Glutamyl Peptides Research Articles

Mammalian Esterase Activity: Implications for Peptide Prodrugs.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

Exploring the Relationships Between Bacterial Community, Taste-Enhancing Peptides and Aroma in Thai Fermented Fish (Pla-ra).

As a traditional Thai condiment, Pla-ra is used to add flavor and richness to dishes. Nine treatment combinations of Pla-ra formulations created from 3 types of fish (Mor fish, Kradee fish, and Mor + Kradee fish) and 4 different carbohydrate sources (none, rice bran, roasted rice, and rice bran─roasted rice mixture) were studied through a 12 month fermentation period (1, 3, 5, 7, 8, 9, 10, 11, and 12 months). 16S rRNA Next Generation Sequencing (NGS) and LC-MS/MS techniques were used to analyze the microbial diversity and identify taste-enhancing peptides. Descriptive sensory analysis was performed on the extracts of the 108 Pla-ra samples mixed in a model broth. Koku perception and saltiness-enhancing attributes were clearly perceived and dominant in all samples, even though glutamyl peptides, including γ-Glu-Val-Gly, were found at subthreshold levels. The samples from mixed fish and Mor fish fermented with roasted ground rice and rice bran for 12 months had the most typical Pla-ra odors and tastes and had high taste-enhancing activities. NGS analysis revealed the presence of bacteria containing a large number of protease and aminopeptidase genes in the samples. Bacillus spp., Gallicola spp., and Proteiniclasticum spp. correlated well with the generation of glutamyl and arginyl peptides and typical odors in the samples. These results confirmed the typical sensory quality of Pla-ra depended on protein sources, carbohydrate sources, and bacteria communities. Further optimization of the microbial composition found could lead to the development of starter cultures to control and promote flavor development in fermented fish products.

Calcium-sensing receptor in GtoPdb v.2023.1

The calcium-sensing receptor (CaS, provisional nomenclature as recommended by NC-IUPHAR [47] and subsequently updated [77]) responds to multiple endogenous ligands, including extracellular calcium and other divalent/trivalent cations, polyamines and polycationic peptides, L-amino acids (particularly L-Trp and L-Phe), glutathione and various peptide analogues, ionic strength and extracellular pH (reviewed in [78]). While divalent/trivalent cations, polyamines and polycations are CaS receptor agonists [14, 110], L-amino acids, glutamyl peptides, ionic strength and pH are allosteric modulators of agonist function [36, 47, 61, 108, 109]. Indeed, L-amino acids have been identified as "co-agonists", with both concomitant calcium and L-amino acid binding required for full receptor activation [149, 54]. The sensitivity of the CaS receptor to primary agonists is increased by elevated extracellular pH [18] or decreased extracellular ionic strength [109] while sensitivity is decreased by pathophysiological phosphate concentrations [20]. This receptor bears no sequence or structural relation to the plant calcium receptor, also called CaS.

Affinity purification, identification, and biochemical characterization of Gamma-glutamyl transpeptidase, a membrane anchored enzyme of Gigantocotyle explanatum.

Gamma-glutamyl transpeptidase is an enzyme that facilitates the transfer of glutamyl groups from glutamyl peptides to other peptides or water. Additionally, it also participates in important processes such as amino acid transport, cellular redox control, drug detoxification, apoptosis, and DNA fragmentation in a various organism. In the present study, GGT activity in Gigantocotyle explanatum was examined in order to characterize the enzyme in the helminth system. GGT is isolated using membrane solubilization and purified through affinity column chromatography (Con-A Sepharose column). Km and Vmax values, as well as the optimal pH, optimal temperature, and incubation period, are also determined using enzyme kinetics. The hetero-dimeric property of the enzyme is demonstrated by the purified GGT, which yielded two subunits of 65.5 and 55kDa. The optimal pH and temperature are found to be 8.0 and 37°C, respectively. While assessing the optimal incubation time of the enzyme, it was observed that the purified GGT not only retained its functional integrity up to 15min but also reflected considerable thermostability at higher temperatures, by retaining 78% and 25% of its initial activities at 50°C and 60°C, respectively. One millimolar concentration of 6-Diazo-5-Oxo Nor-isoleucine (DON), a specific inhibitor of GGT, completely abolished GGT activity. These results suggest that GGT in these worms is a catalytically active enzyme with distinguishing characteristics that can be used for further study to comprehend its function in amphistome biology and in host-parasite relationships, especially since the potential therapeutic candidacy of the GGT enzyme has already been indicated in these groups of organisms.

Impact of axenic and mixed starter cultures on metabolomic and sensory profiles of ripened Italian salami

In this work, the synergistic/antagonistic impact of glucose and mixed starter cultures, namely Latilactobacillus sakei, Pediococcus pentosaceus, and Staphylococcus xylosus, was evaluated in Italian salami in terms of metabolomics and sensory profiles. As expected, Salami manufactured with 0.5 % glucose exhibited a substantial pH drop, showing values close to 5 at 12 days of ripening. Metabolomics revealed 1841 metabolites, mainly belonging to amino acids, peptides, glycerolipids, and nucleic acids, showing a greater hierarchical role of glucose addition when compared with inoculated microbial starters. Distinct metabolomic fingerprints could be observed across treatments, mainly concerning glutamyl peptides like gamma-glutamyl-glutamate (related to the kokumi taste), biogenic amines (spermine), and lipid oxidation products (i.e., the oxylipin 13S-hydroperoxylinolenic acid). Such differences may drive the differences in sensory profiles recorded among treatments. These findings indicate the need to select ad-hoc starter cultures to improve the safety, quality, and sensory traits of salami.

Umami compounds present in umami fraction of acid-hydrolyzed Spirulina (Spirulina platensis)

Spirulina (Spirulina platensis) has been used in food as a protein-rich source. Its umami taste led to the application in meat products, pasta, biscuits and extrusion products. Spirulina hydrolysate has a more intense umami taste. Umami compounds in acid-hydrolysate of the spirulina were investigated in this present study. The spirulina hydrolysate was proven to have a higher umami intensity compared to unhydrolyzed one. Its ultrafiltration fraction with molecular weights <3000 Da was further separated by Sephadex G-25 chromatography, to obtain an umami fraction. The umami fraction was containing succinic acid and free L-glutamic acid at concentrations much higher than their umami taste thresholds. Di- and tripeptides identified as glutamyl peptides were also found abundantly in the fraction.

Calcium-sensing receptor in GtoPdb v.2021.3

The calcium-sensing receptor (CaS, provisional nomenclature as recommended by NC-IUPHAR [47] and subsequently updated [77]) responds to multiple endogenous ligands, including extracellular calcium and other divalent/trivalent cations, polyamines and polycationic peptides, L-amino acids (particularly L-Trp and L-Phe), glutathione and various peptide analogues, ionic strength and extracellular pH (reviewed in [78]). While divalent/trivalent cations, polyamines and polycations are CaS receptor agonists [14, 110], L-amino acids, glutamyl peptides, ionic strength and pH are allosteric modulators of agonist function [36, 47, 61, 108, 109]. Indeed, L-amino acids have been identified as "co-agonists", with both concomitant calcium and L-amino acid binding required for full receptor activation [148, 54]. The sensitivity of the CaS receptor to primary agonists is increased by elevated extracellular pH [18] or decreased extracellular ionic strength [109]. This receptor bears no sequence or structural relation to the plant calcium receptor, also called CaS.

