Recombinant expression and partial characterization of an active soluble histo-aspartic protease from Plasmodium falciparum

Protein Expression and Purification
Huogen XiaoRickey Y Yada

Abstract

Malaria aspartic proteases are attractive drug targets for the treatment of malaria, however, recombinant expression of active histo-aspartic proteinase (HAP) to facilitate its characterization has proven elusive. The present study reports on the first recombinant expression of soluble, active histo-aspartic proteinase from Plasmodium falciparum as a thioredoxin fusion protein. A truncated form of HAP (77p-451) was fused to thioredoxin in the pET32b(+) vector and the fusion protein (Trx-tHAP) was expressed in Escherichia coli Rosetta-gami B (DE3)pLysS. The fusion protein was partially purified from the culture medium using a combination of anion exchange and Ni(2+) affinity chromatography. Soluble tHAP was subsequently purified by enterokinase treatment and removal, followed by gel filtration chromatography. Although truncated HAP was incapable of autocatalytic activation, enterokinase digestion of partially purified fusion protein released the truncated prosegment yielding a mature form of tHAP (mtHAP). N-terminal sequencing of mtHAP indicated that enterokinase cleavage took place at Lys119-Ser120, four residues upstream of the native cleavage site (Gly123-Ser124). Initial activity tests showed that mtHAP was capable of hydrol...Continue Reading

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Citations

Nov 17, 2009·Protein Engineering, Design & Selection : PEDS·Charity L Parr-Vasquez, Rickey Y Yada
Jul 6, 2010·Protein Engineering, Design & Selection : PEDS·Brian C BryksaRickey Y Yada
Jan 23, 2016·Molecular and Biochemical Parasitology·Khomkrit SappakhawNonlawat Boonyalai
Apr 28, 2011·Journal of Structural Biology·Prasenjit BhaumikAlla Gustchina
May 4, 2010·Molecular and Biochemical Parasitology·Huogen XiaoRickey Y Yada
Jun 6, 2009·New Biotechnology·Yasumi HorimotoRickey Y Yada
Mar 17, 2009·Journal of Molecular Biology·Prasenjit BhaumikAlexander Wlodawer
Sep 18, 2008·Phytochemistry·Miguel A Mazorra-Manzano, Rickey Y Yada
May 20, 2015·Molecular and Biochemical Parasitology·Nonlawat BoonyalaiJirundon Yuvaniyama
May 5, 2011·Biochimica Et Biophysica Acta·Prasenjit BhaumikAlexander Wlodawer
Dec 3, 2014·Molecular and Biochemical Parasitology·Huogen XiaoRickey Y Yada
Aug 5, 2008·Bioorganicheskaia khimiia·M E PopovN S Andreeva
May 6, 2020·The Journal of Biological Chemistry·Armiyaw S NasamuDaniel E Goldberg
Oct 17, 2006·The Journal of Biological Chemistry·Jun LiuDaniel E Goldberg
Jul 20, 2011·Archives of Biochemistry and Biophysics·Huogen XiaoRickey Y Yada

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