Production and characterization of the recombinant Islet Neogenesis Associated Protein (rINGAP).
Islet Neogenesis Associated Protein (INGAP) is implicated in pancreatic islet neogenesis. INGAP peptide, a pentadecapeptide comprising amino acids 104-118, reverses diabetes in rodents and improves glucose homeostasis in patients with diabetes. The mechanism of INGAP action is unknown, but such studies would benefit from the availability of the full-length recombinant protein (rINGAP). Here we report the production of rINGAP from 293-SF cells following lentiviral transduction, and its characterization by MALDI-TOF and Q-TOF Mass Spectrometry, and HPLC. Importantly, we show that rINGAP exhibits 100x the bioactivity of INGAP peptide on a molar basis in an in vitro assay of human islet regeneration.
Assouline-Thomas
Béatrice
B
Department of Surgery, McGill University, Montréal, Que., Canada H3G1A4. Beatrice.Assouline@mail.mcgill.ca
Pilotte
Amélie
A
Petropavlovskaia
Maria
M
Makhlin
Julia
J
Ding
Jieping
J
McLeod
David
D
Hanley
Stephen
S
Massie
Bernard
B
Rosenberg
Lawrence
L
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014408:Biomarkers, Tumor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D005786:Gene Expression Regulation; D006801:Humans; D007515:Islets of Langerhans / Q000502:physiology; D037181:Lectins, C-Type / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016086:Lentivirus / Q000235:genetics; D013058:Mass Spectrometry; D008647:Mesocricetus; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000073718:Pancreatitis-Associated Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012038:Regeneration / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism; D014161:Transduction, Genetic
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins