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

Karamitros, C. S., & Konrad, M. (2013). Bacterial co-expression of the alpha and beta protomers of human L-asparaginase-3: Achieving essential N-terminal exposure of a catalytically critical threonine located in the beta-subunit. Protein Expression and Purification, 93, 1-10. doi:10.1016/j.pep.2013.10.007.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0014-CF1F-D Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-CB46-1
Genre: Journal Article

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1878110.pdf (Publisher version), 2MB
 
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 Creators:
Karamitros, C. S.1, Author              
Konrad, M.1, Author              
Affiliations:
1Research Group of Enzyme Biochemistry, MPI for Biophysical Chemistry, Max Planck Society, ou_578612              

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Free keywords: Human L-asparaginase-3; N-terminal hydrolase; alpha/beta Protomers; Catalytic threonine; Co-expression
 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 alpha and beta) which remain non-covalently associated and constitute the catalytically active form of the enzyme. However, recombinant expression of full-length hASNase3 in Escherichia coli results in only partial processing towards the active enzyme. We developed a co-expression system for the two subunits that allowed production of the beta-subunit complexed to the a-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 beta-subunit. The enzyme produced by this co-expression strategy is fully active, thus obviating the necessity of self-activation by slow autoproteolytic cleavage.

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Language(s): eng - English
 Dates: 2013-10-22
 Publication Status: Published online
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.pep.2013.10.007
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Title: Protein Expression and Purification
Source Genre: Journal
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Pages: - Volume / Issue: 93 Sequence Number: - Start / End Page: 1 - 10 Identifier: -