title: Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.
abstract: Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; Department of Biological Sciences, Faculty of Science, University of Jeddah, P.O. Box 80327, Jeddah 21589, Saudi Arabia.
authors: Mohammed N
authors: MN
authors: Baeshen
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Thamer A F
authors: TA
authors: Bouback
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Mubarak A
authors: MA
authors: Alzubaidi
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; Department of Biotechnology, Eternal University, Baru Sahib, Himachal Pradesh 173 101, India.
authors: Roop S
authors: 0000-0002-5586-3829
authors: RS
authors: Bora
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Mohammed A T
authors: 0000-0003-3729-0374
authors: MA
authors: Alotaibi
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Omar T O
authors: OT
authors: Alabbas
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Sultan M
authors: SM
authors: Alshahrani
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Ahmed A M
authors: AA
authors: Aljohani
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Rayan A A
authors: RA
authors: Munshi
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Ahmed
authors: A
authors: Al-Hejin
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; Nucleic Acids Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technology Applications, Alexandria 21934, Egypt.
authors: Mohamed M M
authors: 0000-0001-8316-9714
authors: MM
authors: Ahmed
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology Applications, Alexandria 21934, Egypt.
authors: Elrashdy M
authors: EM
authors: Redwan
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; Cell Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Tahrir Street, Dokki, Cairo 12311, Egypt.
authors: Hassan A I
authors: HA
authors: Ramadan
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; Department of Biotechnology, Eternal University, Baru Sahib, Himachal Pradesh 173 101, India.
authors: Kulvinder S
authors: KS
authors: Saini
authors: Department of Biological Sciences, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia.
authors: Nabih A
authors: NA
authors: Baeshen
mesh_terms: D000284:Administration, Oral; D000818:Animals; D002096:C-Peptide / Q000737:chemistry*; D003001:Cloning, Molecular; D003362:Cost-Benefit Analysis; D018076:DNA, Complementary / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000008:administration & dosage* / Q000096:biosynthesis / Q000627:therapeutic use*; D051379:Mice; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D002096:C-Peptide; D018076:DNA, Complementary; D007328:Insulin; D011994:Recombinant Proteins