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Preparation of uniformly 13C,15N-labeled recombinant human amylin for solid-state NMR investigation
Protein Expression and Purification 99 (2014) 119–130
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Preparation of uniformly 13C,15N-labeled recombinant human amylin for solid-state NMR investigation Iga Kosicka a,b, Torsten Kristensen a,c, Morten Bjerring a,b, Karen Thomsen a, Carsten Scavenius a,c, Jan J. Enghild a,c, Niels Chr. Nielsen a,b,⇑ a b c
Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark Department of Chemistry, Aarhus University, Denmark Department of Molecular Biology and Genetics, Aarhus University, Denmark
a r t i c l e
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Article history: Received 14 October 2013 and in revised form 1 April 2014 Available online 19 April 2014 Keywords: Amylin Amyloid fibrils Protein expression Solid-state NMR
a b s t r a c t A number of diseases are caused by the formation of amyloid fibrils. Detailed understanding of structural features of amyloid fibers is of great importance for our understanding of disease progression and design of agents for diagnostics or potential prevention of protein aggregation. In lack of 3D crystal ordering, solid-state NMR forms the most suited method to determine the structures of the fibrils with atomic resolution. To exploit this potential, large amounts of isotopic-labeled protein need to be obtained through recombinant protein expression. However, expression and purification of amyloidogenic proteins in large amounts remains challenging due to their aggregation potential, toxicity for cells and difficult purification. In this work, we report a method for the production of large amounts of uniformly labeled 13 15 C, N-human amylin, being one of the most amyloidogenic peptides known. This method utilizes inclusion bodies-directed expression and cheap chemical cleavage with cyanogen bromide in order to minimize the cost of the procedure compared to the use of less efficient proteolytic enzymes. We demonstrate the formation of amylin fibrils in vitro characterized using biophysical methods and electron microscopy, show toxicity towards human cells, and demonstrate that produced material may form the basis for structure determination using solid-state NMR. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Amyloid diseases (amyloidoses) are a subgroup of protein conformational diseases, characterized by abnormal protein folding leading to functional impairment, or formation of amyloid – extracellular, insoluble proteinaceous deposits [1]. Numerous conditions have been related to the presence of amyloid – among them a number of neurodegenerative disorders as well as non-neuropathic systemic amyloidoses or localized diseases [2]. Since its discovery in 1987 [3,4] as a major component of pancreatic amyloid deposits [3], amylin has drawn significant attention from the scientific community. Otherwise named islet amyloid polypeptide or IAPP1, amylin consists ⇑ Corresponding author. Address: Nordre Ringgade 1, 8000 Aarhus C, Denmark. Tel.: +45 87155913; fax: +45 86196199. E-mail address: [email protected] (N.Chr. Nielsen). 1 Abbreviations used: AFM, atomic force microscopy; CNBr, cyanogen bromide; EM, electron microscopy; EPR, electron paramagnetic resonance; HFIP, hexafluoro-2propanol; IAPP, islet amyloid polypeptide; MAS ssNMR, magic angle spinning solid state nuclear magnetic resonance; PB, phosphate buffer; rIAPP, recombinant islet amyloid polypeptide; sIAPP, synthetic islet amyloid polypeptide; STEM, scanning transmission electron microscopy; TEM, transmission electron microscopy; TFA, trifluoroacetic acid; ThT, thioflavin T. http://dx.doi.org/10.1016/j.pep.2014.04.002 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
of 37 residues and is a hormone co-secreted with insulin by pancreatic b-cells in response to nutrition [5]. Under normal conditions, amylin plays a role in glycemic regulation by controlling the level of glucose in blood and slowing gastric emptying, thereby promoting satiety [6]. Presence of insoluble amyloid fibrils in pancreas is observed in the majority of patients suffering from diabetes mellitus (diabetes type II), but also in approximately 16% of healthy elderly subjects, and the relation between aging and pancreas fibrosis is often underlined [4,7,8]. Diabetes is a result of decline in b-cells mass or loss of function and is ascribed to several factors such as islet inflammation, glucolipotoxicity, accumulation of cholesterol and formation of amyloid [9–11]. Toxicity of fibrillar amylin has been demonstrated for human and rat islet b-cells [12]. Pancreas amyloid is also a severe obstacle in islet cell transplant [13–15]. The reason for this poisonous effect is not clear yet – among the possible mechanisms the following are often mentioned: physical damage of cell membrane by the growing amyloid; uptake of phospholipid molecules during fibrillogenesis [16–18]; formation of pore arranging oligomers [19–21], or receptor-mediated events [22,23]. The precise mechanism of amylin polymerization into fibrils is not entirely understood, however, amylin shares properties with other amyloidogenic peptides – fibril formation is thought to be a non-covalent assembly process, nucleation-dependent, and
