Gip analog and hybrid polypeptides with selectable properties
An analogue and hybrid technology, applied in the field of hybrid polypeptides containing GIP, can solve the problems that GIP has not made significant progress in clinical application
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[0314] Implementation options include:
[0315] SEQ ID
NO.:
sequence
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
KCNTATCVLGKLSQELHRLQTYPRTNTGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
KCNTATCVLGRLS QELHRLQTLPRTNTGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
KCNTATCVLGRLS QELHRLQTYPPTNTGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
KCNTATCVLGRLSQELHRLQTYPRTNVGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
KCNTATCVLGRLSQELHRLQTLPPTNVGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
KCNTATCVLGRLANFLHRLQTYPRTNTGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
ACNTATCVLGRLSQELHRLQTYPRTNTGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
KCNAATCVLGRLSQELHRLQTYPRTNTGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
KCNTAACVLGRLSQELHRLQTYPRTNTGSNTY
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK-connector-
CANLSTCVLGRLSQELHRLQTYPRTNTGSNTY ...
Embodiment 1
[0763] The preparation of embodiment 1.GIP polypeptide
[0764] Rink amide resin (Novabiochem) at 0.43-0.49 mmol / g loaded at 0.050-0.100 mmol, or 0.63 mmol / g preloaded Wang resin (Fmoc-Tyr(tBu)-Wang resin) (Novabiochem) can be used at The peptides of the present invention were assembled on a Symphony peptide synthesizer (Protein Technologies, Inc.). Fmoc amino acid (5.0 equivalents, 0.250-0.500 mmol) residues were dissolved in 1-methyl-2-pyrrolidone at a concentration of 0.10M. All other reagents (HBTU, 1-hydroxybenzotriazole hydrate and N,N-diisopropylethylamine) were prepared as 0.55M solutions in dimethylformamide. Then use HBTU (2.0 equivalents, 0.100-0.200mmol), 1-hydroxybenzotriazole hydrate (1.8 equivalents, 0.090-0.18mmol), N,N-diisopropylethylamine (2.4 equivalents, 0.120-0.240mmol ), the Fmoc-protected amino acid was coupled to the resin-bound amino acid for 2 hours. After the final amino acid coupling, the peptide was deprotected for 1 hour using 20% (v / v) pi...
Embodiment 2
[0767] Embodiment 2. Binding experiment
[0768] GIP polypeptides of the invention can be tested in various receptor (eg GIPR, GLP-1R, amylin receptor) binding assays using binding assay methods well known to those skilled in the art. Such experiments include those described below.
[0769] Amylin binding assay : Binding of certain exemplary compounds of the invention to amylin receptors can be assessed in nucleus accumbens membranes prepared from rat brain as follows. Male Sprague-Dawley(R) rats (200-250 g) were sacrificed by decapitation. Brains were removed and placed in cold phosphate buffered saline (PBS). Starting on the ventral side, an incision was made from the mouth to the hypothalamus, laterally bounded by the olfactory tracts, and extended from these tracts at a 45° angle to the midline. This basal forebrain tissue, including the nucleus accumbens and surrounding area, was weighed and homogenized in ice-cold 20 mM HEPES buffer (20 mM HEPES acid, pH adjusted ...
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