280 nm using calculatory extinction coefficients (Gill and von Hippel 1989) of 37,530 M–1 cm–1 for wtApoD and of 31,840 M–1 cm–1 for ApoD(W91H), ApoD (W99H), ApoD(W99H/F3N/L5Q), ApoD(W99H/V13T), ApoD (W99H/L101Q), and ApoD(W99H/I118S/L120S).
Gel filtration
GPC was performed at an analytical scale on a Superdex 75 HR 10/30 column (24-mL bed volume, Amersham Pharmacia Biotech) using Dynamax SD-300 HPLC equipment with UV-1 absorbance detector (Rainin). After equilibration with 20 mM Tris/HCl (pH 8.5) (or with other pH values; see Results), the affinity-purified ApoD mutant was applied as a 250-µL sample at a concentration of ~4.5 µM (during inital experiments in the streptavidin affinity chromatography buffer [150 mM NaCl, 1 mM EDTA, 100 mM Tris/HCl at pH 8.0], but later dialyzed against 20 mM Tris/HCl at pH 8.5), and protein elution was monitored at 280 nm using a flow rate of 1 mL/min. The void volume was determined using Blue Dextrane 2000 (Sigma). Molecular size calibration was performed using carbonic anhydrase (29 kDa), myoglobin (17.6 kDa), and aprotinin (6.5 kDa) (Sigma), as well as recombinant BBP (21 kDa) (Schlehuber et al. 2000) as markers. For crystallization of the IMAC-purified engineered ApoD, GPC was carried out on a Superdex 75 HiLoad 16/60 prep grade column (Amersham Pharmacia Bio-tech) using 100 mM NaCl, 1 mM EDTA, 10 mM Tris/HCl (pH 8.0) as running buffer since the His6-tagged protein turned out to be prone to aggregation under low-salt conditions.
CD spectroscopy
Circular dichroism spectra were measured using a Jasco J-810 spectropolarimeter (Jasco). Solutions of wtApoD and its mutant ApoD(W99H/I118S/L120S) in 50 mM K2SO4, 20 mM K-phosphate (pH 7.5), both purified via the Strep-tag II, were applied in a quartz cuvette sealed with a Teflon cap. Spectra were recorded at 20°C from 190 to 250 nm (bandwidth 1 nm, scan speed 100 nm/min, using 20-fold accumulation) and corrected for buffer blank. The molar ellipticity was calculated as 
MRW = x MW/c x d x NA, where denotes the measured ellipticity [deg], MW is the molecular mass (20,341 g/mol for wtApoD and 20,240 g/mol for ApoD[W99H/I118S/L120S]), c is the concentration (0.244 mg/mL and 0.243 mg/mL, respectively), d is the path length of 0.1 cm, and NA corresponds to the number of 178 amino acids for both polypeptides, including the Strep-tag II.
Fluorescence titration
Fluorescence measurements were carried out in an LS 50 B instrument (Perkin-Elmer) essentially as described (Vogt and Skerra 2001). 2 mL of the purified ApoD mutant (1 µM) in citrate/phosphate buffer (100 mM NaCl, 10 mM Na-citrate, 10 mM Na-phosphate at pH 6.0) was titrated in a 1-cm2 quartz cuvette at 25°C with 2–4 µL aliquots of a gravimetrically prepared stock solution of progesterone (500 µM in dioxane/H2O). After equilibration for 1 min, the protein fluorescence was excited at a wavelength of 280 nm, and the Tyr/Trp emission was measured at 344 nm (averaged over 10 sec). After subtraction of the buffer blank, the values were normalized to an initial fluorescence intensity of 100%. Finally, the data were fitted by nonlinear least squares regression according to the Law of Mass Action for bimolecular complex formation.
