Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases

Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX. To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap‐like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15‐fold the IC₅₀ values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.     

A family of aspartic proteases and a novel, dynamic and cell-cycle-dependent protease localization in the secretory pathway of Toxoplasma gondii

Aspartic proteases are important virulence factors in pathogens like HIV, Candida albicans or Plasmodium falciparum . We report here the identification of seven putative aspartic proteases, TgASP1 to TgASP7, in the apicomplexan parasite Toxoplasma gondii . Bioinformatic and phylogenetic analysis of the TgASPs and other aspartic proteases from related Apicomplexa suggests the existence of five distinct groups of aspartic proteases with different evolutionary lineages. The members of each group share predicted biological features that validate the phylogeny. TgASP1 is expressed in tachyzoites, the rapidly dividing asexual stage of T. gondii . We present the proteolytic maturation and subcellular localization of this protease through the cell cycle. TgASP1 shows a novel punctate localization associated with the secretory system in non-dividing cells, and relocalizes dramatically and unambiguously to the nascent inner membrane complex of daughter cells at replication, before coalescing again at the end of division.     

The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells

Toxoplasma gondii and Plasmodium species are obligatory intracellular parasites that export proteins into the infected cells in order to interfere with host‐signalling pathways, acquire nutrients or evade host defense mechanisms. With regard to export mechanism, a wealth of information in Plasmodium spp. is available, while the mechanisms operating in T. gondii remain uncertain. The recent discovery of exported proteins in T. gondii, mainly represented by dense granule resident proteins, might explain this discrepancy and offers a unique opportunity to study the export mechanism in T. gondii. Here, we report that GRA16 export is mediated by two protein elements present in its N‐terminal region. Because the first element contains a putative Plasmodium export element linear motif (RRLAE), we hypothesized that GRA16 export depended on a maturation process involving protein cleavage. Using both N‐ and C‐terminal epitope tags, we provide evidence for protein proteolysis occurring in the N‐terminus of GRA16. We show that TgASP5, the T. gondii homolog of Plasmodium plasmepsin V, is essential for GRA16 export and is directly responsible for its maturation in a Plasmodium export element‐dependent manner. Interestingly, TgASP5 is also involved in GRA24 export, although the GRA24 maturation mechanism is TgASP5‐independent. Our data reveal different modus operandi for protein export, in which TgASP5 should play multiple functions.     

Two palmitoyl acyltransferases involved sequentially in the biogenesis of the inner membrane complex of Toxoplasma gondii

The phylum Apicomplexa includes a number of significant human pathogens like Toxoplasma gondii and Plasmodium species. These obligate intracellular parasites possess a membranous structure, the inner membrane complex (IMC), composed of flattened vesicles apposed to the plasma membrane. Numerous proteins associated with the IMC are anchored via a lipid post‐translational modification termed palmitoylation. This acylation is catalysed by multi‐membrane spanning protein S‐acyl‐transferases (PATs) containing a catalytic Asp‐His‐His‐Cys (DHHC) motif, commonly referred to as DHHCs. Contrasting the redundancy observed in other organisms, several PATs are essential for T. gondii tachyzoite survival; 2 of them, TgDHHC2 and TgDHHC14 being IMC‐resident. Disruption of either of these TgDHHCs results in a rapid collapse of the IMC in the developing daughter cells leading to dramatic morphological defects of the parasites while the impact on the other organelles is limited to their localisation but not to their biogenesis. The acyl‐transferase activity of TgDHHC2 and TgDHHC14 is involved sequentially in the formation of the sub‐compartments of the IMC. Investigation of proteins known to be palmitoylated and localised to these sub‐compartments identified TgISP1/3 as well as TgIAP1/2 to lose their membrane association revealing them as likely substrates of TgDHHC2, while these proteins are not impacted by TgDHHC14 depletion.     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

Toxoplasma gondii Hsp20 is a stripe-arranged chaperone-like protein associated with the outer leaflet of the inner membrane complex

Background information. Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has five sHsps [small Hsps (heat‐shock proteins)] located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual. Results. The localization of Hsp20 was further analysed using high‐resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated with the outer surface of the IMC (inner membrane complex), in a set of discontinuous stripes following the same spiralling trajectories as the subpellicular microtubules. The detergent extraction profile of Hsp20 was similar to that of GAP45 [45 kDa GAP (gliding‐associated protein)], a glideosome protein associated with the IMC, but was different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate phospholipids could be observed. Conclusions. Hsp20 was shown to be associated with a specialized membranous structure of the parasite, the IMC. This discontinuous striped‐arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.     

