Publicação
Cloning and expression of carbohydrate binding module in Pichia pastoris
| 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. |
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| 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 |
| 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. |
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