Protein crystallization
ApoD(W99H/I118S/L120S)-His6 was purified from the fermenter culture of E. coli KS272 harboring both pApoD27 and pTUM4 via IMAC and GPC, followed by ultrafiltration (Amicon Ultra-4 centrifugal filter, cutoff 10,000; Millipore) and dialysis against 100 mM NaCl, 10 mM Tris/HCl (pH 8.0), and subjected to crystallization trials using the sitting or hanging drop vapor-diffusion technique (McPherson 1999). First, sparse matrix crystallization screens (Jancarik and Kim 1991) were performed, and initial conditions were later refined. Drops of 1 µL protein, at a concentration between 14 and 26 mg/mL, and 1 µL of reservoir solution were mixed and equilibrated against 500 µL of the reservoir solution at 20°C. Crystals appeared in the presence of 1.1–1.5 M Li2SO4, 73–100 mM Hepes/NaOH (pH 7.5), 5% (v/v) glycerol after 1 d. Crystals were mounted with a nylon loop (Hampton Research) after soaking them for a few seconds in the reservoir solution supplemented with 30% (v/v) glycerol as cryoprotectant and freezing at 100 K using a Cryostream 700 cooler (Oxford Cryosystems). Diffraction data were collected using a mar345 imaging plate detector (MarResearch) with CuK radiation from a RU-300 rotating anode source (Rigaku) equipped with a confocal Max-Flux optical system (Osmic). The diffraction data were processed with MOSFLM, scaled with SCALA, and reduced with TRUNCATE (CCP4 1994).
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Acknowledgments |
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References |
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Collaborative Computational Suite, Number 4 (CCP4). 1994. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50: 760–763.[CrossRef][Medline]
Dale, G.E., Oefner, C., and D’Arcy, A. 2003. The protein as a variable in protein crystallization. J. Struct. Biol. 142: 88–97.[CrossRef][Medline]
Derewenda, Z.S. 2004. The use of recombinant methods and molecular engineering in protein crystallization. Methods 34: 354–363.[CrossRef][Medline]
Fiedler, M. and Skerra, A. 2001. proBA complementation of an auxotrophic E. coli strain improves plasmid stability and expression yield during fermenter production of a recombinant antibody fragment. Gene 274: 111–118.[CrossRef][Medline]
Fiedler, M., Horn, C., Bandtlow, C., Schwab, M.E., and Skerra, A. 2002. An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity. Protein Eng. 15: 931–941.
Fling, S.P. and Gregerson, D.S. 1986. Peptide and protein molecular weight determination by electrophoresis using a highmolarity tris buffer system without urea. Anal. Biochem. 155: 83–88.[CrossRef][Medline]
Geisselsoder, J., Witney, F., and Yuckenberg, P. 1987. Efficient site-directed in vitro mutagenesis. Biotechniques 5: 786–790.
Gill, S.C. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182: 319–326.[CrossRef][Medline]
Hall, R.E., Horsfall, D.J., Stahl, J., Vivekanandan, S., Ricciardelli, C., Stapleton, A.M., Scardino, P.T., Neufing, P., and Tilley, W.D. 2004. Apolipoprotein-D: A novel cellular marker for HGPIN and prostate cancer. Prostate 58: 103–108.[CrossRef][Medline]
Huber, R., Schneider, M., Mayr, I., Müller, R., Deutzmann, R., Suter, F., Zuber, H., Falk, H., and Kayser, H. 1987. Molecular structure of the bilin binding protein (BBP) from Pieris brassicae after refinement at 2.0 Å resolution. J. Mol. Biol. 198: 499–513.[CrossRef][Medline]
Jancarik, J. and Kim, S.H. 1991. Sparse-matrix sampling—A screening method for crystallization of proteins. J. Appl. Crystallogr. 24: 409–411.[CrossRef]
Jenkins, T.M., Hickman, A.B., Dyda, F., Ghirlando, R., Davies, D.R., and Craigie, R. 1995. Catalytic domain of human immunodeficiency virus type 1 integrase: Identification of a soluble mutant by systematic replacement of hydrophobic residues. Proc. Natl. Acad. Sci. 92: 6057–6061.
