Protein Expression and Purification 109 (2015) 113–119

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Secretagogin, a hexa EF-hand calcium-binding protein: High level bacterial overexpression, one-step purification and properties Anand Kumar Sharma ⇑, Radhika Khandelwal, Yogendra Sharma, Vangipurapu Rajanikanth CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad 500 007, India

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Article history: Received 13 September 2014 and in revised form 22 December 2014 Available online 19 February 2015 Keywords: Secretagogin His-tag Protein expression CaBPs Oligomerization

a b s t r a c t Secretagogin (SCGN), a hexa EF-hand calcium-binding protein, is highly expressed in the endocrine cells (especially in pancreatic islets) and in restricted neuronal sub-populations, albeit at comparatively low level. Since SCGN is predicted to be a potential neuroendocrine marker in carcinoid tumors of lung and gastrointestinal tract, it is of paramount importance to understand the features of this protein in different environment for assigning its crucial functions in different tissues and under pathophysiological conditions. To score out the limitation of protein for in vitro studies, we report a one-step, high purity and high level bacterial purification of secretagogin by refolding from the inclusion bodies yielding about 40 mg protein per litre of bacterial culture. We also report previously undocumented Ca2+/Mg2+ binding and hydrodynamic properties of secretagogin. Ó 2015 Elsevier Inc. All rights reserved.

Introduction Secretagogin (SCGN)1 is a member of hexa EF-hand superfamily of Ca2+-binding proteins (CaBP), expressed in endocrine cells, particularly in pancreatic islets which is evident from abundance of SCGN mRNA [1–3]. Due to its increased expression in some carcinomas, SCGN has been proposed as a biomarker for some types of lung and gastrointestinal tract cancers [4,5], and as a predictive biomarker for traumatic brain injury [6]. SCGN has also been shown to exert neuroprotective role in Alzheimer’s disease where those hippocampal neurons which express SCGN are largely resistant to neurodegeneration [7]. Although, there are six putative EF-hands in the primary structure of SCGN, only four have canonical sequence and thus bind Ca2+ with relatively weaker affinity (represented as [Ca2+]0.5 of approximately 25 lM) [8], contrary to the Ca2+-binding affinities (generally in nanomolar range) of the members of Neuronal Calcium Sensor (NCS) family. Despite comparatively low affinity (lM) towards its ligand, Ca2+ induces conformational changes in tertiary structure of SCGN suggesting that this protein may belong to the calcium sensor class of hexa EF-hand proteins [9,10]. However, one of the regulatory features of the sensor class of CaBP is their participation in a signaling pathway or enzyme modulation, an attribute not yet identified in SCGN. Although there is no experimental evi-

⇑ Corresponding author. Tel.: +91 40 27192562; fax: +91 40 27160591. E-mail address: [email protected] (A.K. Sharma). Abbreviations used: SCGN, secretagogin; BME, b-mercaptoethanol; CaBP, calciumbinding proteins; DTT, dithiothreitol. 1

http://dx.doi.org/10.1016/j.pep.2015.02.011 1046-5928/Ó 2015 Elsevier Inc. All rights reserved.

dence suggesting a role of SCGN in the regulation of enzymes, it is predicted to interact with adenylate cyclase, tyrosine–kinases and neuroendocrine convertase 1 precursor enzymes; however, at low confidence levels (http://www.compbio.dundee.ac.uk/www-pips/ using protein’s uniprot id O76038 and cut off score 0.025) [11,12]. In the light of predicted and proposed multidimensional functions of hexa EF-hand protein SCGN, it seems obvious that it plays important roles in physiological and secretary pathways of endocrine system and yet they largely remain unexplored [7,8,13–16]. In order to assist in deducing the structure and functions of the protein by crystallography, NMR or by other biophysical and biochemical methods without the limitation of protein quantity, we report a one-step high level bacterial expression and purification of SCGN with a His-tag. Existing protocols described by Rogstam et al. [8] or Bitto et al. [16] involve expression of SCGN fusion with glutathione S-transferase (GST) or mannose-binding protein (MBP) and subsequent cleavage of the fusion protein with appropriate protease, which makes these approaches more expensive than the Ni-NTA affinity purification described here. However, the yields achieved by Rogstam et al. and Bitto et al. were sufficient (49 mg from two-litre of bacterial culture) [16] for the extensive characterization and crystallization of the recombinant SCGN. The yield by the protocol described here is apparently more (40 mg per litre of bacterial culture) and is convenient which could boost in vitro research concerning this protein. Further, we also report some of the essential characteristics of mouse SCGN which are not known yet.