Screening of characteristic umami substances in preserved egg yolk based on the electronic tongue and UHPLC-MS/MS

In this study, the electronic tongue, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and multivariate statistical methods were used to explore the characteristic taste of preserved egg yolk. The results of the electronic tongue showed that the prominent characteristic taste of preserved egg yolk was umami, and there existed significant differences in metabolites between preserved egg yolk and fresh raw yolk according to UHPLC-MS/MS results. Combined with principal component analysis (PCA), orthogonal partial least squares-discriminate analysis (OPLS-DA) and partial least square analysis (PLS), the umami flavor profile of preserved egg yolk was obtained. Glutamic acid (Glu), aspartic acid (Asp), nucleotides, succinic acid, betaine, glutamyl peptides and their derivatives were considered to be the important components of umami substances in preserved egg yolk. Other substances such as carnosine, glutamine and lipids may promote its umami taste retention. Interestingly, it was also found that the change of lipids had a certain effect on the umami taste of preserved egg yolk. Consequently, we explored the characteristic flavor of preserved egg yolk and provided metabolite information of its related umami-producing substances.

Calcium-sensing receptor (version 2020.5) in the IUPHAR/BPS Guide to Pharmacology Database

The calcium-sensing receptor (CaS, provisional nomenclature as recommended by NC-IUPHAR [46] and subsequently updated [76]) responds to multiple endogenous ligands, including extracellular calcium and other divalent/trivalent cations, polyamines and polycationic peptides, L-amino acids (particularly L-Trp and L-Phe), glutathione and various peptide analogues, ionic strength and extracellular pH (reviewed in [77]). While divalent/trivalent cations, polyamines and polycations are CaS receptor agonists [14, 109], L-amino acids, glutamyl peptides, ionic strength and pH are allosteric modulators of agonist function [35, 46, 60, 107, 108]. Indeed, L-amino acids have been identified as "co-agonists", with both concomitant calcium and L-amino acid binding required for full receptor activation [147, 53]. The sensitivity of the CaS receptor to primary agonists is increased by elevated extracellular pH [17] or decreased extracellular ionic strength [108]. This receptor bears no sequence or structural relation to the plant calcium receptor, also called CaS.

Investigation of Kokumi Substances and Bacteria in Thai Fermented Freshwater Fish (Pla-ra).

A 16S rRNA next-generation sequencing technique was applied to investigate the microbial diversity and liquid chromatography-tandem mass spectrometry was used to identify glutamyl peptide profiles of 10 Thai fermented freshwater fish (Pla-ra) samples. A total of 12 genera of bacteria were able to be detected, with Tetragenococcus spp., Staphylococcus spp., and Lactobacillus spp. dominating. Of the 18 glutamyl peptides analyzed, 17 were found, even though the amounts detected were lower than the taste threshold. Despite this, an increase in mouthfulness sensation, reflecting kokumi activity, was clearly identified in most of the samples, which might be because of a synergistic effect of different sub-threshold compounds present in the samples. In principle component analysis, the relationship between microorganisms and glutamyl peptide generation was observed, especially between Tetragenococcus spp. and Lentibacillus spp. and the generation of γ-Glu-Val-Gly. Correlations between microbial diversity and the generation of taste enhancers were identified in this study.

Calcium-sensing receptor (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The calcium-sensing receptor (CaS, provisional nomenclature as recommended by NC-IUPHAR [44]) responds to multiple endogenous ligands, including extracellular calcium and other divalent/trivalent cations, polyamines and polycationic peptides, L-amino acids (particularly L-Trp and L-Phe), glutathione and various peptide analogues, ionic strength and extracellular pH (reviewed in [74]). While divalent/trivalent cations, polyamines and polycations are CaS receptor agonists [14, 106], L-amino acids, glutamyl peptides, ionic strength and pH are allosteric modulators of agonist function [34, 44, 58, 104, 105]. Indeed, L-amino acids have been identified as "co-agonists", with both concomitant calcium and L-amino acid binding required for full receptor activation [143, 51]. The sensitivity of the CaS receptor to primary agonists is increased by elevated extracellular pH [17] or decreased extracellular ionic strength [105]. This receptor bears no sequence or structural relation to the plant calcium receptor, also called CaS.

Diversity of γ- glutamyl peptides and oligosaccharides, the "kokumi" taste enhancers, in seeds from soybean mini core collections.

