Display options
Cite
Karamitros CS, Konrad M. Bacterial co-expression of the α and β protomers of human l-asparaginase-3: Achieving essential N-terminal exposure of a catalytically critical threonine located in the β-subunit. Protein Expr Purif. 2013;93:1-10doi: 10.1016/j.pep.2013.10.007.
Karamitros, C. S., & Konrad, M. (2014). Bacterial co-expression of the α and β protomers of human l-asparaginase-3: Achieving essential N-terminal exposure of a catalytically critical threonine located in the β-subunit. Protein expression and purification, 931-10. https://doi.org/10.1016/j.pep.2013.10.007
Karamitros, Christos S, and Konrad, Manfred. "Bacterial co-expression of the α and β protomers of human l-asparaginase-3: Achieving essential N-terminal exposure of a catalytically critical threonine located in the β-subunit." Protein expression and purification vol. 93 (2014): 1-10. doi: https://doi.org/10.1016/j.pep.2013.10.007
Karamitros CS, Konrad M. Bacterial co-expression of the α and β protomers of human l-asparaginase-3: Achieving essential N-terminal exposure of a catalytically critical threonine located in the β-subunit. Protein Expr Purif. 2014 Jan;93:1-10. doi: 10.1016/j.pep.2013.10.007. Epub 2013 Oct 22. PMID: 24157738.
Copy Download .nbib Format: NLM
Share it on

Protein Expr Purif. 2014 Jan;93:1-10. doi: 10.1016/j.pep.2013.10.007. Epub 2013 Oct 22.

Bacterial co-expression of the α and β protomers of human l-asparaginase-3: Achieving essential N-terminal exposure of a catalytically critical threonine located in the β-subunit.

Protein expression and purification

Christos S Karamitros, Manfred Konrad

Affiliations

  1. Enzyme Biochemistry Group, Max Planck Institute for Biophysical Chemistry, Göttingen D-37077, Germany.

PMID: 24157738 DOI: 10.1016/j.pep.2013.10.007

Abstract

l-asparaginases hydrolyze l-asparagine to l-aspartic acid and ammonia. Enzymes of bacterial origin are used as therapeutic agents for the treatment of acute lymphoblastic leukemia. Recently, the structure of a human homolog, hASNase3, which possesses l-asparaginase activity, was solved setting the basis for the development of an anti-leukemic protein drug of human origin. Being an N-terminal hydrolase, hASNase3 undergoes intramolecular self-cleavage generating two protomers (subunits α and β) which remain non-covalently associated and constitute the catalytically active form of the enzyme. However, recombinant expression of full-length hASNase3 in Escherichiacoli results in only partial processing towards the active enzyme. We developed a co-expression system for the two subunits that allowed production of the β-subunit complexed to the α-subunit such that the N-terminal methionine is removed by endogenous methionine aminopeptidase to expose the catalytically essential threonine residue at the N-terminus of the β-subunit. The enzyme produced by this co-expression strategy is fully active, thus obviating the necessity of self-activation by slow autoproteolytic cleavage.

Copyright © 2013 Elsevier Inc. All rights reserved.

Keywords: Catalytic threonine; Co-expression; Human l-asparaginase-3; N-terminal hydrolase; α/β Protomers

Publication Types