Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals.
Human interferon-alpha 2b (IFN-alpha2b) was cloned and expressed in Pichia pastoris under the control of alcohol oxidase promoter (AOX1) using three different secretion signals. Native secretion signal of IFN-alpha2b, Saccharomycescerevisiae MF-alpha factor prepro sequence and a mutated alpha prepro sequence without the Glu-Ala (EAEA) repeats were used separately for directing the secretion of IFN-alpha2b into the culture medium of P. pastoris. The native secretion signal of IFN-alpha2b did not secrete protein into the culture medium of P. pastoris. The alpha prepro sequence without the EAEA repeats directed the secretion of maximum amount of IFN-alpha2b (200 mg/l) into the culture medium, with the same amino acid sequence as that of the native IFN-alpha2b secreted by human lymphocytes. The full alpha prepro sequence, having both the protease cleavage sites for KEX2 and STE13 gene products, also secreted an equivalent amount of IFN-alpha2b into the culture medium. However, two interferon bands with similar molecular masses were observed, when full alpha prepro sequence was used for the secretion of IFN-alpha2b. The difference in the molecular masses of the two bands was found to arise due to the difference in the molecular masses of the N-terminal fragment, and the inefficient processing of secretion signal.
Ghosalkar
Anand
A
Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
Sahai
Vikram
V
Srivastava
Aradhana
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003470:Culture Media; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005822:Genetic Vectors; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
D003470:Culture Media; D017931:DNA Primers; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins