WO2005089038A2

WO2005089038A2 - Method for cloning, expression and purification of the amyloid polypeptide from human pancreatic islets

Info

Publication number: WO2005089038A2
Application number: PCT/BR2005/000039
Authority: WO
Inventors: Sergio Teixeira Ferreira; Mari Cleide Sogayar; Theri Leica Degaki; Christian Colin; Dahabada Helena José LOPES
Original assignee: Universidade Federal Do Rio De Janeiro-Ufrj
Priority date: 2004-03-23
Filing date: 2005-03-23
Publication date: 2005-09-29
Also published as: BRPI0400735A8; BRPI0400735A

Abstract

Method for cloning and expression of the mature pancreatic islets amyloid polypeptide (IAPP) in the recombinant form, utilizing an Escherichia coli polymerase, in which a DNA fragment containing the sequence that codes for the mature IAPP is amplified by reverse transcription and polymerase chain reaction (RT-PCR) from total RNA derived from purified human pancreatic islets, cloned into an expression vector, as described above, and expressed in bacteria, and method for rh-IAPP purification, by lysing the cells, centrifuging the cell lysate, ressuspending the precipitate in the presence of a denaturing agent, centrifuging the suspension, removing the insoluble material, collecting and filtration the clarified supernatant, cleaving with protease and eluting the rh-IAPP in the purified form.

