Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase

Kód výsledku v 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>

Výsledek na webu

<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>

Jazyk výsledku

angličtina

Název v původním jazyce

Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Protein Expression and Purification

ISSN

1046-5928

e-ISSN

1096-0279

Svazek periodika

183

Číslo periodika v rámci svazku

JUL 2021

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

12

Strana od-do

105877

Kód UT WoS článku

000639854700004

EID výsledku v databázi Scopus

2-s2.0-85103387079