Easy and efficient protocol for purification of recombinant peptides

Protein Expression and Purification 95 (2014) 129–135

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Easy and efficient protocol for purification of recombinant peptides Prashant Kumar, Gopala K. Aradhyam ⇑ Department of Biotechnology, IIT Madras, Chennai 600 036, India

a r t i c l e

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Article history: Received 26 September 2013 and in revised form 27 November 2013 Available online 19 December 2013 Keywords: Peptides Acetonitrile Glutathione S-transferase (GST) PreScission protease Volatile buffer

a b s t r a c t Peptide synthesis and purification remains a challenge. Low abundance leads to small yields when peptides are purified from natural sources. On the other hand, synthetic methods are limited by the chemical properties of the amino acids and the concurrent aggregation of peptides. In this paper, we report a versatile, high yielding and general purification method for randomly chosen recombinant peptides of variable sizes (ranging from 1.7 kDa to 10 kDa). Expressed as fusion proteins with commonly used tag proteins, these peptides are cleaved by ‘PreScission protease’ in a volatile buffer that makes concentration and recovery of the peptide easy. Separation of the cleaved peptide is achieved by selective precipitation of the larger tag protein with acetonitrile; leaving the peptide in solution. Our protocol can be used to generate a wide variety of peptides in significant quantities for biochemical, biophysical and physiological studies. Ó 2013 Elsevier Inc. All rights reserved.

Introduction Peptides are bioactive molecules that act as ligands (e.g. apelin, angiotensin, insulin, glucagon) [1–4], inhibitors (e.g., cyclicpeptides, peptide analogues) [5], toxins (e.g., conotoxin and snake toxins) [6,7], biomarkers [8], molecules of therapeutic importance (e.g., bacteriocin) [9] and have often been employed for probing the structure–function relationship of proteins [10]. Peptides are obtained either through chemical synthesis, using solid phase chemistry [11], or purified from natural sources [9]. Despite the popularity of solid phase method, peptide synthesis has several limitations e.g., steric hindrance due to bulkier side chains and/or amino acid protecting groups [12–15] and problems with coupling reaction efficiency [16,17]. While recombinant DNA technology helps overcome many of these problems, purification of the peptide remains a major hurdle for large-scale requirements. ⇑ Corresponding author. Address: Signal Transduction Lab, Department of Biotechnology, IIT Madras, Chennai 600 036, India. Tel.: +91 4422574112; fax: +91 4422574102. E-mail addresses: [email protected] (P. Kumar), [email protected] (G.K. Aradhyam). 1 Abbreviations used: MBP, maltose binding protein; GST, glutathione S-transferase; CNBr, cyanogen bromide; APJ C-ter, APJ C-terminal; IPTG, isopropyl b-D-1-thiogalactopyranoside; PMSF, phenylmethanesulfonylfluoride; GdmCl, guanidine hydrochloride; CV, column volumes; DTT, dithiothreitol; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; CCA, alphacyanohydroxycinnamic acid; TFE, 1,1,1-trifluoroethanol; GFP, green fluorescent protein; GPCR, G-protein coupled receptor; DMEM, Dulbecco’s modified eagle medium; FBS, foetal bovine serum. 1046-5928/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2013.12.004