Description

METHOD FOR CLONING, EXPRESSION AND PURIFICATION OF THE AMYLOID POLYPEPTIDE FROM HUMAN PANCREATIC ISLETS The present invention refers to a method for cloning, expression and purification of the amyloid polypeptide from human pancreatic islets (IAPP) , more specifically to a method for ' cloning, expression and purification of the mature, native, human pancreatic islets polypeptide (IAPP) in the recom inant form, utilizing a heterologous expression system in Eschericchia coli, based on the acl- T7 RNA polymerase. State of the Art Deposition of proteins in the form of insoluble amyloid aggregates is a common characteristics in a large number of important human diseases, including Alzheimer, Parkinson's and Huntinton's diseases, type II diabetes and the transmissible spongiform encephalopathies (Stefani, M. & Dobson CM. (2003) . Protein aggrega tion and aggrega te toxicity: new insights into protein folding _r misfolding diseases and biological evolution . J. Mol. Med. 81, 678- 699) . These amyloid deposits of protein nature result from polymerization of peptides and/or proteins that aggregate forming crosses anti-parallel β-sheets. Through still not completely understood mechanisms, the amyloid deposition leads to disfunction and death of the cells, leading, in turn, to severe damage to the tissue to which the disease is related (Stefani, M. & Dobson CM. (2003) . Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution . J. Mol. Med. 81, 678-699). The amyloid deposits formed by the pancreatic islets amyloid polypeptide (IAPP) are present in greater than 95% of the type II diabetic patients (T2DM) and it is believed that its abundance is directly correlated with the pathogenesis (Westermark, P.; Wersnstedt, C; Wilander, E.; Hayden, D.W.; O'Brien, T.D. & Johnson, K.H. (1987). Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic ca t are derived from a neuropeptide-like protein also present in normal islets cells . Proc. Natl. Acad. Sci. USA 84, 3381-3385; Lorenzo, A.; Razzaboni, B.; Weir, G.C. & Yankner, B.A. (1994). Pancreatic islet cell toxicity of amylin associated with type-2 diabetes melli tus . Nature 368, 756-60) . The transgenic mice experimental models which express the human IAPP (h-IAPP) present a diabetes phenotype that is directly associated with tissue deposition of hIAPP in the amyloid form (Soeller, W.C; Janson, J.; Hart, S.E.; Parker, J.C.; Carty, M.D.; Stevenson, R.W.; Kreutter, D.K. & Butler, P.C (1998) . Islet amyloid-associated diabetes melli tus in obese A (vw) /a mice expressing human IAPP. Diabetes 47, 743- 750) . In vi tro, IAPP exhibits a marked tendency to aggregate in amyloid fibers, following a kinetics which is consistent with a nucleation-dependent polymerization mechanism (Krampert, M.; Bernhagen, J.; Schmucker, J.; Horn, A.; Schmauder, A.; Brunner, H.; Voelter, W. & Kapurniotu, A. (2000) . Amyloidogeni ci ty of recombinant human pro-islet amyloid polypeptide (ProIAPP) . Chem. Biol. 7, 855-871; Padrick, S.B. & Miranker, A.D. (2002). Islet amyloid: Phase partitioning and secondary nucleations are central to the mechanism of fibrillogenesis. Biochemistry 9, 4694-703). The pancreatic islets amyloid polypeptide (IAPP), or amylin, is a polypeptide composed of 37 (thirty seven) aminoacid residues, which display significant homology with a inoacid sequences related to the calcitonin gene (CGRPs) . Both possess the same number of aminoacids, in addition to displaying dissulfate bonds between cysteine residues in positions 2 (two) and 7 (seven) and being amidated at the carbi-terminal end. IAPP is synthesized, processed in the Golgi complex, stored in the interior of secretory granules and, finally, releaed into the circulation, along with insulin, by the pancreatic β cells, in response to elevations of the plasmatic levels of glucose (Kapurniotu, A. (2001) . Amyloidogenicity and cytotoxicity of islet amyloid polypeptide. Biopolymers 60, 438-459) . This polypeptide has been considered as the third regulator of carbohydrate metabolism in the liver and in skeletal muscle (Hayden, M.R. & Tyagi, S.C (2001). ^λ\A" for amylin and amyloid in type 2 diabetes mellitus. J. Pancreas 2, 129- 139) . In humans, the gene coding for IAPP is localized in the small arm of chromosome 12 (twelve) , being transcribed as a fragment of approximately 1.5kb, whose product is a precursor peptide of 89 (eighty nine) aminoacids ( (Sanke, T.; Bell, G.I.; Sample, C; Rubenstein, A.H. & Steiner, D.F. (1988). An Islet Amyloid peptide is derived from an 89-amino acid precursor by proteolytic processing. J. Biol. Chem. 263 (33): 17243-17246; Krampert, M.; Bernhagen, J.; Schmucker, J.; Horn, A.; Schmauder, A.; Brunner, H.; Voelter, W. & Kapurniotu, A. (2000) . Amyloidogenici ty of recombinant human pro-islet amyloid polypeptide (ProIAPP) . Chem. Biol. 7, 855-871) . Like other secreted polypeptides, IAPP is synthesized as a large precursor molecule, nemd pre-pro-pancreatic amyloid polypeptide (preproIAPP) . Both the possible physiological role of these precursors and the mechanisms incolved in processing of the preproIAPP to yield the active mature pancreatic amyloid polypeptide (IAPP) are still largely unknown (Nishi, M.; Sanke, T.; Seino, S.; Eddy, R.L.; Fan, Y.S.; Byers, M.G.; Shows, J.B.; Bell, G.I. & Steiner, D.F. (1989). Human islet amyloid polypeptide gene : complete nucleotlde sequence, chromosomal localiza tion, and evolutionary history. Mol. Endocrinol. 