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

Research output: Chapter in Book/Report/Conference proceedingConference contribution

1 Citation (Scopus)

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 protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Original languageEnglish
Title of host publication2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium
PublisherAmerican Institute of Physics Inc.
Volume1678
ISBN (Electronic)9780735413252
DOIs
Publication statusPublished - 25 Sep 2015
Event2015 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2015 - Selangor, Malaysia
Duration: 15 Apr 201516 Apr 2015

Other

Other2015 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2015
CountryMalaysia
CitySelangor
Period15/4/1516/4/15

Fingerprint

Niger
Aspergillus
Escherichia
isolation
polypeptides
proteins
modules
homology
disulfides
cellulose
genes
strands
folding
peptides
amino acids
coding
deoxyribonucleic acid
inclusions
cells

ASJC Scopus subject areas

  • Physics and Astronomy(all)

Cite this

Woon, J. S. K., Abd. Murad, A. M., & Abu Bakar, F. D. (2015). Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus Niger in Escherichia coli. In 2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium (Vol. 1678). [030004] American Institute of Physics Inc.. https://doi.org/10.1063/1.4931225

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus Niger in Escherichia coli. / Woon, J. S K; Abd. Murad, Abdul Munir; Abu Bakar, Farah Diba.

2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium. Vol. 1678 American Institute of Physics Inc., 2015. 030004.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Woon, JSK, Abd. Murad, AM & Abu Bakar, FD 2015, Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus Niger in Escherichia coli. in 2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium. vol. 1678, 030004, American Institute of Physics Inc., 2015 Postgraduate Colloquium of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology, UKM FST 2015, Selangor, Malaysia, 15/4/15. https://doi.org/10.1063/1.4931225
Woon JSK, Abd. Murad AM, Abu Bakar FD. Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus Niger in Escherichia coli. In 2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium. Vol. 1678. American Institute of Physics Inc. 2015. 030004 https://doi.org/10.1063/1.4931225
Woon, J. S K ; Abd. Murad, Abdul Munir ; Abu Bakar, Farah Diba. / Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus Niger in Escherichia coli. 2015 UKM FST Postgraduate Colloquium: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium. Vol. 1678 American Institute of Physics Inc., 2015.
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AB - 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 protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

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