Soybeans (Glycine max (L,) Merr,) contain γ-glutamyl peptides and oligosaccharides, and these components play an important role in imparting the "kokumi" taste to foods. To gain insight into the genetic diversities and molecular mechanisms of accumulation of γ-glutamyl peptides and oligosaccharides in soybean, we measured the contents of these components using the Japan and World mini core collections. Similar to other previously reported traits, wide variations were detected among the accessions in the core collections with respect to the content of γ-glutamyl peptides and oligosaccharides. We found a positive relationship between the content of γ-glutamyl tyrosine and γ-glutamyl phenylalanine and between the content of raffinose and stachyose. Furthermore, there were unique accessions that included high levels of γ-glutamyl peptides and oligosaccharides. These accessions may be helpful in understanding the accumulation mechanism of γ-glutamyl peptides and oligosaccharides and to increase the "kokumi" taste components in soybean by performing a genetic analysis.

Analysis of Umami Taste Compounds in a Fermented Corn Sauce by Means of Sensory-Guided Fractionation.

Corn sauce, an ingredient obtained from the fermentation of enzymatically hydrolyzed corn starch and used in culinary applications to provide savory taste, was investigated in this study. The links between its sensory properties and taste compounds were assessed using a combination of analytical and sensory approaches. The analyses revealed that glutamic acid, sodium chloride, and acetic acid were the most abundant compounds, but they could not explain entirely the savory taste. The addition of other compounds, found at subthreshold concentrations (alanine, glutamyl peptides, and one Amadori compound), contributed partly to close the sensory gap between the re-engineered sample and the original product. Further chemical breakdown, by a sensory-guided fractionation approach, led to the isolation of two fractions with taste-modulating effects. Analyses by mass spectrometry and nuclear magnetic resonance showed that the fractions contained glutamyl peptides, pyroglutamic acid, glutamic acid, valine, N-formyl-glutamic acid, and N-acetyl-glutamine.

Development of a LC-MS/MS method for the determination of isomeric glutamyl peptides in food ingredients.

A liquid chromatography with tandem mass spectrometry method was developed for the determination of 27 glutamyl di- and tripeptides in food ingredients. Such compounds are of importance for the food industry, as they can modulate the perception of basic tastes (sweet, salty, and umami). Due to their high polarity, the hydrophilic interaction chromatography mode was selected to have sufficient retention on the column and the best separation was obtained on an amide hybrid silica stationary phase packed with 1.7μm particles. Thorough optimization of the mobile phase was performed as the start-composition had to be free of ammonium to avoid on-column cis-trans isomerization of the first eluting proline dipeptide. A baseline separation was achieved for all α and γ isomers whereas only a partial resolution was obtained for γ-Glu-Leu and γ-Glu-Ile, for which only the position of a methyl group differs. A fast sample preparation, based on successive dilutions, was performed before injection into the liquid chromatography with tandem mass spectrometry system. The developed method was then applied for the semi-quantification of glutamyl di- and tri-peptides in four different food ingredients. The methodology will further support the optimization of production processes to select the conditions for which the peptide concentrations would be the highest.

How does conserved dopamine neurotrophic factor protect against and rescue neurodegeneration of PC12 cells?

