Production and secretion of biologically active recombinant canine growth hormone by Pichia pastoris.
Production of recombinant canine (Canis familiaris) growth hormone (rCFGH) by two expression systems, methanol utilization slow (Muts) and methanol utilization plus (Mut+) based on Pichia pastoris. Led by the Saccharomyces cerevisiae alpha-mating type signal sequence (SS), the hormone was secreted into the culture medium in its mature and active form. The level of total proteins secreted into the medium achieved at 25 ml working volume using Erlenmeyer flasks was approximately 40 and 15 microg/ml for Muts and Mut+ constructs, respectively. As judged by densitometry of proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the hormone produced by the fermented Mut(s) strain upon induction with methanol reached 24 microg/ml, representing around 60% of the total secreted proteins and being eight times more abundant than in its Mut+ counterpart. Finally, the recombinant hormone showed activity when tested in the Nb2 cell proliferation assay.
Ascacio-Martínez
Jorge A
JA
Laboratorio de Biotecnología, de la Unidad de Laboratorios de Ingeniería y Expresión Genéticas del Departamento de Bioquímica. Facultad de Medicina de la Universidad Autónoma de Nuevo León, Monterrey, NL, México.
Barrera-Saldaña
Hugo A
HA
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors / Q000235:genetics; D013006:Growth Hormone / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D013006:Growth Hormone