characterized by sigmoidal reaction profiles [24,25]. Probably both fibril-independent (nucleation-dependent) and fibril-dependent (fibrils acting as nuclei) events are involved during fibrils growth [26,27]. Although the accurate mechanism of the polymerization remains to be elucidated, it is commonly accepted that amyloid fibril formation is accompanied by transition from disordered or partially ordered peptide conformation into a b-sheet rich structure [25,28]. A common feature of amyloids is a helical array of b-strands perpendicular to the fibril axis [29,30]. The structure of amylin fibrils has been partly revealed. Goldsbury et al. [31] utilized TEM and STEM to characterize the morphology of amylin fibrils and showed its ability to form fibrils of higher order structures, which display distinct polymorphism into striated ribbons or coiled fibrils. Tycko and co-workers (Luca et al. [32]) deduced a low resolution structural model of striated ribbons consistent with experimental data derived from imaging techniques (AFM, TEM) and solid-state NMR on selectively 13C,15N-isotope labeled IAPP. According to this model the fibers consist of four layers of parallel b-sheets formed by two asymmetric layers of amylin molecules. The authors place the amino acid region 18–27 as the connection between two b-strands. This model was indirectly confirmed by biophysical studies conducted by Abedini and Raleigh [33]. The structure from [32] is in contrast to an older study of Glenner et al. [34], who, based on the ability of the IAPP 20-29 fragment to form fibrils, suggested that this region may be the fibrillation core of the molecule. A high-resolution supramolecular structure of the 20-29 fragment of IAPP was determined with MAS ssNMR by Nielsen et al. [35]. Structures of other two fibril forming fragments, residues 21–27 and 28–33, were determined with atomic resolution with X-ray crystallography by Wiltzius and Sievers [36]. These models are in close agreement with the structure presented in [32], except from minor differences in the packaging of amino acid side chains. Regarding the twisted morphology of amylin fibrils, Bedrood et al. [37] used wave and pulsed EPR and EM imaging to derive an atomistic model in which amylin monomers have the shape of a horseshoe consisting of two b-strands, spanning about 15 Å. Despite extensive studies, the high-resolution three-dimensional structures of IAPP in diverse aggregation states still require further investigations. Even less information is available about the relation between the structural variants of amylin aggregates and their role in b-cell toxicity. Structural insight into the aggregation mechanisms of IAPP is in acute need to guide therapeutic design, giving a perspective for prevention and more efficient treatment of the disorder, which according to the data of The International Diabetes Federation every year kills 4.8 million people worldwide. The access to structural information has been hampered by the fact that amylin is the most amyloidogenic peptide ever characterized [38]. Synthesis and purification of the full length peptide are troublesome [39], additionally pure amylin has low solubility in aqueous solutions, and once solubilized, it aggregates
instantaneously. Furthermore, amylin fibrils display high degree of polymorphism and to prepare morphologically uniform preparations consumes significant quantities of peptide. Overall for these reasons it is difficult to prepare large amounts of pure peptide required for structural studies using methods like magic-anglespinning (MAS) solid-state NMR [40–44]. Usually, in the order of 5–30 mg of isotope-labeled (typically 13C and 15N) pure fibrils is necessary for such investigations [45]. The wish for uniform or extensive labeling with 13C to establish sufficient restraints to derive a detailed structural model renders peptide synthesis costly and impractical. On the other hand, cheap and efficient production of recombinant proteins in biological systems require many purification steps and may involve use of expensive reagents, for example proteases used to liberate the pure peptide from its fusion partner [46–48]. In this work, we report a method of recombinant amylin production for solid-state NMR investigations. Escherichia coli expression in a bioreactor using thioredoxin (trxA) as a fusion partner for IAPP and efficient CNBr-based liberation of the pure peptide comprise an efficient and cheap method for producing milligram amounts of uniformly 13C,15N-labeled human islet amyloid polypeptide. We have characterized fibrils formed by recombinant peptide with TEM imaging, ThT fluorescence assay, circular dichroism and MAS solid-state NMR spectroscopy, and demonstrated toxicity towards human cells in vitro.