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A.K. Sharma et al. / Protein Expression and Purification 109 (2015) 113–119

Materials and methods Cloning Mouse brain cDNA (a kind gift from Salil Saurav Pathak, CCMB) was used as template to amplify full-length SCGN (accession No. BC016093) using forward primer with NdeI restriction site (sequence 50 -ACCAAGCATATGGACAACGCACGC-30 ) and reverse primer with XhoI site (50 -GGATCACTCGAGGGGGTTTATTTTTAGCCC30 ). The PCR amplified gene product was digested with NdeI and XhoI and ligated in pET21b vector. The resulting clone was transformed in Escherichia coli DH5a competent cells and SCGN gene sequence was confirmed by DNA sequencing. The confirmed clone was finally transformed into expression host E. coli BL-21(DE3) for protein overexpression. Overexpression and purification E. coli BL-21 (DE3) cells, harboring the plasmid of interest, were grown in LB media. Various temperatures and IPTG concentrations were optimized for best expression. After OD600 reached 0.6, protein expression was induced with various concentrations of IPTG in bacterial culture and allowed it to grow at 37 °C for varying incubation times. Induction by 0.5 mM IPTG followed by 7 h post-induction incubation at 37 °C was found to yield best overexpression. Cells were harvested and after initial check, the overexpressed protein was found in the insoluble fraction and none was in soluble fraction. Briefly, bacterial cell pellet was suspended in 7 M urea prepared in 50 mM Tris buffer, pH 7.5 and 150 mM KCl. Suspension was sonicated for 40 min with 50% efficiency. Lysate was centrifuged for 1 h at 18,000 rpm at 4 °C and supernatant was loaded onto a pre-equilibrated (50 mM Tris buffer, pH 7.5 and 150 mM KCl) Ni-NTA column. After removal of urea from the column by washing with 8 volumes of wash buffer (50 mM Tris pH 7.5, 150 mM KCl, 1 mM DTT, 20 mM imidazole), bound protein was eluted with a gradient flow of elution buffer (50 mM Tris, 150 mM KCl, 1 mM DTT) containing imidazole concentrations of 50 and 250 mM. The fractions were analyzed by SDS–PAGE on a 12% gel. Fractions containing pure protein were pooled and concentrated to 5 ml using Amicon ultrafiltration device with a 10 kDa molecular weight cut-off membrane. The concentrated protein was loaded onto a Superdex-75 column (GE-Wipro), pre-equilibrated with 50 mM Tris, 150 mM KCl and 1 mM DTT for the final step of purification. The pure fractions were pooled, and concentrated to desired level by Amicon concentrator. For preparing the Ca2+ free protein, solution was incubated with 0.1 mM EDTA for half an hour followed by buffer exchange with Chelex-purified buffer (50 mM Tris, pH 7.5, 150 mM KCl) to remove EDTA from the protein solution. We further evaluated if solubilization of inclusion bodies by urea and subsequently on-column refolding could yield protein in native form. Analytical gel filtration The hydrodynamic volume and oligomeric properties of SCGN (1 mg/ml sample) were studied on a Superose 12 (10/300) column (Wipro GE Healthcare) under reducing (1 mM DTT) or non-reducing (without DTT) conditions. Column was pre-equilibrated with buffer (50 mM Tris, pH 7.5, 100 or 150 mM KCl) containing either 0.05 mM EGTA or 2 mM Ca2+ for apo or holo conditions respectively. Protein elution was performed with a flow rate of 0.5 ml/min. Native/semi-native PAGE Protein samples (prepared in appropriate buffer containing either 0.05 mM EGTA or 2 mM Ca2+) were incubated at room

temperature for 30 min in reducing (with 1 mM DTT) or non-reducing conditions. Native acrylamide gels were prepared as for SDS–PAGE except the absence of SDS both in acrylamide gel and in running buffer. Loading dye was also free from any denaturing/reducing agent (b-mercaptoethanol). Isothermal titration calorimetry (ITC) Ca2+/Mg2+ binding affinities were evaluated by ITC experiments at 30 °C on a Microcal VP-ITC instrument. Protein samples (30 lM concentration) were prepared in Chelex-treated 50 mM Tris, pH 7.5, and 150 mM KCl buffer. The protein sample in the cell was titrated against appropriate ligand (either 3 mM CaCl2 or 10 mM MgCl2) loaded in the syringe. The blanks obtained by titrating the buffer with respective ligand under identical conditions were subtracted from the respective sample titrations. Data were evaluated using the program Origin 8, supplied by the vendor. Fluorescence spectroscopy Intrinsic tryptophan fluorescence emission spectra of protein samples (0.1 mg/ml concentration in 50 mM Tris–HCl, pH 7.5, 150 mM KCl) were recorded after excitation at 295 nm in the correct spectrum mode of the instrument on a F-4500 fluorescence spectrometer (Hitachi Inc., Japan). The spectra (300–450 nm) were recorded using excitation and emission band slits set at 5 nm each. The protein was titrated with increasing concentration of Ca2+ (500 nM–3 mM) or Mg2+ (1 lM–5 mM). Mg2+-saturated protein was titrated with 500 nM to 1 mM Ca2+. Circular dichroism spectroscopy Circular dichroism (CD) spectra were recorded on a Jasco J-815 spectropolarimeter in far-UV as well as in near-UV regions using cuvette of appropriate path length. Protein samples (1 mg/ml) were prepared in 50 mM Tris (pH 7.5) containing 150 mM KCl and titrated with increasing Ca2+ or Mg2+ concentrations. All spectra were corrected for buffer baseline. Results Bacterial overexpression and purification of SCGN Optimal overexpression of histidine-tagged SCGN was standardized using different concentrations of IPTG (up to 1 mM) maintaining the post-induction incubation temperature at 37 °C. The best expression was obtained at 0.5 mM IPTG (and above) concentration with 7 h incubation, which we used for further preparations (Fig. 1a). In our overexpression experiments under different IPTG conditions, protein formed inclusion bodies with almost no protein in the soluble fraction at 0.5 mM IPTG concentration (Fig. 1a, lanes 2 and 3). At low temperature (18 °C) and IPTG concentration of