Few examples exist of cloning and expression of peptides in the bacterial system, mostly as fusion partners to maltose binding protein (MBP1) or glutathione S-transferase (GST) for ease of purification, that either report low yield or have not been applied to purification of peptides of variable sizes and structures [18–23]. Peptides are cleaved from the tag protein either by cyanogen bromide (CNBr) [24] or with enzymes (to cleave at engineered enzyme sites), and are subsequently separated by reversed phase liquid chromatography [25]. Two drawbacks with CNBr cleavage method are (i) its inapplicability to peptides containing methionine and (ii) generation of unwanted homoserine lactone as a by-product [26]. Another problem, emanating from the small size of most peptides, is getting salt-free concentrated peptide. In order to overcome these limitations we have developed a simple, efficient and versatile strategy to purify peptides of varying sizes (in salt-free condition) expressed as fusion partners to MBPHis6 or GST in bacterial system. Our procedure involves two steps: (i) cleavage of the peptide from its fusion partner in a volatile buffer (0.15 M NH4HCO3) and (ii) selective precipitation of the larger tag protein with acetonitrile. Use of this protocol not only makes desalting redundant but also reduces a purification step. We report the use of this protocol in purification of peptides of variable sizes (from 1.70 kDa to 10 kDa) unlike previous attempts [19,21,22,24]. The procedure is quantifiable and amenable to scale up processes. To prove our point we chose peptides from (a) soluble protein (b) an intra-cellular peptide of a trans-membrane protein and (c) a peptide ligand (with varying sizes). We have characterized the secondary structure of these peptides and also demonstrated functional activity of the peptide ligand apelin (1.70 kDa).

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Methods Cloning, expression and purification of rat nesfatin1, APJ C-terminal domain, apelin23⁄ and apelin The genes for rat nesfatin1, APJ C-terminal (APJ C-ter), apelin23⁄ and apelin peptides were cloned in bacterial expression vectors. Rat nesfatin1 gene was chemically synthesized (Eurofins-MWG) and inserted into pCR2.1, a TA cloning vector using primers with adaptors for NheI and XhoI restriction sites. It was sub-cloned into a modified pMAL-c5E vector to give MBP-His6-rat nesfatin1. The vector was modified to have 6 Histidine for purification and PreScission protease cleavage site at the N-terminal of rat nesfatin1. The other peptide gene (APJ C-ter) was amplified by PCR using pcDNA3.0APJ clone as template and primers containing BamHI and EcoRI restriction site adaptors (Supplementary Table 1). It was inserted into pGEM-T vector (a TA cloning vector) and subsequently sub-cloned into pGEX-6P-1 expression vector to generate GST–APJ C-ter. Apelin23⁄ gene was chemically synthesized and inserted into another TA cloning vector (pGEM-T Easy) and subsequently sub-cloned into a single EcoRI site of pGEX-6P-1 vector to generate GST-apelin23⁄. Extra 8 amino acids at N-terminal domain of apelin23⁄, generated as a consequence of this protocol, were deleted using site directed mutagenesis kit from Stratagene as per the manufacturer’s protocol to generate GST-apelin (Supplementary Table 1). All clones and their orientations were confirmed by nucleotide sequencing. All the peptides with their respective tags were expressed in bacteria (see below) for purification. MBP-His6-rat nesfatin1 gene was expressed in Escherichia coli BL21 (DE3). Cells were grown at 37 °C until A600  0.9 was achieved. Cells were then induced with 500 lM isopropyl b-D-1-thiogalactopyranoside (IPTG) in LB media at 37 °C for 6 h. All other peptides with GST tag (APJ C-ter, apelin23⁄ and apelin) were expressed in E. coli ER2566 cells. These cells were grown at 37 °C to achieve A600  0.9. Cells were then induced with 200 lM IPTG at 24 °C for 12 h. All cells were harvested by centrifuging them at 18,000g and subsequently stored at 80 °C. Purification by affinity chromatography: Cells expressing MBPHis6-rat nesfatin1 were re-suspended in high salt buffer [50 mM Tris–Cl (pH 8.0) and 500 mM NaCl, containing 2 mM b-mercaptoethanol, 1 mM phenylmethanesulfonylfluoride (PMSF) and 2 M guanidine hydrochloride (GdmCl)]. Cells were mechanically lysed in a ‘vibrasonic’ cell disrupter at ultrahigh (amplitude of 30 db) frequency with 2 s ‘on’ and 4 s ‘off’ cycle, for 2 min. Soluble protein fractions were obtained by centrifugation at 18,000g for 45 min at 4 °C and purified by passing through a 4 ml Ni–NTA resin at a flow rate of 0.1 ml/min. The resin was then washed with 8 column volumes (CV) of high salt buffer containing 0.6 M GdmCl (flow rate of 0.4 ml/min) followed by another wash of 6 CV with high salt buffer without GdmCl. The fusion protein was finally eluted with 2 CV of Ni–NTA elution buffer (high salt buffer with 300 mM imidazole). Peptides with GST tag were purified by GSH resin. GST–APJ C-ter failed to bind to GSH resin efficiently in cleavage buffer alone [20 mM Tris–Cl (pH 7.4), 150 mM NaCl, 1 mM PMSF and 1 mM dithiothreitol (DTT)]. Hence, a variety of conditions were attempted with different concentrations of urea (e.g., 0.5 M, 1.0 M and 2 M) in the cleavage buffer. The presence of 2 M urea yielded the best binding conditions. The entire cellular protein (12 ml) was incubated with 4 ml GSH resin for 12 h at 4 °C with end-to-end mixing (batch method). The resin was washed thrice with 4 CV of the cleavage buffer in 2 M urea. The bound protein was eluted with 4 CV of elution buffer (10 mM glutathione in 50 mM Tris–Cl, pH 8.0). Bacterial cells over-expressing the other tagged proteins (GST-apelin23⁄,

