Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase
The result's identifiers
Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F21%3A00548867" target="_blank" >RIV/60077344:_____/21:00548867 - isvavai.cz</a>
Result on the web
<a href="https://www.sciencedirect.com/science/article/pii/S1046592821000607?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/pii/S1046592821000607?via%3Dihub</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.1016/j.pep.2021.105877" target="_blank" >10.1016/j.pep.2021.105877</a>
Alternative languages
Result language
angličtina
Original language name
Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase
Original language description
The Leishmania major leucyl-aminopeptidase (LAPLm), a member of the M17 family of proteases, is a potential drug target for treatment of leishmaniasis. To better characterize enzyme properties, recombinant LAPLm (rLAPLm) was expressed in Escherichia coli. A LAPLm gene was designed, codon-optimized for expression in E. coli, synthesized and cloned into the pET-15b vector. Production of rLAPLm in E. coli Lemo21(DE3), induced for 4 h at 37 ?C with 400 ?M IPTG and 250 ?M L-rhamnose, yielded insoluble enzyme with a low proportion of soluble and active protein, only detected by an anti-His antibody-based western-blot. rLAPLm was purified in a single step by immobilized metal ion affinity chromatography. rLAPLm was obtained with a purity of10% and a volumetric yield of 2.5 mg per liter, sufficient for further characterization. The aminopeptidase exhibits optimal activity at pH 7.0 and a substrate preference for Leu-p-nitroanilide (appKM = 30 ?M, appkcat = 14.7 s- 1). Optimal temperature is 50 ?C, and the enzyme is insensitive to 4 mM Co2+, Mg2+, Ca2+ and Ba2+. However, rLAPLm was activated by Zn2+, Mn2+ and Cd2+ but is insensitive towards the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, being inhibited by EDTA and bestatin. Bestatin is a potent, non-competitive inhibitor of the enzyme with a Ki value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10608 - Biochemistry and molecular biology
Result continuities
Project
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Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Others
Publication year
2021
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
Protein Expression and Purification
ISSN
1046-5928
e-ISSN
1096-0279
Volume of the periodical
183
Issue of the periodical within the volume
JUL 2021
Country of publishing house
US - UNITED STATES
Number of pages
12
Pages from-to
105877
UT code for WoS article
000639854700004
EID of the result in the Scopus database
2-s2.0-85103387079