[Constitutive expression of human angiostatin in Pichia pastoris using glycerol as only carbon source].
Carbon source plays an important role in the constitutive expression of foreign proteins in Pichia pastoris. In present study, glucose , glycerol , methanol and oil acid, was used respectively as the only carbon source to constitutively express hAS in Pichia pastoris GS115 (pGAP9K-AS)in shaking flask. The result shows that oleic acid is the best (163 mg/L) compared with glycerol (83mg/L), glucose (76 mg/L)and methanol (57 mg/L). Since oleic acid is insoluble in water, glycerol was used as the carbon source in the high-density cell culture of GS115 (pGAP9K-AS) in a 30 liter bioreactor and 169 mg/L of angiostatin was obtained after 48h of culture. The expressed angiostatin is immunologically active as shown by Western blotting. The recombinant hAS inhibits bFGF induced CAM angiogenesis and suppresses the growth of B16 melanoma in C57BL/6J mice. The tumor inhibition rate is 90% after 12 days of treatment. Statistics analysis revealed that the tumor volume difference of mice between the hAS group and PBS group is prominent (P < 0.01).
Tu
Fa-Zhi
FZ
1 Institute of Tropical Bioscience and Biotechnology/National Key Biotechnology Laboratory for Tropical Crops, CATAS, Haikou 571101, China.
Fu
Ce-Yi
CY
Zhang
Tian-Yuan
TY
Luo
Jin-Xian
JX
Zhang
Ai-Lian
AL
D020533:Angiogenesis Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use; D043168:Angiostatins / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use; D000818:Animals; D019149:Bioreactors / Q000382:microbiology*; D003470:Culture Media / Q000494:pharmacology; D005285:Fermentation; D005990:Glycerol / Q000494:pharmacology*; D006801:Humans; D008546:Melanoma, Experimental / Q000188:drug therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use
D020533:Angiogenesis Inhibitors; D003470:Culture Media; D011994:Recombinant Proteins; D043168:Angiostatins; D005990:Glycerol