GST-apelin and Pre-Scission protease) were lysed in the cleavage buffer alone and the resultant soluble cellular protein of GSTapelin23⁄, GST-apelin and PreScission protease were passed through GSH resin at a flow rate of 0.1 ml/min, subsequently washed with 10 CV of cleavage buffer at a flow rate of 1 ml/min. The protein was then eluted with 3 CV of elution buffer. The eluted samples obtained of all the purified proteins were pooled and divided into two parts and buffer exchanged into either cleavage buffer or volatile buffer. The purity of all the tagged proteins was checked on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) [27]. The fusion proteins yields were estimated by Bradford method [28]. Peptide cleavage Peptides were cleaved by PreScission protease to release them from their affinity tags. PreScission protease (with a unique octamer cleavage sequence, LEVLFQ/GP) tagged to GST used throughout this study, was also purified to 99.0% by GSH affinity chromatography [29]. The purified fusion proteins were digested either in cleavage buffer or volatile buffer with PreScission protease. The concentration of the fusion proteins was kept at 0.5 mg/ ml for GST–APJ C-ter, and 1 mg/ml for MBP-His6 rat nesfatin1, GST-apelin23⁄ and GST-apelin respectively. The cleavage of the fusion proteins was done at 8 °C for 16 h with protein-to-protease ratio at 25:1 (w/w) in both the buffers. Peptide purification All the peptides released from tags by protease cleavage were purified in the following manner. The cleaved tags, GST of APJ C-ter peptide and MBP-His6 of rat nesfatin1 peptide were precipitated with acetonitrile in ratio of either 1:1 or 2:1 (acetonitrile: buffer; v/v) in cleavage or volatile buffers at 25 °C for 20 min. The solution was mixed by inverting it 3–4 times and kept standing for 20 min to complete the precipitation. The soluble fractions containing the peptides were separated by centrifugation at 19,000g for 20 min at 4 °C. The supernatants were freeze-dried and stored for further analysis. The purity of the peptides was checked by analyzing the samples on Tricine–SDS–PAGE [30]. In fact, acetonitrile precipitation in the ratio of 2:1 (acetonitrile: volatile buffer; v/v) was also tested for its efficiency on purification of rat nesfatin1 at different fusion protein concentrations 1 mg/ml, 0.5 mg/ml and 0.25 mg/ml. For large-scale purification of the peptides, 50 mg each of GSTapelin23⁄ and GST-apelin fusion proteins were digested in volatile buffer and precipitated with acetonitrile in the ratio 2:1 (acetonitrile: buffer; v/v) in buffer as mentioned above. The soluble fractions containing the peptides were freeze-dried and reconstituted in 1 ml of 30% acetonitrile and 0.1% trifluoroacetic acid (TFA) to check purity. Peptide fractions were loaded on size-exclusion ‘‘peptide column’’ (Superdex peptide 10/300 GL column, GE Life Sciences). The column was run at 0.5 ml/min and fractions of 1-ml size were collected (monitored by absorbance at 280 nm). The fractions corresponding to peaks were analyzed by MALDI-MS. The purified peptides rat nesfatin1, apelin23⁄ and apelin were estimated by fluorescamine method [31]. MALDI-MS analysis of apelin23⁄ and apelin The purity of apelin23⁄ and apelin peptides, and their sizes were analyzed on MALDI-MS for which the samples were initially mixed in 1:1 ratio of CCA resin (alpha-cyanohydroxycinnamic acid). Subsequently, the resin (10 mg/ml) was prepared by re-suspending it in 1:1:1 ratio of water/acetonitrile/0.1% TFA. Spectra were recorded

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Fig. 1. (a) MBP-His6-rat nesfatin1 digestion by PreScission protease in volatile buffer and peptide rat nesfatin1 (starred) purification by acetonitrile treatment: L1-MBP-His6 rat nesfatin1 purified chimera, L2-MBP-His6 tag and released nesfatin1 peptide after protease cleavage, L3-Protein molecular marker, L4-Soluble fraction had both MBP-His6 tag (partially precipitated) and rat nesfatin1 peptide after precipitation in 1:1 (v/v) ratio of acetonitrile to volatile buffer and L5-Soluble fraction had only pure peptide rat nesfatin1 fraction (shown by ‘‘’’) after precipitation in 2:1 (v/v) ratio of acetonitrile to volatile buffer. (b) MBP-His6-rat nesfatin1 digestion by PreScission protease in cleavage buffer and peptide rat nesfatin1 purification (starred) by acetonitrile treatment: L1 and L3-Protein molecular marker, L2-MBP-His6 nesfatin1 purified chimera, L4-MBP-His6 tag and released nesfatin1 peptide after cleavage, L5-Soluble fraction had both MBP-His6 tag (partially precipitated) and rat nesfatin1 peptide after precipitation with 1:1 (v/v) ratio of acetonitrile to volatile buffer and L6-Soluble fraction had only pure peptide rat nesfatin1 fraction (shown by ‘‘’’) after precipitation with 2:1 (v/v) ratio of acetonitrile to volatile buffer. (c) GST–APJ C-ter digestion by PreScission protease in volatile buffer and APJ C-ter peptide (starred) purification by acetonitrile treatment: L1Molecular marker, L2-GST–APJ C-ter purified chimera, L3-GST tag and released APJ C-ter peptide after protease cleavage, L4-Soluble fraction had both GST tag (partially precipitated) and APJ C-ter peptide after precipitation with 1:1 (v/v) ratio of acetonitrile to volatile buffer, and L5-Soluble fraction had only pure peptide APJ C-ter fraction (shown by ‘‘’’) after precipitation with 2:1 (v/v) ratio of acetonitrile to volatile buffer. (d) GST–APJ C-ter digestion by PreScission in volatile buffer and APJ C-ter peptide (starred) purification: L1-Protein marker, L2-GST–APJ C-ter purified chimera, L3-GST tag and released APJ C-ter peptide after protease cleavage, L4-Soluble fraction had both GST tag (partially precipitated) and APJ C-ter peptide after precipitation with 1:1 (v/v) ratio of acetonitrile to volatile buffer, and L5-Soluble fraction had only pure APJ C-ter peptide fraction (shown by ‘‘’’) after precipitation with 2:1 (v/v) ratio of acetonitrile to volatile buffer.

Table 1 Purification and yields of fusion proteins and peptides: Total culture volume size was 200 ml LB broth in each case. The proteins were estimated by Bradford method [28] using bovine serum albumin (in lg) as standard. The peptides apelin23⁄ and apelin were estimated by fluorescamine method [31] using glutathione (in nano molar range) as standard. Fluorescamine binds to primary amine (amino terminal of peptides) and e amino group of lysine. We have considered 1 mol of glutathione (N-terminus and no lysines) is equivalent to 7 mol of rat nesfatin1 (N-terminus and 6 lysine residues), 2 mol of apelin23⁄ and 2 mol of apelin (N-terminus and 1 lysine for both) as reported earlier [31].

