title: Constitutive expression of a rhIL-2-HSA fusion protein in Pichia pastoris using glucose as carbon source.
abstract: A constitutive expression vector for rhIL-2-HSA fusion protein production in yeast Pichia pastoris was constructed. The coding gene was placed in frame with the Saccharomyces cerevisiae α-factor secretion signal sequence under the control of the GAP promoter. The recombinant plasmid pGAPZαA-rhIL-2-HSA was integrated into the genome of the P. pastoris GS115. The effect of different carbon sources on rhIL-2-HSA fusion protein expression was evaluated in shaking flask cultures. We found that recombinant P. pastoris grew well and efficiently secreted rhIL-2-HSA fusion protein into the medium when using glucose as carbon source. To achieve higher production, the influence of initial pH and culture temperature was also evaluated. Fed-batch fermentation strategy using glucose as carbon source for constitutive expression of rhIL-2-HSA fusion protein was investigated in 5-L bioreactor and the expression level of rhIL-2-HSA could reach about 250 mg/L after 60-h fermentation. The rhIL-2-HSA fusion protein produced by this constitutive expression system was purified and exhibited a specific bioactivity of 1.040 × 10(6) IU/mg in vitro. This study described constitutive expression of rhIL-2-HSA fusion protein by P. pastoris and development of a simple high-cell density fermentation strategy for biologically active rhIL-2-HSA fusion protein using glucose as sole carbon source.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Guan
authors.forename: Fengxiang
authors.initials: F
authors.lastname: Chen
authors.forename: Jianyong
authors.initials: J
authors.lastname: Lei
authors.forename: Yunhua
authors.initials: Y
authors.lastname: Li
authors.forename: Zuoying
authors.initials: Z
authors.lastname: Duan
authors.forename: Ruiyu
authors.initials: R
authors.lastname: Zhu
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.forename: Huazhong
authors.initials: H
authors.lastname: Li
authors.forename: Jian
authors.initials: J
authors.lastname: Jin
mesh_terms: D061249:Batch Cell Culture Techniques; D002244:Carbon / Q000378:metabolism*; D002452:Cell Count; D005240:Feasibility Studies; D005285:Fermentation; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D005947:Glucose / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010843:Pichia / Q000166:cytology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012709:Serum Albumin / Q000235:genetics*; D013696:Temperature
chemical_list: D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D002244:Carbon; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005947:Glucose