Chymostatin can combine with pepstatin to eliminate extracellular protease activity in cultures of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL-3

Aspergillus strains are being considered as potential hosts for recombinant heterologous protein production because of their excellent extracellular enzyme production characteristics. However, Aspergillus proteases are problematic in that they modify and degrade the heterologous proteins in the extracellular medium. In previous studies we observed that media adjustments and maintenance of a filamentous morphology greatly reduced protease activity and that a low concentration of the aspartic protease inhibitor pepstatin inhibited the latter protease activity to the extent of approximately 90%. In this paper we report that when the serine protease inhibitor chymostatin is used in combination with pepstatin 99–100% of total protease activity in Aspergillus cultures is inhibited. In protease assays a concentration of 30 μM chymostatin combined with 0.075 μM pepstatin was required for maximum inhibition. Inhibitor concentrations of chymostatin and pepstatin of 120 and 0.3 μM, respectively, when added to Aspergillus cultures, has no significant effect on biomass production, glucose utilization or culture pH pattern. The potential of using these protease inhibitors in cultures of recombinant Aspergillus strains producing heterologous proteins will now be investigated to determine if the previously observed recombinant protein denaturing effects of Aspergillus proteases can be negated.     

The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

Secreted aspartic proteases (Saps) are extracellular proteolytic enzymes that enhance the virulence of Candida pathogens. These enzymes therefore represent possible targets for therapeutic drug design. Saps are inhibited by nanomolar concentrations of the classical inhibitor of aspartic proteases pepstatin A and also by the inhibitors of the HIV protease, but with the K_i of micromolar values or higher. To contribute to the discussion regarding whether HIV protease inhibitors can act against opportunistic mycoses by the inhibition of Saps, we determined the structure of Sapp1p from Candida parapsilosis in complex with ritonavir (RTV), a clinically used inhibitor of the HIV protease. The crystal structure refined at resolution 2.4 Å proved binding of RTV into the active site of Sapp1p and provided the structural information necessary to evaluate the stability and specificity of the protein-inhibitor interaction.     

Analysis of Two Aspergillus nidulans Genes Encoding Extracellular Proteases

vanKuyk, P. A., Cheetham, B. F., and Katz, M. E. 2000. Analysis of two Aspergillus nidulans genes encoding extracellular proteases. Characterization of prtAΔ mutants, generated by gene disruption, showed that the prtA gene is responsible for the majority of extracellular protease activity secreted by Aspergillus nidulans at both neutral and acid pH. The prtAΔ mutation was used to map the prtA gene to chromosome V. Though aspartic protease activity has never been reported in A. nidulans and the prtAΔ mutants appear to lack detectable acid protease activity, a gene (prtB) encoding a putative aspartic protease was isolated from this species. Comparison of the deduced amino acid sequence of PrtB to the sequence of other aspergillopepsins suggests that the putative prtB gene product contains an eight-amino-acid deletion prior to the second active site Asp residue of the protease. RT-PCR experiments showed that the prtB gene is expressed, albeit at a low level.     

Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases

Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino‐acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.     

A Structural Biology Approach to Understand Human Lymphatic Filarial Infection

The presence of aspartic protease inhibitor in filarial parasite Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO₂)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from ¹⁵N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method.     