Materials and methods Preparation of the pET32-rIAPP1-37 expression vector The pET32-rIAPP1-37 expression vector is presented in Fig. 1. pET32 (Novagen) plasmid DNA was prepared for cloning by digestion with BglII and SalI. A 142-bp IAPP DNA fragment including the BglII and SalI sites as well as a methionine codon immediately upstream of the Lys1 codon in IAPP and a stop codon following the codon for Tyr37 was amplified by PCR using human chromosomal DNA as template. The PCR reaction conditions were as follows: denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s. Forward and reverse primers were: AmCNBrF: 50 -GGT GGA AGA TCT GAT GAA ATG CAA CAC TGC CAC ATG TG-30 , and AmstR: 50 -GGT GGT GTC GAC TCA ATA TGT ATT GGA TCC CAC GTT G-30 (BglII and SalI sites are underlined). PCR products were separated on 2% agarose gels and purified using the Nucleospin Extract II kit (Macherey–Nagel GmbH & Co. KG). The ends of the PCR product were trimmed with BglII and SalI (Fermentas), the resulting fragment purified on agarose gels, and ligated into the pET32 vector using T4 DNA ligase (Fermentas). The ligation mixture was transformed into competent E. coli TOP10F0 cells and plated onto LB agar plates containing ampicillin. Plasmids from single colonies were purified and confirmed by
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Fig. 10. Solid-state NMR spectra of U-13C,15N amylin fibrils acquired with 12 kHz MAS at 16.4 T. (A) 1D 13C CP/MAS spectrum. (B) 2D 13C–13C DARR correlation spectrum showing aliphatic and carbonyl regions. The spectrum is obtained using a 20 ms period for 13C–13C mixing. Certain signals which can be unambiguously assigned to a single amino acid type are highlighted.
than 1 ppm indicate a well-structured peptide, however, some degree of overlap is seen in the spectrum, and higher dimensional spectra are necessary for resolving all signals. Nonetheless, the system seems well suited for further solid-state NMR investigations, and a full set of 2- and 3-dimensional spectra will be acquired for a more complete structural analysis of the amylin fibrils. Preliminary analysis of the chemical shifts can be performed on the basis of the spectra in Fig. 10, where signals from some amino acid types have been indicated based on typical chemical shift values and chemical shift patterns. Chemical shift values for Ca, Cb, and C’ may be compared with random coil chemical shift values and using the principle of correlation between the chemical shifts and the backbone secondary structure introduced by Spera et al. [72], it is confirmed that the peptide adopts a b-sheet conformation, as expected. A single threonine spin system (indicated by a dotted line circle in Fig. 10B) shows chemical shift values which indicate random coil conformation rather than b-sheet and this spin system may be assigned to the C-terminal threonine. Conclusion The present work addresses the challenge of high-yield production of recombinant peptide amylin. The rIAPP1-37 peptide was expressed as a fusion protein with an N-terminal thioredoxin A fusion tag separated from rIAPP1-37 by a methionine residue, which is used as a target for cyanogen bromide digestion. The fusion protein is directed to inclusion bodies within the cell, which prevents undesired aggregation and enables easier purification under
denaturating conditions. Experiments using unlabeled recombinant IAPP1-37 were conducted in order to characterize fibril growth kinetics and verify toxicity of the produced material towards human cells. A yield of 16 mg of pure amylin per liter of culture was obtained and the fibrils were subjected to TEM imaging and preliminary solid-state NMR experiments. Fibrils dimensions and morphology correspond to other available data concerning amylin fibrils structure. Furthermore, the recombinant fibrils resemble the synthetic amylin fibrils, widely used in structural studies. Solidstate NMR data indicate that the recombinant peptide comprising amyloid fibrils is well structured. Nevertheless, due to signal overlap, sample requires further experiments utilizing two and three dimensional spectra, which are underway. The work presented provides an effective method of production of labeled amylin in large amounts, suited for solid-state NMR spectroscopy.
