ABSTRACT
The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the leader peptide and pro I region of alkaline extracellular protease in the yeast Yarrowia lipolytica. hEGF was purified from culture supernatant by reverse-phase chromatography and analysed by Western-blot hybridisations. The biologically active hEGF in the purified sample was assayed using the radioreceptor assay and estimated to be 100 microg/l. However, the level of expression was found to be substantially low compared to the levels of homologous protein, alkaline extracellular protease (AEP), possibly due to degradation by secreted acid protease(s). A novel and sensitive bioassay was developed to determine the biological activity of hEGF produced at low levels and is based on the effect produced by hEGF in the regenerating tails of the wall lizard. Intramuscular injections of culture supernatant from the recombinant yeast and the standard hEGF led to a drastic reduction in tail regeneration confirming the biological activity of the recombinant hEGF.
Subject(s)
Ascomycota/genetics , Epidermal Growth Factor/biosynthesis , Epidermal Growth Factor/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Epidermal Growth Factor/genetics , Epidermal Growth Factor/pharmacology , Gene Dosage , Genetic Vectors/genetics , Humans , Lizards , Protein Sorting Signals/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/pharmacology , Regeneration/drug effects , Serine Endopeptidases/genetics , Tail/physiologyABSTRACT
Expression of the gene encoding the hepatitis B virus middle surface antigen (pre-HBsAg) in the yeast Yarrowia lipolytica has been studied. The preS2-HBsAg gene was expressed from the alkaline extracellular protease-encoding gene (XPR2) promoter. In the fusion construct, the membrane-spanning 'a' domain of preS2-HBsAg has been replaced by the leader peptide and the proI region of the alkaline protease, thus eliminating the epitope responsible for the immune escape mechanism. Expression has been found to be growth-stage dependent with the highest protein accumulation during the stationary phase, accounting for around 2.35% of the total soluble intracellular proteins. The produced protein was assembled into Dane particles and was immunogenic in mice. The expression vector was found to be stable for at least 100 generations under non-selective conditions.