3, 1775-17810) . This polypeptide was recently described as the most amyloidogenic known so far (Kayed, R. ; Bernhagen, J.; Greenfield, N.; Al-Abed, Y.; Teichberg, S.; Frank, R.W.; Voelter, W. & Bucala, R. (1999). Conforma tional transitions of islet amyloid polypeptide (IAPP) in amyloid formation in vi tro. J. Mol. Biol. 287, 781-796) . However, the molecular determinants of the IAPP aggregation process have not been elucidated yet, lending support to the crucial importance of characterizing its molecular and conformational properties, as well as elucidating the mechanisms of amyloid formation. Since its discovery, in 1987, several studies have focused on the seach for analogs of this polypeptide, which could be employed in treatment of type I diebetes mellitus (T1DM) patients, that is, patients who undergo a process of autoimmune destruction of insulin- and IAPP-secreting β cells, which, consequently, do not secrete these hormones (Bailey, C.J. (2001) . New pharmacologic agents for diabetes. Curr. Diab. Rep. 1 (2): 119-26). In 1997, a french company (Amylin Pharmaceuticals, San Diego, California, USA) developped the first synthetic IAPP analog, named amlintide, to treat T1DM patients. However, like IAPP, this analog also aggregated forming an amyloid material. For this reason, a new analog was developed with three point aminoacids modifications, which was called pramlitide (USAN council. List No. 392. New names . Amlintide. (1997). Clin. Pharmacol. Ther. 61, 500.; Kleppinger, E.L. & Vivian, E.M. (2003) . Pramlintide for the treatment of diabetes mellitus. Ann Pharmacother. 37 (7-8), 1082-9).. Since then, this analog has been commercialized to be used in the control of TlDM and T2DM, in conjunction with the insulin therapy. The first results of utilization of pramlitide in humansshowed that administration of this IAPP analog seems to be effective in decreasing the levels of glycated hemoglobin A (HbAlc) and in preventing the gain loss, when used in conjunction with insulin (Hayden, M.R. & Tyagi, S.C (2001). "A" for amylin and amyloid in type 2 diabetes melli tus . J. Pancreas 2, 129-139) . Other results also indicate that administration of this IAPP analog, in combinantion with insulin, has been effective to control the glucose post-prandial levels, through decreased food ingestion, reduction in the rate of gastric flow and suppression of glucagon secretion (Kleppinger, E.L. & Vivian, E.M. (2003) . Pramlintide for the trea tment of diabetes mellitus . Ann Pharmacother. 37 (7-8), 1082-9). Other IAPP analogs have been developed and some are in the testing phase for commercialization (Baron, A.D.; Kim, D. & Weyer, C (2002) . Novel peptides under development for the treatment of type 1 and type 2 diabetes melli tus . Curr. Drug Targets Immune Endocr. Metabol. Disord. 2 (1) : 63-82) . It is important to point out that hormone replacement has been an ever increasing practice in treatment of endocrine defficiencies. Several patents related to this subject are available. However, none of them refer to cloning, expression and purification of the human IAPP in the recombinant form. Among these patents, the following american patents can be cited: 5,116,948, which reports the preparation of the islets-elated polypeptide (IAPP) through purification of amyloids isolated from mammalians and through chemical synthesis, as well as preparation of antibodies to LAPP; 5,124,314, which reports pharmaceutical formulations containing amylin or peptides related to the calcitonin gene, for treatment of diabetes mellitus; 5,175,145, that refers to treatment of diabetes mellitus with amylin agonists; 5,266,561, which refers to development of compounds and methods to block the effects of IAPP during the course of T2DM pathogenesis; 5,716,619, that refers to the utilization of antibodies to amylin in treatment of T2DM and 6,610,824, reporting the preparation, by chemical synthesis, and use of amylin analogs. From the above mentioned evidence, it is possible to conclude that to this date, the pancreatic amyloid polypeptide (IAPP) or amylin could only be obtained by chemical synthesis or by purification from amyloid plaques obtained from human pancreas post-mortem or from insulinomas (β cell tumor) . Chemical synthesis of amyloidogenic peptides is often difficult (Nilsson, M.R.; Nguyen, L.L. & Raleigh, D.P. (2001) Synthesis and purification of amyloidogenic peptides . Anal. Biochem. 288, 76-82) and production of the majority of them, including IAPP, in the recombinant form, has not been achieved yet. A recent study by Krampert and cols. (Krampert, M.; Bernhagen, J.; Schmucker, J. ; Horn, A.; Schmauder, A.; Brunner, H.; Voelter, W. & Kapurniotu, A. (2000) . Amyloidogenici ty of recombinant human pro-islet amyloid polypeptide (ProIAPP). Chem. Biol. 7, 855-871; Sanke, T.) reported the expression of recombinant human proIAPP conjugated with a histidine tail. However, this product is significantly less amyloidogenic than native IAPP. Another recent study decribed the expression and purification of recombinant IAPP fused to the maltose binding protein (MBP) , however, this study failed in their attempts to express intact mature human IAPP (Mazor, Y.; Gilead, S.; Benhar, I. & Gazit, E. (2002). Identification and characterization of a novel molecular-recogni tion and self-assembly domain wi thin the islet amyloid polypeptide. J. Mol. Biol. 322, 1013-1024). The chemical synthesis of amyloidogenic peptides is hampered by their tendency to aggregate. In addition, the cost of production of amyloidogenic proteins throgh chemical synthesis is usually vey high. Syntheisis of these proteins and peptides not always can be performed in a totally automated manner. Several steps of the chemical synthesis process are carried out manually, requiring both material and well trained human resources. On the other hand, extraction of amyloid plaques from human pancreas or from insulinomas, is greatly hampered by ethical issues and by difficulties in obtaining this rare tumor, in addition to resulting in low yields of a product which does not display optimal purity. Therefore, development of efficient, low cost and high yield methods for the production of these amyloidogenic peptides and proteins, in the recombinant form, represents a great advantage and a biotechnological achievement. Particularly, the perspective to obtain IAPP with poin mutations in aminoacid residues which are relevant for the aggregation of this protein, will allow a number of investigations on the molecular aspects of the aggregation process. Moreover, production of recombinant of recombinant IAPP should also allow the development of recombinant IAPP analogs, which can be utilized in hormone replacement therapy for both type I and type II diabetic patients who are regularly subjected to insulin therapy. As a state of the art novelty, the present invention describes the cloning, expression and purification of human mature IAPP in the recombinant form, utilizing, to this end, an Escherichia coli heterologous expression system based on the Lacl-T7 RNA polymerase promoter. The amyloidogenic properties and the cytotoxic potential of the recombinant human IAPP (rhlAPP) were characterized using a variety of biochemical and biophysical methods, incluiding right angle light scattering, thioflavin fluorescence measurements, transmission electron microscopy and cytotoxicity assays using human pancreatic isletcell cultures. In addition to applying to IAPP, the methods described in the present invention may also be useful in expression and purification of other amyloidogenic proteins and peptides, which are difficul to obtain by chemical synthesis. Description of the Invention For illustrative purposes, the methods of cloning and expression, as well as purification, which are the object of this patent request, will be described using the examples and figures that follow. Cloning of mature human IAPP - a 151pb DNA fragment containing the sequence that codes for the mature IAPP polypeptide, was amplified by reverse transcription and polymerase chain reaction (RT-PCR), according to Fig. 1, from 5ug of total RNA derived from human pancreatic islets purified at the Human Pancreatic Islet Unit (UIPH) of the Chemistry Institute of the University of Sao Paulo. Total RNA was purified, after cell lysis, with guanidine isothiocyanate and β-mercaptoetanol, followed by ultracentrifugation in a cesium chloride cushion (Chirgwin, J.M.; Przybyla, A.E.; Mc Donald, R.J. & Rutter, W.J. (1979) . Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-9) . Reverse transcription was carried out utilizing Superscript II (Invitrogen) and oligo dT oligonucleotide primers, according to manufactirer's instructions. Amplification of the fragment of interest was carried out by PCR, using the Platinum Taq High Fidelity (Invitrogen), following a program that involves a 95 °C denaturing phase for lmin, followed by 35 cycles of incubation at 95 °C for 30sec, 55 °C for 30sec, 68 °C por 30 sec and a final extension phase of 5 min at 68 °C The transcription primers in the sense (IAPP-F) and antisense (IAPP-R) orientations, which correspond to, respectively, the 5' e 3' ends of the sequence coding for the mature human IAPP (IAPP-F: 5'-AGA TCT GGG TAC CGA CGA CGA CGA CAA GAA ATG CAA CAC TGC CAC AT-3'; IAPP-R: 5'-GTC GAC TCA ATA TGT ATT GGA TCC CAC GTT GGT AG-3' ) , were designed to contain the restriction sites to Bglll and Sail, for in-frame directional cloning, as well as a termination codon aftr the last residue. In the IAPP-F oligonucleotide, a cleavage site for enterokinase (Ek) was also inserted, before the first IAPP aminoacid residue, to allow cleavage to obtain the mature IAPP. The resulting PCR product was digested and ligated to the pET-32a vector, which had been previously digested with the same enzymes and dephosphorylated. The cleavage site for IAPP was removed from the vector and replaced by thye insert generated by PCR, which already contained the Ek site. The IAPP was thyen expressed as a fusion protein, coupled to a poly-histidine tail, with a thioredoxin domain and a fragment of RnaseA (HT-TRX-S) according to Fig. 2. The ligated product was then electroporated into competent DH10B E. coli. The recombinant clone (pET32a-IAPP) was isolated and characterized by digestion with restriction enzymes and the product was sequenced bu automatic sequencing. Expression in bacteria and purification - the , pET32a-IAPP plasmid, containing the IAPP fusion protein, was used for transformation of competent BL21 (DE3)pLysS bacteria, by electroporation. These cells were grown at 37 °C in a bioreactor, in 1L of Super Broth medium (20g triptone, 12g yeast extrat, 5ml NaOH, 5g NaCl) contendo ampicilina (150 μg por ml). When the Absorbance at 600nm reached 0.6 (-1.7 x 10⁸ cells/ml), isopropil-1-thio-β-D-galactopiranoside (IPTG) was added to a final concentration of ImM. After 6h of induction at 37 °C, the medium was centrifuged (5,000g at 4 °C por 30min) and the precipitate was ressuspended in 500mL of buffer containing 20 rtiM Tris-HCl, 500 mM NaCl, 1 rtiM irnidazol, pH 7.4. The cells were then lysed using a sonicator (Vibracell 72412; Bioblock, Illkirch, France) at 20kHz, with a 19mm probe, appying 10 one-minute pulses in ice, with 2min intervals between pulses. The suspension was centrifuged (18,000g, at 4 °C for 40min) to separate the inclusion bodies containing IAPP. The precipitate was ressuspended in 250mL of the same buffer containing 6M urea and incubated overnight at 4 °C for solubilization of the inclusion bodies. In order to remove the insoluble material leftover, the sample was centrifuged (39.000g at 4 °C for 20 in) . The clarified supernatant was collected, filtered through a 0.45μm pore membrane and used for affinity purification of IAPP by chromatography through a column containing immobilized metallic ions, using nitrilotriacetic acid (Ni-NTA) coupled to agarose and charged with nickel (Amersham Pharmacia Biotech, Sweden) , in the presence of 6M urea. The Ni-NTA column (ImL volume) was washed with three volumes of the buffer described above, in the absence of urea, followed by three washes with the same buffer containing 6M ures, using a lmL/min flow rate. The column was then charged with the filtered supernatant containing the ecombinant human IAPP (rh-IAPP), utilizing a 0.25mL/min flow rate. After charging the column was washed with three volumes of washing buffer (20mM Tris-HCl, 500mM NaCl, 5mM irnidazol, pH 7.4) containing 6M urea. The urea was slowly removed, by utilizing a discontinuous urea gradient (from 6 to 0M) in the same washing buffer. The rh-IAPP was cleaved with enterokinase inside the column to remove the poly-histidine and thioredoxin tails. To this end, one international unit (IU) of Ek was introduced into the column in a buffer containing 10 mMTris-HCl e lO M CaCl₂, pH 8,0 and the cleavage reaction was allowed to proceed for 18h at 25 °C The purified rh-IAPP was eluted in a solution containing 50% (V/V) 2-2-2-trifluoroetanol (TFE) diluted into the cleavage buffer. The concentration of purified rh- IAPP was determined by Absorbance at 280nm, considering as the molar extinction coefficient the value of 1,330 M^~1cm^~1. Electrophoresis systems - Analysis of the fractions obtained throughout the different stages of protein expression and purification was carried out by polyacrylamide gel electrophoresis in the presence of duodecil-sodium-sulfate (SDS-PAGE) (Shapiro, A.L.; Vinuela, E. & Maizel, J.V. Jr. (1967) . Molecular weight estimation of polypeptide chains by electrophoresis in SDS- polyacrylamide gels . Biochem. Biophys . Res. Commun. 7, 28 (5): 815-20.) ou o de eletroforese com tricina (Schagger, H. & Jagow, G.V. (1987) . Tricine-sodium dodecyl sulfa te- polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa . Anal. Biochem. 166, 368-379) . The protein bands were visualized by staining with Coomassie Brilliant Blue R-250 or silver. Description of the above mentioned invention was presented for illustrative and descriptive purposes. Moreover, the description is not intended to limit the invention to the form here described. Consequently, variations and modifications compatible with the knowledge above and ability or knowledge of the relevant technology are within the scope of the present invention. The present invention intends to include all modifications and variations of the same, within the scope described in the report and in the claims enclosed.

Claims

CLAIMS 1. Method for cloning and expression of the amyloid peptide from human pancreatic islets (IAPP) characterized to be in the recombinant form, and use of a heterologous expression system in Escherichia coli cells, based on the iacI-T7 RNA polymerase promoter, in which a DNA fragment containing the coding sequence for the mature IAPP peptide is amplified by reverse transcription and polymerase chain reaction (RT-PCR) from total RNA derived from human pancreatic islets.

2. Method according to claim No. 1, characterized by the fact that the DNA fragment is preferentially 151-bp in length and amplified from 5 μg of total RNA derived from human pancreatic islets.

3. Method according to claim No. 1, characterized by the fact that amplification by PCR reaction is performed using an enzyme through denaturation steps followed by cycles of annealing and extension.

4. Method according to claim No. 3, characterized by the fact that the enzyme is preferentially Platinum Taq

High Fidelity.

5. Method according to claim No. 3, characterized by the fact that the denaturing step is preferentially performed at 95°C for 1 minute.

6. Method according to claim No. 3, characterized by performing preferentially 35 cycles of incubation at 95°C for 30 seconds.

7. Method according to claim No. 3, characterized by performing preferentially 35 cycles of incubation at 55°C for 30 seconds.

8. Method according to claim No. 3, characterized by performing preferentially 35 cycles of incubation at 68°C for 30 seconds.

9. Method according to claim No. 3, characterized by performing an extension step at 68°C for 5 minutes.

10. Method according to claim No. 1, characterized by the fact that the sense and antisense amplification primers (IAPP-F and IAPP-R), corresponding to the 5' and 3' ends of the coding sequence for the mature polypeptide for human IAPP (IAPP-F: 5'-AGA TCT GGG TAG CGA CGA CGA CGA CAA GAA ATG CAA CAC TGC CAC AT-3' ; IAPP-R: 5'-GTC GAC TCA ATA TGT ATT GGA TCC CAC GTT GGT AG-3'), were designed to contain the Bglll and Sail restriction sites for in-frame directional cloning, and also containing a translation termination codon inserted juxtaposed immediately after the last IAPP aminoacid residue.

11. Method according to claim No. 1, characterized by the fact that a cleavage site for enterokinase (Ek) has been inserted into the IAPP-F oligonucleotide juxtaposed to the first aminoacid of IAPP.

12. Method according to claim No. 1, characterized by the fact that the product resulting from the PCR reaction is digested and ligated to the expression vector which has been previously digested with restriction enzymes and dephosphorylated.

13. Method according to claim No. 1, characterized by by the fact the fragment coding for the cleavage site for Ek is removed from the vector and replaced by the PCR- generated DNA insert.

14. Method according to claim No. 1, characterized by the fact that mature IAPP is expressed as a fusion protein preferentially bound to a polyhistidine tag, a thioredoxin domain and a pancreatic RNAse A fragment (HT-TRX-S), in which the ligation product is used to electroporate competent DH10B E. coli cells.

15. Method according to claim No. 1, characterized by the fact that the recombinant clone (pET32a-IAPP) is isolated and characterized through restriction enzyme digestion and sequenced by automatic DNA sequencing.