McElroy, H.E., Sisson, G.W., Schoettlin, W.E., Aust, R.M., and Villa-franca, J.E. 1992. Studies on engineering crystallizability by mutation of surface residues of human thymidylate synthase. J. Cryst. Growth 122: 265–272.[CrossRef]
McPherson, A. 1999. Crystallization of biological macromolecules. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Milne, R.W., Rassart, E., and Marcel, Y.L. 1993. Molecular biology of apolipoprotein D. Curr. Opin. Lipidol. 4: 100–106.[CrossRef]
Peitsch, M.C. and Boguski, M.S. 1990. Is apolipoprotein D a mammalian bilin-binding protein? New Biol. 2: 197–206.[Medline]
Rassart, E., Bedirian, A., Do Carmo, S., Guinard, O., Sirois, J., Terrisse, L., and Milne, R. 2000. Apolipoprotein D. Biochim. Biophys. Acta 1482: 185–198.[CrossRef][Medline]
Schindler, P.A., Settineri, C.A., Collet, X., Fielding, C.J., and Burlingame, A.L. 1995. Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion. Protein Sci. 4: 791–803.[Abstract]
Schiweck, W. and Skerra, A. 1995. Fermenter production of an artificial Fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling. Proteins 23: 561–565.[CrossRef][Medline]
Schlehuber, S., Beste, G., and Skerra, A. 2000. A novel type of receptor protein, based on the lipocalin scaffold, with specificity for digoxigenin. J. Mol. Biol. 297: 1105–1120.[CrossRef][Medline]
Skerra, A. 1994a. A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene 141: 79–84.[CrossRef][Medline]
———. 1994b. Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151: 131–135.[CrossRef][Medline]
———. 2000. Lipocalins as a scaffold. Biochim. Biophys. Acta 1482: 337–350.[CrossRef][Medline]
Skerra, A. and Schmidt, T.G. 2000. Use of the Strep-tag and streptavidin for detection and purification of recombinant proteins. Methods Enzymol. 326: 271–304.[Medline]
Sreerama, N. and Woody, R.W. 2000. Circular dichroism of peptides and proteins. In Circular dichroism—Principles and applications (eds. N. Berova et al.), pp. 601–620. John Wiley & Sons, New York.
Strauch, K.L. and Beckwith, J. 1988. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc. Natl. Acad. Sci. 85: 1576–1580.
Thomas, E.A., Copolov, D.L., and Sutcliffe, J.G. 2003. From pharma-cotherapy to pathophysiology: Emerging mechanisms of apolipoprotein D in psychiatric disorders. Curr. Mol. Med. 3: 408–418.[CrossRef][Medline]
Vogt, M. and Skerra, A. 2001. Bacterially produced apolipoprotein D binds progesterone and arachidonic acid, but not bilirubin or E-3M2H. J. Mol. Recognit. 14: 79–86.[CrossRef][Medline]
———. 2004. Construction of an artificial receptor protein ("anticalin") based on the human apolipoprotein D. Chembiochem 5: 191–199.[CrossRef][Medline]
Yang, C.Y., Gu, Z.W., Blanco-Vaca, F., Gaskell, S.J., Yang, M., Massey, J.B., Gotto Jr., A.M., and Pownall, H.J. 1994. Structure of human apolipoprotein D: Locations of the intermolecular and intramolecular disulfide links. Biochemistry 33: 12451–12455.[CrossRef][Medline]
Yanisch-Perron, C., Vieira, J., and Messing, J. 1985. Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119.[CrossRef][Medline]
Zeng, C., Spielman, A.I., Vowels, B.R., Leyden, J.J., Biemann, K., and Preti, G. 1996. A human axillary odorant is carried by apolipoprotein D. Proc. Natl. Acad. Sci. 93: 6626–6630.
Zhang, F., Basinski, M.B., Beals, J.M., Briggs, S.L., Churgay, L.M., Clawson, D.K., DiMarchi, R.D., Furman, T.C., Hale, J.E., Hsiung, H.M., et al. 1997. Crystal structure of the obese protein leptin-E100. Nature 387: 206–209.[CrossRef][Medline]
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Ex = 280 nm/