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Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

Lookup NU author(s): Dr James Woon

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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).


Abstract

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the proteinstructure which may have contributed to the improper folding of the proteinand thus, resulting in inclusion bodies in E. coli.


Publication metadata

Author(s): Woon JSK, Murad AMA, Bakar FDA

Publication type: Conference Proceedings (inc. Abstract)

Publication status: Published

Conference Name: 2015 UKM FST Postgraduate Colloquium

Year of Conference: 2015

Pages: 030004

Print publication date: 25/09/2015

Acceptance date: 31/08/2015

Publisher: AIP Publishing

URL: http://aip.scitation.org/doi/abs/10.1063/1.4931225

DOI: 10.1063/1.4931225

Series Title: AIP Conference Proceedings


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