Conserved dopamine neurotrophic factor protects and rescues dopaminergic neurodegeneration induced by 6-hydroxydopamine in vivo, but its potential value in treating Parkinson's disease remains controversial. Here, we used the proteasome inhibitors lactacystin and MG132 to induce neurodegeneration of PC12 cells. Afterwards, conserved dopamine neurotrophic factor was administrated as a therapeutic factor, both pretreatment and posttreatment. Our results showed that (1) conserved dopamine neurotrophic factor enhanced lactacystin/MG132-induced cell viability and morphology, and attenuated alpha-synuclein accumulation in differentiated PC12 cells. (2) Enzyme linked immunosorbent assay showed up-regulated 26S proteasomal activity in MG132-induced PC12 cells after pre- and posttreatment with conserved dopamine neurotrophic factor. Similarly, 26S proteasome activity was upregulated in lactacystin-induced PC12 cells pretreated with conserved dopamine neurotrophic factor. (3) With regard proteolytic enzymes (specifically, glutamyl peptide hydrolase, chymotrypsin, and trypsin), glutamyl peptide hydrolase activity was up-regulated in lactacystin/MG132-administered PC12 cells after pre- and posttreatment with conserved dopamine neurotrophic factor. However, upregulation of chymotrypsin activity was only observed in MG132-administered PC12 cells pretreated with conserved dopamine neurotrophic factor. There was no change in trypsin expression. We conclude that conserved dopamine neurotrophic factor develops its neurotrophic effects by modulating proteasomal activities, and thereby protects and rescues PC12 cells against neurodegeneration.

Modulation of sweet taste by umami compounds via sweet taste receptor subunit hT1R2.

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.

The mathematical modelling of cheese ripening

A mathematical model is developed for the ripening of cheese. Such models may assist predicting final cheese quality using measured initial composition. The main constituent chemical reactions are described with ordinary differential equations. Numerical solutions to the model equations are found using Matlab. Unknown parameter values have been fitted using experimental data available in the literature. The results from the numerical fitting are in good agreement with the data. Statistical analysis is performed on near infrared data provided to the MISG. However, due to the inhomogeneity and limited nature of the data, not many conclusions can be drawn from the analysis. A simple model of the potential changes in acidity of cheese is also considered. The results from this model are consistent with cheese manufacturing knowledge, in that the pH of cheddar cheese does not significantly change during ripening. References D. Dawson and J. Feagan, Bacteriology of cheddar cheese: A study of starter organisms in manufacture and maturing. J. Dairy Res. 24 , 210–224, 1957. M.A. Drake, R.E. Miracle and D.J. McMahon, Impact of fat reduction on flavor and flavor chemistry of Cheddar cheeses. Journal of Dairy Science 93 , 5069–5081, 2010. M. Fenelon and T. Guinee, Primary proteolysis and textural changes during ripening in cheddar cheeses manufactured to different fat contents. Int. Dairy J. 10 , 151–158, 2000. R. Hutkins and N. Nannen, pH homeostasis in lactic acid bacteria. J. Dairy Sci. 76 , 2354–2365, 1992. M. Johnson, Cheese pH–-what's behind the rise and fall? Technical Report 14, Wisconsin Center for Dairy Research, December 2002. J.K. Kim, K. Starzak, G.W. Preckshot, R. Marshall and R.K. Bajpai, Critical reactions in ripening of cheeses–-a kinetic analysis. Appl. Biochem. Biotech. 45 , 51–68, 1994. B.A. Law and A.S. Wigmore, Microbial proteinases as agents for accelerated cheese ripening. Int. J. Dairy Tech. 35 , 75–76, 2007. R. Lawrence, L. Creamer, and J. Gilles, Texture development during cheese ripening. J. Dairy Sci. 70 , 1748–1760, 1987. R. Marsili, Monitoring chemical changes in cheddar cheese during aging by high performance liquid chromatography and gas chromatography techniques. Journal of Dairy Science 68 , 3155–3161, 1985. K. Presser, D. Ratkowsky, and T. Ross, Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration. Appl. Environ. Microbiol. 63 , 2355–2360, 1997. S. Rehman, D. Waldron, and P. Fox, Effect of modifying lactose concentration in cheese curd on proteolysis and in quality of Cheddar cheese. Int. Dairy J. 14 , 591–597, 2004. T. Singh, M. Drake, and K. Cadwallader, Flavour of cheddar cheese: A chemical and sensory perspective. Compr. Rev. Food Sci. F. 2 , 166–189, 2003. S. Toelstede and T. Hofmann, Kokumi-active glutamyl peptides in cheeses and their biogeneration by Penicillium roquefortii. Agric. Food Chem. 13 , 3738–3748, 2009.