16. Method according to claim No. 1, characterized by the fact that the pET32a-rh-IAPP plasmid containing the recombinant human IAPP fusion protein coding sequence is used for the transformation of competent BL21 (DE3)pLysS E. coli cells through electroporation.

17. Method according to claim No. 1, characterized by the fact that cell growth is carried out in a medium containing preferentially 20g/L of tryptone, 12g/L of yeast extract, 5 mL/L of 1M NaOH solution, 5g/L of NaCl and 150 μg/mL ampicillin.

18. Method according to claim No. 17, characterized by the fact that cell growth is carried out preferentially at 37°C.

19. Method for purification of the recombinant amyloid peptide from human pancreatic islets (IAPP) characterized by including the steps for cell lysis, cell suspension centrifugation and resuspension of the pellet in the presence of the denaturing agent, centrifugation of the suspension, removal of the insoluble material, harvesting and filtration of the clarified supernatant, binding of the recombinant protein to an affinity column containing imobilized metallic ions, cleavage with protease and elution of rh-IAPP in its purified form.

20. Method according to claim No. 19, characterized by the fact that isopropyl-1-thio-β-D-galactopyranoside (IPTG) is added preferentially to 1 mM final concentration when the bacterial culture reaches an Absorbance at 600 nm of 0.6.

21. Method according to claim No. 19, characterized by the fact that medium centrifugation is performed preferentially at 5,000g, 4°C and for 30 minutes.

22. Method according to claim No. 19, characterized by the fact that ressuspension of the pellet is performed preferentially in 500 mL of buffer containing 20mM Tris- HC1, 500mM NaCl and 1 mM irnidazol, at pH 7.4.

23. Method according to claim No. 22, characterized by the fact that cell lysis is performed preferentially after 6 hours of induction and at 37°C

24. Method according to claim No. 9, characterized by the fact that cell lysis is performed preferentially after ten 1-minute pulses of sonication in ice, with a 2-minute interval between the pulses.

25. Method according to claim No. 24, characterized by the fact that the lysate should be preferentially kept on ice throughout the lysis step.

26. Method according to claim No. 19, characterized by the fact that centrifugation of the lysate suspension is performed at 18,000g, 4°C and for 40 minutes.

27. Method according to claim No. 19, characterized by the fact that pellet resuspension is performed at 4°C using

250mL of buffer containing 20mM Tris-HCl, 500mM NaCl, lmM irnidazol and 6M urea.

28. Method according to claim No. 19, characterized by the fact that removal of the insoluble material is obtained by centrifugation of the sample preferentially at 39,000g, 4 °C and for 20 minutes.

29. Method according to claim No. 19, characterized by the fact that harvest and filtration of the clarified supernatant is performed preferentially through a 0.45 μm- pore membrane, in which the flow-through is employed for the purification of IAPP by using column chromatography based on affinity to imobilized metallic ions (nickel ions complexed to agarose-coupled nitrileacetic acid - Ni-NTA) , in the presence of 6M of urea.

30. Method according to claim No. 19, characterized by the fact that cleavage of rh-IAPP is performed with enterokinase (Ek) , through preferential introduction into the column of one International Unit (UI) of Ek in buffer containing lOmM Tris-HCl, lOmM CaCl₂ at pH 8.0.

31. Method according to claim No. 30, characterized by the fact that cleavage is preferentially performed for 18 hours at 25°C 32. Method according to claim No. 19, characterized by the fact that elution of purified rh-IAPP is performed preferentially using a 50% (v/v) 2-2' -2' ' -trifluoroethanol (TFE) solution diluted in the cleavage buffer. 33. Sense and antisense amplification primers (IAPP-F: 5'-AGA TCT GGG TAC CGA CGA CGA CGA CAA GAA ATG CAA CAC TGC CAC AT-3' and IAPP-R: 5'-GTC GAC TCA ATA TGT ATT GGA TCC CAC GTT GGT AG-3' ) characterized by the fact that have been designed, to possess the Bglll and Sail restriction sites for in-frame directional cloning, and also to contain a translation termination codon inserted juxtaposed immediately after the last IAPP aminoacid residue.