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Cloning and expression of carbohydrate binding module in Pichia pastoris

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Resumo:The main goal of this work is the production of recombinant biologically active peptides fused with a Carbohydrate Binding Module (CBM). Aiming at the optimization of large scale expression, CBM peptide production was done by cloning CBM coding sequence in two different systems of Pichia pastoris: pGAPZC which has a constitutive promoter and pPICZC which has an inductive promoter. The integration of the CBM coding sequence, in yeast genome, was confirmed by slot-blot for both expression systems. Transcription was analysed by northern-blot and SDS-PAGE. The results obtained with these two expression systems were different. Apparently, there were no clones of P. pastoris transformed with pGAPZC-CBM that had produced any protein with starch affinity, under the batch and fed-batch conditions tested in this work. On the other hand, only one studied clone of P. pastoris transformed with pPICZC-CBM vector had produced, in batch conditions, a protein with affinity for starch. However, under fed-batch conditions, the results obtained with this clone were not conclusive, suggesting that conditions for large scale production must be optimized.
Autores principais:Moreira, Susana
Outros Autores:Domingues, Lucília; Gama, F. M.; Casal, Margarida
Ano:2005
País:Portugal
Tipo de documento:outro
Tipo de acesso:acesso aberto
Instituição associada:Universidade do Minho
Idioma:inglês
Origem:RepositóriUM - Universidade do Minho

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