Acknowledgments This work is supported by Danish National Research Foundation (DNRF59). The authors would like to thank Anne Gylling for technical assistance with human cell cultures, Ida B. Thøgersen for performing the Edman degradation and Prof. Daniel Otzen for access to biophysical instrumentation. The authors would also like to thank Assoc. Prof. Kamille Smidt Rasmussen for supplying the INS-1E cells and the medium for the cell culture. We also thank Pierre Maechler and Prof. Claes Wollheim, University Medical Centre Geneva, Switzerland for permission to use the INS-1E cells for our project.
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Appendix A.
Table A1. Composition of media for recombinant amylin expression.
Component
M9 batch medium
M9 feeding medium
Na2HPO4 KH2PO4 NaCl FeCl3 MnCl2 ZnCl2 CoCl2 CuSO4 NiCl2 H3BO3 MgSO4 CaCl2 Thiamine (vit. B1) 12 C- or 13C-glucose 13 N- or 15N-NH4Cl
7.4 g/L 3 g/L 0.5 g/L 0.1 mM 1 mM 1 mM 0.2 mM 0.1 mM 0.2 mM 0.1 mM 1 mM 0.2 mM 100 lg/mL 8 g/L 2 g/L
22.2 g/L 9 g/L 0.5 g/L 0.2 mM 2 mM 2 mM 0.4 mM 0.2 mM 0.4 mM 0.2 mM 2 mM – 200 lg/mL 13.3 g/L 3.3 g/L
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Preparation of uniformly 13C,15N-labeled recombinant human amylin for solid-state NMR investigation Iga Kosicka a,b, Torsten Kristensen a,c, Morten Bjerring a,b, Karen Thomsen a, Carsten Scavenius a,c, Jan J. Enghild a,c, Niels Chr. Nielsen a,b,⇑ a b c
Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark Department of Chemistry, Aarhus University, Denmark Department of Molecular Biology and Genetics, Aarhus University, Denmark
a r t i c l e
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Article history: Received 14 October 2013 and in revised form 1 April 2014 Available online 19 April 2014 Keywords: Amylin Amyloid fibrils Protein expression Solid-state NMR
a b s t r a c t A number of diseases are caused by the formation of amyloid fibrils. Detailed understanding of structural features of amyloid fibers is of great importance for our understanding of disease progression and design of agents for diagnostics or potential prevention of protein aggregation. In lack of 3D crystal ordering, solid-state NMR forms the most suited method to determine the structures of the fibrils with atomic resolution. To exploit this potential, large amounts of isotopic-labeled protein need to be obtained through recombinant protein expression. However, expression and purification of amyloidogenic proteins in large amounts remains challenging due to their aggregation potential, toxicity for cells and difficult purification. In this work, we report a method for the production of large amounts of uniformly labeled 13 15 C, N-human amylin, being one of the most amyloidogenic peptides known. This method utilizes inclusion bodies-directed expression and cheap chemical cleavage with cyanogen bromide in order to minimize the cost of the procedure compared to the use of less efficient proteolytic enzymes. We demonstrate the formation of amylin fibrils in vitro characterized using biophysical methods and electron microscopy, show toxicity towards human cells, and demonstrate that produced material may form the basis for structure determination using solid-state NMR. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Amyloid diseases (amyloidoses) are a subgroup of protein conformational diseases, characterized by abnormal protein folding leading to functional impairment, or formation of amyloid – extracellular, insoluble proteinaceous deposits [1]. Numerous conditions have been related to the presence of amyloid – among them a number of neurodegenerative disorders as well as non-neuropathic systemic amyloidoses or localized diseases [2]. Since its discovery in 1987 [3,4] as a major component of pancreatic amyloid deposits [3], amylin has drawn significant attention from the scientific community. Otherwise named islet amyloid polypeptide or IAPP1, amylin consists ⇑ Corresponding author. Address: Nordre Ringgade 1, 8000 Aarhus C, Denmark. Tel.: +45 87155913; fax: +45 86196199. E-mail address: [email protected] (N.Chr. Nielsen). 1 Abbreviations used: AFM, atomic force microscopy; CNBr, cyanogen bromide; EM, electron microscopy; EPR, electron paramagnetic resonance; HFIP, hexafluoro-2propanol; IAPP, islet amyloid polypeptide; MAS ssNMR, magic angle spinning solid state nuclear magnetic resonance; PB, phosphate buffer; rIAPP, recombinant islet amyloid polypeptide; sIAPP, synthetic islet amyloid polypeptide; STEM, scanning transmission electron microscopy; TEM, transmission electron microscopy; TFA, trifluoroacetic acid; ThT, thioflavin T. http://dx.doi.org/10.1016/j.pep.2014.04.002 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
of 37 residues and is a hormone co-secreted with insulin by pancreatic b-cells in response to nutrition [5]. Under normal conditions, amylin plays a role in glycemic regulation by controlling the level of glucose in blood and slowing gastric emptying, thereby promoting satiety [6]. Presence of insoluble amyloid fibrils in pancreas is observed in the majority of patients suffering from diabetes mellitus (diabetes type II), but also in approximately 16% of healthy elderly subjects, and the relation between aging and pancreas fibrosis is often underlined [4,7,8]. Diabetes is a result of decline in b-cells mass or loss of function and is ascribed to several factors such as islet inflammation, glucolipotoxicity, accumulation of cholesterol and formation of amyloid [9–11]. Toxicity of fibrillar amylin has been demonstrated for human and rat islet b-cells [12]. Pancreas amyloid is also a severe obstacle in islet cell transplant [13–15]. The reason for this poisonous effect is not clear yet – among the possible mechanisms the following are often mentioned: physical damage of cell membrane by the growing amyloid; uptake of phospholipid molecules during fibrillogenesis [16–18]; formation of pore arranging oligomers [19–21], or receptor-mediated events [22,23]. The precise mechanism of amylin polymerization into fibrils is not entirely understood, however, amylin shares properties with other amyloidogenic peptides – fibril formation is thought to be a non-covalent assembly process, nucleation-dependent, and
characterized by sigmoidal reaction profiles [24,25]. Probably both fibril-independent (nucleation-dependent) and fibril-dependent (fibrils acting as nuclei) events are involved during fibrils growth [26,27]. Although the accurate mechanism of the polymerization remains to be elucidated, it is commonly accepted that amyloid fibril formation is accompanied by transition from disordered or partially ordered peptide conformation into a b-sheet rich structure [25,28]. A common feature of amyloids is a helical array of b-strands perpendicular to the fibril axis [29,30]. The structure of amylin fibrils has been partly revealed. Goldsbury et al. [31] utilized TEM and STEM to characterize the morphology of amylin fibrils and showed its ability to form fibrils of higher order structures, which display distinct polymorphism into striated ribbons or coiled fibrils. Tycko and co-workers (Luca et al. [32]) deduced a low resolution structural model of striated ribbons consistent with experimental data derived from imaging techniques (AFM, TEM) and solid-state NMR on selectively 13C,15N-isotope labeled IAPP. According to this model the fibers consist of four layers of parallel b-sheets formed by two asymmetric layers of amylin molecules. The authors place the amino acid region 18–27 as the connection between two b-strands. This model was indirectly confirmed by biophysical studies conducted by Abedini and Raleigh [33]. The structure from [32] is in contrast to an older study of Glenner et al. [34], who, based on the ability of the IAPP 20-29 fragment to form fibrils, suggested that this region may be the fibrillation core of the molecule. A high-resolution supramolecular structure of the 20-29 fragment of IAPP was determined with MAS ssNMR by Nielsen et al. [35]. Structures of other two fibril forming fragments, residues 21–27 and 28–33, were determined with atomic resolution with X-ray crystallography by Wiltzius and Sievers [36]. These models are in close agreement with the structure presented in [32], except from minor differences in the packaging of amino acid side chains. Regarding the twisted morphology of amylin fibrils, Bedrood et al. [37] used wave and pulsed EPR and EM imaging to derive an atomistic model in which