Over-expression, purification, and characterization of metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria

Protein Expression and Purification
Patrick A CrawfordMichael W Crowder

Abstract

The gene from Aeromonas veronii bv. sobria encoding the metallo-beta-lactamase ImiS was subcloned into pET-26b, and ImiS was over-expressed in BL21(DE3) Escherichia coli and purified using SP-Sepharose chromatography. This protocol yielded over 5 mg of ImiS per liter of growth culture under optimum conditions. The biochemical properties of recombinant ImiS were compared with those of native ImiS. Recombinant and native ImiS have the same N-terminus of A-G-M-S-L, and CD spectroscopy was used to show that the enzymes have similar secondary structures. Gel filtration chromatography revealed that both enzymes exist as monomers in solution. MALDI-TOF mass spectra showed that the enzymes have a molecular mass of 25,247 Da, and metal analyses demonstrated that both as-isolated enzymes bind ca. 0.7 mol of Zn(II). Metal titrations demonstrate that the maximum activity of recombinant ImiS occurs when the enzyme binds one equivalent of zinc. Steady-state kinetic studies reveal that recombinant ImiS is a carbapenemase like native ImiS and that the recombinant enzyme exhibits similar kcat and K(m) values for the substrates tested, as compared to the native enzyme. This over-expression protocol now allows for detailed spectroscopic and mecha...Continue Reading

References

Nov 1, 1992·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·K WatanabeK Shimizu
Aug 21, 1992·Science·H C Neu
Apr 1, 1992·The Journal of Antibiotics·K BandohK Ueno
Nov 1, 1989·Analytical Biochemistry·S C Gill, P H von Hippel
Jun 1, 1995·Antimicrobial Agents and Chemotherapy·A FeliciG Amicosante
Aug 1, 1993·Journal of Medical Microbiology·D J Payne
Apr 1, 1993·The Biochemical Journal·A FeliciJ M Frère
Apr 30, 1996·Proceedings of the National Academy of Sciences of the United States of America·T HesterkampB Bukau
Jan 1, 1997·Antimicrobial Agents and Chemotherapy·D J PayneJ Marchand-Brynaert
Jul 1, 1996·Microbial Drug Resistance : MDR : Mechanisms, Epidemiology, and Disease·M Hernandez VilladaresG Amicosante
Mar 1, 1996·The Journal of Antimicrobial Chemotherapy·T R WalshP M Bennett
Apr 4, 1998·Antimicrobial Agents and Chemotherapy·T R WalshP M Bennett
Jun 17, 1998·FEMS Microbiology Letters·K O'HaraS Iyobe
Aug 26, 1998·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·K Bush
Feb 4, 1999·Biochemistry·S McManus-Munoz, M W Crowder
Jul 23, 1999·Archives of Biochemistry and Biophysics·K W Yang, M W Crowder
Oct 20, 1999·International Journal of Antimicrobial Agents·J D Williams
Feb 22, 2001·Antimicrobial Agents and Chemotherapy·M GalleniUNKNOWN Metallo-beta-lactamases Working Group
Feb 24, 2001·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·M W CrowderT R Walsh
Mar 10, 2001·JAMA : the Journal of the American Medical Association·N L LeeC R Kumana
Mar 16, 2001·Bioorganic & Medicinal Chemistry·S BounagaM I Page
Jun 8, 2001·The Journal of Biological Chemistry·J H ToneyY D Gao

❮ Previous
Next ❯

Citations

Aug 31, 2011·Antimicrobial Agents and Chemotherapy·Fátima FonsecaJames Spencer
Jun 14, 2008·Bioinorganic Chemistry and Applications·Michael I Page, Adriana Badarau
Jan 1, 2009·International Journal of Cell Biology·Payal KhandelwalRamareddy V Guntaka
Feb 21, 2006·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Alison CostelloDavid L Tierney
Apr 22, 2015·ACS Medicinal Chemistry Letters·Shao-Kang YangKe-Wu Yang
Sep 26, 2015·Biomacromolecules·Erik J LiuShaoyi Jiang
May 1, 2008·Analytical Biochemistry·Zhenxin HuMichael W Crowder
Apr 2, 2015·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Mahesh AithaMichael W Crowder
Jul 14, 2004·Protein Expression and Purification·Patrick A CrawfordMichael W Crowder
Feb 14, 2016·Applied and Environmental Microbiology·Cornelia U WelteMike S M Jetten
Jun 7, 2020·Biomolecules·Antonella R PalaciosAlejandro J Vila
Aug 25, 2015·FEBS Letters·María-Rocío MeiniAlejandro J Vila
Apr 3, 2019·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Yilin ZhangKewu Yang
Nov 3, 2021·Environmental Microbiology·Juan Hilario CafieroMaría Florencia Del Papa
Dec 5, 2021·Chemical Biology & Drug Design·Jia-Zhu ChiganKe-Wu Yang

❮ Previous
Next ❯

Related Concepts

Related Feeds

Aminoglycosides (ASM)

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.

Carbapenems

Carbapenems are members of the beta lactam class of antibiotics and are used for the treatment of severe or high-risk bacterial infections. Discover the latest research on carbapenems here.

Aminoglycosides

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.

Carbapenems (ASM)

Carbapenems are members of the beta lactam class of antibiotics and are used for the treatment of severe or high-risk bacterial infections. Discover the latest research on carbapenems here.