Biotechnology1682-296x1682-2978Asian Network for Scientific Information10.3923/biotech.2017.174.181Pichia pastoris]]>MadadiNasrin GhasemiFaezeh SoukhtanlooMohammad MojaradMajid AvvalFarnaz Zahedi MashkaniBaratali 4-62017164-6Background and Objectives: Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factors involved in proliferation and differentiation of bone marrow precursor cells into granulocytes, monocyte that used in treatment of patient with cancer, therefore, it was decided to express murine GM-CSF as a recombinant protein in Pichia pastoris (P. pastoris). Materials and Methods: The protein sequence of mouse GM-CSF was obtained from Uniprot database and ordered for synthesis after back translation and optimization of coding sequence. It was subsequently cloned into pPICZαA expression vector expressed in Pichia pastoris. Expression and activity of GM-CSF was confirmed by SDS-PAGE, dot blot, Western blotting in the culture supernatant. Biological activity of the product was confirmed by promoting FDC-P1 cell growth. Non-linear regression analysis was used for fitting a curve onto the cell proliferation data plotted against the growth factor dilutions and calculation of EC50 value. Results: Sequence optimization improved CAI from 0.66 in the native to 0.86 in the optimized GM-CSF sequence through replacing 24% of the nucleotides. In dot blot analysis, GM-CSF expression reached its peak on 5 days and started to decline after 7 days. The GM-CSF protein was appeared as bands with apparent molecular mass nearly 16-17 kDa. It was estimated that 18.5 μg mL1 of active murine GM-CSF was secreted into the culture supernatant. Conclusion: The recombinant GM-CSF protein was expressed in Pichia pastoris yeast and was biologically active in the FDC-P1 cell line proliferation.]]>Srour, E.F., J.E. Brandt, R.A. Briddell, S. Grigsby, T. Leemhuis and R. Hoffman,1993in vitro.]]>81661669Foss, D.L., A.M. Bennaars, C.A. Pennell, M.D. Moody and M.P. Murtaugh,2003Pichia pastoris.]]>91205215Qiu, J.T., T.C. Chang, C.T. Lin, Y.M. Chen, F.Q. Li, Y.K. Soong and C.H. Lai,200725253263Mitsuyasu, R.T. and D.W. Golde,19893411425Ehsani, M.A., M. Ordouee, P. Salamati, E. Shahgholi and K. Sotoudeh et al.,200616319324Groopman, J.E., R.T. Mitsuyasu, M.J. DeLeo, D.H. Oette and D.W. Golde,1987317593598Baldwin, G.C., J.C. Gasson, S.G. Quan, J. Fleischmann and R. Weisbart et al.,19888527632766Yamauchi, T., K. Yada, A. Umemura, E. Asakura, T. Hanamura and T. Tanabe,199659296301Cregg, J.M., I. Tolstorukov, A. Kusari, J. Sunga, K. Madden and T. Chappell,2009Pichia pastoris.]]>463169189Mashkani, B., M.H. Tanipour, M. Saadatmandzadeh, L.K. Ashman and R. Griffith,2016776156166Nuc, P. and K. Nuc,2006Escherichia coli.]]>52448456, (In Polish)Mashkani, B., A.F. Odell, E.M. Byrnes, R. Griffith and L.K. Ashman,2013Pichia pastoris.]]>889397Karimi, E., H. Faraji, D.H. Alamdari, M. Souktanloo, M. Mojarrad, L.K. Ashman and B. Mashkani,2017Pichia pastoris.]]>53421428Faraji, H., M. Ramezani, H.R. Sadeghnia, K. Abnous, F. Soltani and B. Mashkani,2017Pichia pastoris.]]>47379387Donahue, R.E., M.M. Johnson, L.I. Zon, S.C. Clark and J.E. Groopman,1987in vitro haematopoiesis following human immunodeficiency virus infection.]]>326200203Daly, R. and M.T.W. Hearn,2005Pichia pastoris: A useful experimental tool in protein engineering and production.]]>18119138Tang, H., S. Wang, J. Wang, M. Song and M. Xu et al.,2016Saccharomyces cerevisiae.]]>2016Brondyk, W.H.,2009463131147Laukens, B., C. de Wachter and N. Callewaert,2015Pichia pastoris N-glycosylation pathway using the glycoswitch technology.]]>1321103122