Functional characterization of recombinant bromelain of Ananas comosus expressed in a prokaryotic system.
Bromelain (BRM) is a defense protein present in the fruit and stem of pineapple (Ananas comosus) and it is grouped as a cysteine protease enzyme with diversified medicinal uses. Based on its therapeutic applications, bromelain has got sufficient attention in pharmaceutical industries. In the present study, the full coding gene of bromelain in pineapple stem (1,093 bp) was amplified by RT-PCR. The PCR product was cloned, sequenced, and characterized. The sequence analysis of the gene revealed the single nucleotide polymorphism and its phylogenetic relatedness. The peptide sequence deduced from the gene showed the amino acid variations, physicochemical properties and secondary and tertiary structural features of the protein. The full BRM gene was transformed to prokaryotic vector pET32b and expressed in Escherichia coli BL21 DE3pLysS host cells successfully. The identity of the recombinant bromelain (rBRM) protein was confirmed by Western blot analysis using anti-BRM-rabbit IgG antibody. The activity of recombinant bromelain compared with purified native bromelain was determined by protease assay. The inhibitory effect of rBRM compared with native BRM in the growth of Gram-positive and Gram-negative strains of Streptococcus agalactiae and Escherichia coli O111 was evident from the antibacterial sensitivity test. To the best of our knowledge, this is the first report showing the bactericidal property of rBRM expressed in a prokaryotic system.
SCMS Institute of Bioscience and Biotechnology Research and Development, Management House, South Kalamassery, Cochin, 682033, Kerala, India.
Susan
S
George
Salini
S
Bhasker
Harish
H
Madhav
Archana
A
Nair
Mohankumar
M
Chinnamma
D031230:Ananas / Q000201:enzymology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001963:Bromelains / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D014644:Genetic Variation; D010802:Phylogeny; D020641:Polymorphism, Single Nucleotide; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000494:pharmacology*; D013292:Streptococcus agalactiae / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D001963:Bromelains