amylin monomers have the shape of a horseshoe consisting of two b-strands, spanning about 15 Å. Despite extensive studies, the high-resolution three-dimensional structures of IAPP in diverse aggregation states still require further investigations. Even less information is available about the relation between the structural variants of amylin aggregates and their role in b-cell toxicity. Structural insight into the aggregation mechanisms of IAPP is in acute need to guide therapeutic design, giving a perspective for prevention and more efficient treatment of the disorder, which according to the data of The International Diabetes Federation every year kills 4.8 million people worldwide. The access to structural information has been hampered by the fact that amylin is the most amyloidogenic peptide ever characterized [38]. Synthesis and purification of the full length peptide are troublesome [39], additionally pure amylin has low solubility in aqueous solutions, and once solubilized, it aggregates
instantaneously. Furthermore, amylin fibrils display high degree of polymorphism and to prepare morphologically uniform preparations consumes significant quantities of peptide. Overall for these reasons it is difficult to prepare large amounts of pure peptide required for structural studies using methods like magic-anglespinning (MAS) solid-state NMR [40–44]. Usually, in the order of 5–30 mg of isotope-labeled (typically 13C and 15N) pure fibrils is necessary for such investigations [45]. The wish for uniform or extensive labeling with 13C to establish sufficient restraints to derive a detailed structural model renders peptide synthesis costly and impractical. On the other hand, cheap and efficient production of recombinant proteins in biological systems require many purification steps and may involve use of expensive reagents, for example proteases used to liberate the pure peptide from its fusion partner [46–48]. In this work, we report a method of recombinant amylin production for solid-state NMR investigations. Escherichia coli expression in a bioreactor using thioredoxin (trxA) as a fusion partner for IAPP and efficient CNBr-based liberation of the pure peptide comprise an efficient and cheap method for producing milligram amounts of uniformly 13C,15N-labeled human islet amyloid polypeptide. We have characterized fibrils formed by recombinant peptide with TEM imaging, ThT fluorescence assay, circular dichroism and MAS solid-state NMR spectroscopy, and demonstrated toxicity towards human cells in vitro.
Materials and methods Preparation of the pET32-rIAPP1-37 expression vector The pET32-rIAPP1-37 expression vector is presented in Fig. 1. pET32 (Novagen) plasmid DNA was prepared for cloning by digestion with BglII and SalI. A 142-bp IAPP DNA fragment including the BglII and SalI sites as well as a methionine codon immediately upstream of the Lys1 codon in IAPP and a stop codon following the codon for Tyr37 was amplified by PCR using human chromosomal DNA as template. The PCR reaction conditions were as follows: denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s. Forward and reverse primers were: AmCNBrF: 50 -GGT GGA AGA TCT GAT GAA ATG CAA CAC TGC CAC ATG TG-30 , and AmstR: 50 -GGT GGT GTC GAC TCA ATA TGT ATT GGA TCC CAC GTT G-30 (BglII and SalI sites are underlined). PCR products were separated on 2% agarose gels and purified using the Nucleospin Extract II kit (Macherey–Nagel GmbH & Co. KG). The ends of the PCR product were trimmed with BglII and SalI (Fermentas), the resulting fragment purified on agarose gels, and ligated into the pET32 vector using T4 DNA ligase (Fermentas). The ligation mixture was transformed into competent E. coli TOP10F0 cells and plated onto LB agar plates containing ampicillin. Plasmids from single colonies were purified and confirmed by
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Fig. 10. Solid-state NMR spectra of U-13C,15N amylin fibrils acquired with 12 kHz MAS at 16.4 T. (A) 1D 13C CP/MAS spectrum. (B) 2D 13C–13C DARR correlation spectrum showing aliphatic and carbonyl regions. The spectrum is obtained using a 20 ms period for 13C–13C mixing. Certain signals which can be unambiguously assigned to a single amino acid type are highlighted.