Hydrophilic di- and tripeptides are not a precondition for savoury flavour in mature Cheddar cheese

In the literature hydrophilic glutamyl di- and tripeptides have been described to induce an umami or savoury taste. In this study, HILIC-ESI-MS was applied for analysis of low-molecular-weight peptides in the ultra-filtrated (cut-off 10 kg · mol−1) water-soluble extract of two mature Cheddar cheeses, selected because of their pronounced savoury flavour. A reference mixture of amino acids and di- and tripeptides was successfully separated by HILIC using ammonium acetate buffer (0.50 g·L−1, pH 5.5) as the mobile phase. Unambiguous identification of the compounds was based on retention time and characteristic fragmentation patterns in MS/MS and MS3. However, no low-molecular-weight glutamyl peptides were identified in the extracts of the mature Cheddar cheeses. It was concluded that hydrophilic glutamyl di- and tripeptides are not a precondition for savoury flavour in mature Cheddar cheese.

Alterations of cerebral cortex and hippocampal proteasome subunit expression and function in a traumatic brain injury rat model

Following cellular stress or tissue injury, the proteasome plays a critical role in protein degradation and signal transduction. The present study examined the beta-subunit expression of constitutive proteasomes (beta1, beta2, and beta5), immunoproteasomes (beta1i, beta2i, and beta5i) and the 11S proteasome activator, PA28alpha, in the rat CNS after traumatic brain injury (TBI). Concomitant measures assessed changes in proteasome activities. Quantitative real time PCR results indicated that beta1 and beta2 mRNA levels were not changed, while beta5 mRNA levels were significantly decreased in injured CNS following TBI. However, beta1i, beta2i, beta5i, and PA28alpha mRNA levels were significantly increased in the injured CNS. Western blotting studies found that beta1, beta2, beta5, beta2i, and beta5i subunit protein levels remained unchanged in the injured CNS, but beta1i and PA28alpha protein levels were significantly elevated in ipsilateral cerebral cortex and hippocampus. Proteasome activity assays found that peptidyl glutamyl peptide hydrolase-like and chymotrypsin-like activity were significantly reduced in the CNS after TBI, and that trypsin-like proteasome activity was increased in the injured cerebral cortex. Our results demonstrated that both proteasome composition and function in the CNS were affected by trauma. Treatments that preserve proteasome function following CNS injury may be beneficial as an approach to cerebral neuroprotection.

Characterization and localization of Plasmodium falciparum homolog of prokaryotic ClpQ/HslV protease

The β subunits (β1, β2, and β5) of 20S proteasome and HslV/ClpQ are ATP-dependent threonine proteases present in eukaryotes and prokaryotes, respectively that control levels of key regulatory proteins in the cell. The orthologue of prokaryotic HslV protease in Plasmodium falciparum (PfHslV) is a novel drug target candidate that has no homolog in the human host. In the present study, the PfHslV was expressed, localized and biochemically characterized. The recombinant PfHslV harbored threonine protease specific activity as well as chymotrypsin like and peptidyl glutamyl peptide hydrolase activities. All the three activities could be inhibited by respective specific inhibitors. The protein was localized in the cytosol of the parasite as a soluble protein by Western immunoblotting of parasite fractions and by immuno-fluorescence microscopy. Activity of the protease in the parasite was ascertained by following the degradation of GFP in a transgenic parasite line expressing fusion protein of GFP and Arc-repressor gene, a known target of HslV protease in the prokaryotes. A model structure of PfHslV was constructed based on the crystal structure of Escherichia coli HslV to assess the structural homology. Availability of the structure model of PfHslV may facilitate identification or designing of novel and specific drugs against PfHslV. The in vitro protease assays with recombinant PfHslV and the transgenic parasite line generated in the present study may be exploited in the screening of novel inhibitors to evaluate their anti-malarial activity.