Expression, purification, and characterization of a recombinant stem bromelain from Ananas comosus

► We cloned stem bromelain gene in Escherichia coli host system. ► Vector used provide good expression system and suitable the host used. ► A single protein band appeared suggested stem bromelain was successfully cloned and expressed. ► High activity also suggest expressed bromelain is pure. Commerc...

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Bibliographic Details
Published in:Process biochemistry (1991) 2011-12, Vol.46 (12), p.2232-2239
Main Authors: Amid, Azura, Ismail, Nurul Azira, Yusof, Faridah, Salleh, Hamzah Mohd
Format: Article
Language:English
Subjects:
affinity chromatography
Ananas comosus
Bromelain
Cloning
Escherichia coli
gelatin
genes
polymerase chain reaction
Protease
Protein expression
protein synthesis
purification methods
Recombinant enzyme
sequence analysis
stem bromelain
Western blotting
yields
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Summary:► We cloned stem bromelain gene in Escherichia coli host system. ► Vector used provide good expression system and suitable the host used. ► A single protein band appeared suggested stem bromelain was successfully cloned and expressed. ► High activity also suggest expressed bromelain is pure. Commercially available bromelain is prepared by performing a tedious and costly purification method that yields bromelain at different degrees of purity. In the current study, a gene encoding stem bromelain from Ananas comosus was amplified using polymerase chain reaction. This bromelain gene was initially cloned into the pENTR/TEV/D-TOPO vector before being sub-cloned into the pDEST17 expression vector. DNA sequencing of the amplified products exhibited a high level of homology to the corresponding gene from the NCBI public database. Protein expression was conducted in the BL21-AI Escherichia coli strain. The recombinant bromelain was then purified in a single step using immobilized metal affinity chromatography, specifically a Ni-NTA spin column. The purified recombinant bromelain was detected by Western blotting. In addition, the purified enzyme exhibited hydrolytic activity towards gelatin and a synthetic substrate, LNPE. The purified recombinant bromelain exhibited optimum activity at pH 4.6 and 45°C.
ISSN:1359-5113
1873-3298
DOI:10.1016/j.procbio.2011.08.018