than 1 ppm indicate a well-structured peptide, however, some degree of overlap is seen in the spectrum, and higher dimensional spectra are necessary for resolving all signals. Nonetheless, the system seems well suited for further solid-state NMR investigations, and a full set of 2- and 3-dimensional spectra will be acquired for a more complete structural analysis of the amylin fibrils. Preliminary analysis of the chemical shifts can be performed on the basis of the spectra in Fig. 10, where signals from some amino acid types have been indicated based on typical chemical shift values and chemical shift patterns. Chemical shift values for Ca, Cb, and C’ may be compared with random coil chemical shift values and using the principle of correlation between the chemical shifts and the backbone secondary structure introduced by Spera et al. [72], it is confirmed that the peptide adopts a b-sheet conformation, as expected. A single threonine spin system (indicated by a dotted line circle in Fig. 10B) shows chemical shift values which indicate random coil conformation rather than b-sheet and this spin system may be assigned to the C-terminal threonine. Conclusion The present work addresses the challenge of high-yield production of recombinant peptide amylin. The rIAPP1-37 peptide was expressed as a fusion protein with an N-terminal thioredoxin A fusion tag separated from rIAPP1-37 by a methionine residue, which is used as a target for cyanogen bromide digestion. The fusion protein is directed to inclusion bodies within the cell, which prevents undesired aggregation and enables easier purification under
denaturating conditions. Experiments using unlabeled recombinant IAPP1-37 were conducted in order to characterize fibril growth kinetics and verify toxicity of the produced material towards human cells. A yield of 16 mg of pure amylin per liter of culture was obtained and the fibrils were subjected to TEM imaging and preliminary solid-state NMR experiments. Fibrils dimensions and morphology correspond to other available data concerning amylin fibrils structure. Furthermore, the recombinant fibrils resemble the synthetic amylin fibrils, widely used in structural studies. Solidstate NMR data indicate that the recombinant peptide comprising amyloid fibrils is well structured. Nevertheless, due to signal overlap, sample requires further experiments utilizing two and three dimensional spectra, which are underway. The work presented provides an effective method of production of labeled amylin in large amounts, suited for solid-state NMR spectroscopy.
Acknowledgments This work is supported by Danish National Research Foundation (DNRF59). The authors would like to thank Anne Gylling for technical assistance with human cell cultures, Ida B. Thøgersen for performing the Edman degradation and Prof. Daniel Otzen for access to biophysical instrumentation. The authors would also like to thank Assoc. Prof. Kamille Smidt Rasmussen for supplying the INS-1E cells and the medium for the cell culture. We also thank Pierre Maechler and Prof. Claes Wollheim, University Medical Centre Geneva, Switzerland for permission to use the INS-1E cells for our project.
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Appendix A.
Table A1. Composition of media for recombinant amylin expression.
Component
M9 batch medium
M9 feeding medium
Na2HPO4 KH2PO4 NaCl FeCl3 MnCl2 ZnCl2 CoCl2 CuSO4 NiCl2 H3BO3 MgSO4 CaCl2 Thiamine (vit. B1) 12 C- or 13C-glucose 13 N- or 15N-NH4Cl
7.4 g/L 3 g/L 0.5 g/L 0.1 mM 1 mM 1 mM 0.2 mM 0.1 mM 0.2 mM 0.1 mM 1 mM 0.2 mM 100 lg/mL 8 g/L 2 g/L
22.2 g/L 9 g/L 0.5 g/L 0.2 mM 2 mM 2 mM 0.4 mM 0.2 mM 0.4 mM 0.2 mM 2 mM – 200 lg/mL 13.3 g/L 3.3 g/L
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