Weight biomass Column type and size Fusion protein yields Peptides and yield

MBP-His6 rat nesfatin1

GST-apelin23⁄

GST-apelin

0.6 g Ni–NTA column 4 ml 14 mg Rat nesfatin11.85 mg

1.42 g GSH column 4 ml 45 mg Apelin23⁄  4.00 mg

1.43 g GSH column 4 ml 50 mg Apelin  2.03 mg

in positive ion reflectron mode. Finally, external calibration was done using nine peptides with m/z ranging from 757.39 to 3147.47 Da. Study of the secondary structures of the purified peptides by circular dichroism Far-UV CD spectra of the peptides were recorded on a Jasco J815 CD spectrometer and data were collected by Spectrum Manager Software. The spectra for rat nesfatin-1, APJ C-ter, Apelin23⁄ and apelin were recorded in different concentrations of 1,1,1-trifluoroethanol (TFE: 0–80%) and buffer [10 mM phosphate buffer (pH 7.0) and 20 mM NaCl]. The molar ellipticities of all the purified peptides were calculated using the formula [h] = 100  h/(C  l) where C is the molar concentration, l is the path length in cm and a factor of 100 converts the path length in meter.

Apelin ligand activity assay by internalization Apelin is an endogenous ligand for APJ receptor, a cardiac G-Protein Coupled Receptor (GPCR). The activity of the purified apelin peptide was checked on green fluorescent protein (GFP) tagged APJ receptor stably expressed in HEK293 cells. HEK293 cells were grown and maintained in Dulbecco’s modified eagle medium (DMEM) with 10% foetal bovine serum (FBS) at 37 °C in a humidified chamber containing 5% CO2. For stable expression, HEK293 cells were transfected with DNA encoding GFP-tagged APJ receptor using lipofectamine2000 in accordance with the manufacturer’s (Invitrogen) protocol. After 24 h, transfected cells expressing the GFP tagged APJ receptor were split into 60 mm cell tissue culture plates to attain 20–25% confluence. The cells were allowed to adhere and grow in DMEM and 10% FBS for another 24 h. The media was then changed to DMEM, 10% FBS and 2 mg/ml of

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Fig. 3. Mass spectra of purified apelin23⁄ and apelin peptides after peptide column: (a) Apelin23⁄ fraction showed peak maxima at 2622.396 (m/z) corresponding to calculated molecular weight of the peptide. (b) Apelin showed peak maxima at 1704.974 (m/z) corresponding to the calculated molecular weight of the peptide. Fig. 2. Chromatogram is showing purity of apelin23⁄ and apelin peptide by peptide column: The soluble fractions of apelin23⁄ and apelin was precipitated with acetonitrile in 2:1 (v/v) ratio of acetonitrile to volatile buffer mixture and were loaded on ‘peptide column’ (Superdex peptide 10/300 GL column, GE Life sciences) pre-equilibrated with 30% acetonitrile + 0.1% trifluoroacetic acid. The eluent was monitored by UV absorbance (at 280 nm). The flow rate was maintained at 0.5 ml/ min and 1- ml fraction were collected (after the void volume of 8 ml) (a) apelin23⁄ peptide showed a single elution peak at 24 ml and (b) apelin showed a single peak at 24 ml.

G418 antibiotic (selective antibiotic for neomycin resistance gene) for selection. The cells were maintained in the same media with change of media at every 72 h. Cells were grown as such until pure colonies of stably expressing APJ receptor were found. The stable cells were maintained in DMEM with 10% FBS and 1 mg/ml G418 for further experiments. For internalization assay, the stable cell lines were grown in DMEM with 10% FBS and 1 mg/ml G418 for 24–48 h until they were 60–70% confluent. These cells were then treated with varying apelin concentrations of 50 nM, 0.5 lM and 5 lM and incubated further at 37 °C for 45 min in a humidified chamber with 5% CO2. The GFP-tagged receptor trafficking/internalization was analyzed with fluorescence microscope.

to bind GSH resin in the cleavage buffer. The binding conditions of the tagged protein were checked in varying amounts (0.5 M, 1 M and 2 M) of urea, in cleavage buffer (Supplementary Fig. 2). 2 M urea vastly improved the binding of GST–APJ C-ter to GSH resin. Subsequently, GST–APJ C-ter was purified to 90% purity by GSH affinity in cleavage buffer with 2 M urea (Fig. 1c and d). GST-apelin23⁄ and GST-apelin proteins were over-expressed at 24 °C and purified to 95–99% purity (Supplementary Figs. 3 and 4 respectively) by GSH affinity chromatography. The yields of all fusion proteins are listed in Table 1. Peptide cleavage

Results

All the peptides were cleaved from their tags by ‘PreScission protease’ (Supplementary Fig. 5). The MBP-His6-rat nesfatin1 (70 lg) was cleaved completely and rat nesfatin1 was released from the tag in both, cleavage buffer (L2 of Fig. 1a) and the volatile buffer (L2 of Fig. 1b) as is evident on 10% Tricine–SDS–PAGE gel. Similarly, GST–APJ C-ter (35 lg) was also cleaved both in cleavage buffer (L3 of Fig. 1c) and volatile buffer (L3 of Fig. 1d). The cleavage led to release of APJ C-ter of 7.8 kDa from GST tag as seen on 10% Tricine–SDS–PAGE.

Purification of tagged peptides with affinity tags

Peptide purification

The MBP-His6-rat nesfatin1 purification was done in the presence of high salt buffer containing 2 M GdmCl to prevent its degradation (Supplementary Fig. 1). The protein was purified up to 90% homogeneity (L1 of Fig. 1a and L2 of Fig. 1b) by Ni–NTA affinity chromatography. The other tagged protein, GST–APJ C-ter failed

Desalting is a major problem in peptide/small molecular weight protein purification process. In order to avoid this, we have demonstrated the use of a volatile buffer that does not affect the activity of PreScission protease. Additionally, we employed precipitation with acetonitrile to selectively remove the ‘tag proteins’ while

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Fig. 4. Secondary structure of peptides by CD spectroscopy: (a) Rat nesfatin1 peptide was unstructured in buffer and helicity was induced by 20% TFE, (b) APJ C-ter, (c) Apelin23⁄ and (d) Apelin demonstrated unstructured behavior, not affected by different concentrations of TFE (0–80%).

Fig. 5. APJ receptor internalization assay: Apelin ligand activity was analyzed for GFP tagged APJ receptor stably expressed in HEK293 cells. The GFP tagged APJ receptor showed internalization, as seen by punctuate appearance, at different concentrations of the ligand (a) without ligand, (b) 50 nM apelin,(c) 0.5 mM apelin and (d) 5 mM apelin.

the peptides remain soluble. A ratio of 2:1 (v/v; acetonitrile:cleavage/volatile buffer) precipitates MBP-His6 tag and GST tag completely thereby yielding pure rat nesfatin1 (L5 of Fig. 1a and L6 of Fig. 1b) and APJ C-ter (L5 of Fig. 1c and d) peptides respectively. The use of lower acetonitrile solvent, at the ratio of 1:1 (v/v; acetonitrile to cleavage/volatile buffer) failed to show good purification (L4 of Fig. 1a, L5 of Fig. 1b, L4 of Fig. 1c and L4 of Fig. 1d). This

acetonitrile purification protocol is also effective at lower protein concentrations (0.25 mg/ml, 0.5 mg/ml and 1 mg/ml) shown by MBP-His6-rat nefatin1 as an example (Supplementary Fig. 6). Similar results were also obtained with apelin23⁄ (2.62 kDa) and apelin (1.70 kDa), both cloned as GST tag. Purity of apelin23⁄ and apelin peptides was confirmed by the appearance of single peak while performing chromatography on peptide column using an

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Discussion

Akta purification system (GE Life Sciences) (Fig. 2a and b respectively). Apelin peptide though showed a broad peak (Fig. 2b) probably due to conformational heterogeneity.

