Part:BBa_K2933006
subclass B1 metallo-beta-lactamase IND-10, codon optimized in E. coli
This part encodes a protein called IND-10, which is a metallo-beta-lactamase of subclass B1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
IND-10 is a type of subclass B1 metal beta-lactamases, which is found from Chryseobacterium indologenes. The beta lactamases of subclass B1 family can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.
Chryseobacterium indologenes, of which the taxonomic characteristics were first described in 1983, is a common gram-negative bacterium and belongs to the genus Chryseobacterium.
Chryseobacterium indologenes has been proved to be intrinsically resistant to most antimicrobial agents often used to treat gram-negative bacteria, but the mechanism of the multidrug resistance is not clear. The metallo-β-lactamase (MBL), which can cleave the β-lactam ring of antibiotics of the penicillin family, is thought to be closely associated with multidrug resistance of this bacterium. Since the first type of class B MBL gene, bla IND, was identified in C. indologenes, there are fifteen IND variants have been deposited in Genbank to date. These enzymes share 27–92 % identity with that of IND-1 at the amino acid sequence level.
References
[1]Yabuuchi E, Kaneko T, Yano I, Moss CW, Miyoshi N. Sphingobacterium gen. nov., Sphingobacterium spiritivorum comb. nov., Sphingobacterium multivorum comb. nov., Sphingobacterium mizutae sp. nov., and Flavobacterium indologenes sp. nov.: glucose-nonfermenting gram-negative rods in CDC groups IIK-2 and IIb. Int J Syst Bacteriol. 1983;33:580–98.
[2]Chang Y-C, Lo H-H, Hsieh H-Y, Chang S-M. Identification, epidemiological relatedness, and biofilm formation of clinical Chryseobacterium indologenes isolates from central Taiwan. J Microbiol Immunol Infect. 2015;48:559–64.
[3]Chen F-L, Wang G-C, Teng S-O, Ou T-Y, Yu F-L, Lee W-S. Clinical and epidemiological features of Chryseobacterium indologenes infections: analysis of 215 cases. J Microbiol Immunol Infect. 2013;46:425–32.
[4]Bebrone C. Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem Pharmacol. 2007;74:1686–701.
[5]Zeba B, De Luca F, Dubus A, Delmarcelle M, Simporé J, Nacoulma OG, et al. IND-6, a highly divergent IND-type metallo-beta-lactamase from Chryseobacterium indologenes strain 597 isolated in Burkina Faso. Antimicrob Agents Chemother. 2009;53:4320–6.
[6]Yamaguchi Y, Takashio N, Wachino J, Yamagata Y, Arakawa Y, Matsuda K, et al. Structure of metallo-beta-lactamase IND-7 from a Chryseobacterium indologenes clinical isolate at 1.65-A resolution. J Biochem. 2010;147:905–15.
[7]Perilli M, Caporale B, Celenza G, Pellegrini C, Docquier JD, Mezzatesta M, et al. Identification and characterization of a new metallo-beta-lactamase, IND-5, from a clinical isolate of Chryseobacterium indologenes. Antimicrob Agents Chemother. 2007;51:2988–90.
[8]Bellais S, Léotard S, Poirel L, Naas T, Nordmann P. Molecular characterization of a carbapenem-hydrolyzing beta-lactamase from Chryseobacterium (Flavobacterium) indologenes. FEMS Microbiol Lett. 1999;171:127–32.
Molecular cloning
First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. Left: The PCR result of IND-10. Right: The verification results by enzyme digestion.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
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