Over the years peptides have become indispensable products/ reagents, with an ever increasing variety of uses in biochemistry and medicine [32–34]. Generation of peptides of specific sequence and their purification remains a major challenge. In order to address this difficulty, we have developed a general two-step purification protocol (summarized in Fig. 6) for recombinant peptides using four different model peptides, chosen without any bias. Acetonitrile has previously been employed to precipitate high abundance proteins in the process of identifying biomarkers like neuro-peptides from spinal cord fluid [35] and in proteomic/metabolic analysis of folate in blood serum [36]. We have extended this concept to purify recombinant tagged peptides expressed with affinity tags like GST, MBP-His6 (using a PreScission protease site separating them). The tag and the peptides were subsequently treated with acetonitrile resulting in the precipitation of the larger GST or MBP-His6 tags, leaving the relevant peptides in soluble fraction. Acetonitrile, owing to its hydrophobic nature leads to loss of interactions amongst charged residues and hydrogen bonding interactions with water molecules on the surface of the protein, in turn, leading to exposure of hydrophobic residues to the surface. Hydrophobic interactions among protein molecules causes their aggregation and precipitation [37–39]. Peptides, on the other hand, owing to their small size, do not exhibit large hydrophobic interactions and remain soluble. Our method will be immensely useful in purifying recombinant peptides, their mutants in large amounts and also aid in obtaining isotopically labeled peptides for structural studies [40]. We have determined the secondary structure of these purified peptides, rat nesfatin1 adopting alpha helical secondary structure in 20% TFE. Recombinant apelin peptide (1.70 kDa) purified by our method induces internalization of APJ receptor in HEK293 cells, thereby demonstrating the retention of biological activity of peptides.

MALDI-MS of apelin23⁄ and apelin

Conclusion

Apelin23⁄ and apelin were further analyzed by MALDI-MS to confirm their purity and molecular size. The purified fractions demonstrated a major peak of 2.62 kDa (mass to charge: m/z) (Fig. 3a) and another of 1.70 kDa (m/z) (Fig. 3b) corresponding to the calculated molecular weights of the respective peptides. The additional peaks 1720.974 (m/z) observed in Fig. 3b may correspond to oxidation of methionine and the other additional major peak 1690.315 due to nitrogen atom loss (NH3 from any of the amino acids R, Q, K or N) during ionization steps.

By this simple method, we have succeeded in purifying recombinant peptides of different sizes (1.7 kDa to 10.12 kDa) with ease and without being limited by the size, primary sequence or secondary structure, a problem inherent in most existing protocols for peptide purification. Our procedure has an edge over the existing protocols and circumvents many of the problems.

Fig. 6. Scheme showing generalized recombinant peptide purification by acetonitrile treatment method.

Secondary structure of the purified peptides Rat nesfatin1 was unstructured in phosphate buffer, attaining alpha helicity in 20% TFE with negative peaks at 208 nm and 222 nm and a positive peak at 195 nm (Fig. 4a). None of the other peptides exhibited any secondary structure.

Acknowledgments We thank Dr. Robert W. Doms, University of Pennsylvania, for providing human APJ receptor clone in pCDNA3. We also thank Dr. Sai Krishna for making apelin23⁄ clone in pGEM-T Easy vector and generating modified pMALc5e vector with 6 histidine tag for Ni-NTA affinity purification and having PreScission protease cleavage site. We thank Mr. Pavan Mujawdiya for making rat nesfatin1 gene clone in modified pMALc5e vector system. We also thank DBT, New Delhi and IIT Madras for financial support.

APJ receptor internalization assay Appendix A. Supplementary data APJ receptor is a cardiac GPCR known to be activated by the peptide ligand apelin leading to its internalization. The apelin ligand mediated activation of GFP tagged APJ receptor was analyzed by fluorescence microscopy. The purified apelin ligand (1.7 kDa) obtained by us was biologically active and was able to induce internalization of green fluorescent tagged APJ receptor, demonstrated by punctuate appearances in HEK293 cells in a dose dependent manner (Fig. 5).

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