北黄海沉积物可培养产蛋白酶细菌分离鉴定

【目的】揭示北黄海沉积物中可培养产胞外蛋白酶细菌及蛋白酶多样性,增加人们对北黄海生态系统中产蛋白酶菌多样性的认识,为海洋产蛋白酶微生物的挖掘提供菌群资源。【方法】分别将5个北黄海沉积物样品梯度稀释涂布至酪蛋白明胶筛选平板,选择性分离产蛋白酶细菌;并通过分析基于16S rRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;分别测定胞外蛋白酶活性并对酶活较高的39株菌进行基于苯甲基磺酰氟(PMSF,丝氨酸蛋白酶抑制剂)、邻菲罗啉(o-phenanthroline,O-P,金属蛋白酶抑制剂)、E-64(半胱氨酸蛋白酶抑制剂)和pepstatin A(天冬氨酸蛋白酶抑制剂)4种抑制剂的酶活抑制实验以及所有菌株对3种底物(酪蛋白、明胶、弹性蛋白)的水解能力;分析这些细菌所产胞外蛋白酶的特性及多样性。【结果】从5个北黄海沉积物样品中分离获得66株产蛋白酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria、Actinobacteria和Firmicutes 4个门的7个属,其中Pseudoalteromonas(69.9%)、Sulfitobacter(12.1%)和Salegentibacter(10.6%)是优势菌群;沉积物中可培养的产蛋白酶细菌的丰度为104 CFU/g;蛋白酶酶活抑制实验表明所有测定菌株产生的胞外蛋白酶属于丝氨酸蛋白酶和/或金属蛋白酶,仅有少数菌株所产蛋白酶具有半胱氨酸蛋白酶或天冬氨酸蛋白酶活性。【结论】北黄海沉积物中可培养产蛋白酶细菌类群较为丰富,Pseudoalteromonas、Sulfitobacter和Salegentibacter菌株是优势菌群,测定菌株所产胞外蛋白酶主要是丝氨酸蛋白酶和/或金属蛋白酶。     

Toxoplasma gondii protease TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites

Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full‐length TgSUB1 restores processing, complementation of Δsub1 parasites with TgSUB1 lacking the GPI anchor (Δsub1::ΔGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Δsub1 and Δsub1::ΔGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.     

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable ‘enzyme-product’ complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (K_b) values between 10.23?×?10³ and 6.52?×?10³ M^?1. The binding reactions were enthalpy driven with ΔH_b values between ?50.99 and ?46.07 kJ mol^?1. From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.     

Identification and characterization of Toxoplasma SIP,a conserved apicomplexan cytoskeleton protein involved in maintaining the shape,motility and virulence of the parasite

Apicomplexa possess a complex pellicle that is composed of a plasma membrane and a closely apposed inner membrane complex (IMC) that serves as a support for the actin‐myosin motor required for motility and host cell invasion. The IMC consists of longitudinal plates of flattened vesicles, fused together and lined on the cytoplasmic side by a subpellicular network of intermediate filament‐like proteins. The spatial organization of the IMC has been well described by electron microscopy, but its composition and molecular organization is largely unknown. Here, we identify a novel protein of the IMC cytoskeletal network in Toxoplasma gondii, called TgSIP, and conserved among apicomplexan parasites. To finely pinpoint the localization of TgSIP, we used structured illumination super‐resolution microscopy and revealed that it likely decorates the transverse sutures of the plates and the basal end of the IMC. This suggests that TgSIP might contribute to the organization or physical connection among the different components of the IMC. We generated a T.gondii SIP deletion mutant and showed that parasites lacking TgSIP are significantly shorter than wild‐type parasites and show defects in gliding motility, invasion and reduced infectivity in mice.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> glycosylphosphatidylinositols are not involved in <Emphasis Type="Italic">T. gondii</Emphasis>-induced host cell survival

Toxoplasma gondii is an intracellular parasite able to both promote and inhibit apoptosis. T. gondii renders infected cells resistant to programmed cell death induced by multiple apoptotic triggers. On the other hand, increased apoptosis of immune cells after in vivo infection with T. gondii may suppress the immune response to the parasite. Glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of T. gondii tachyzoites and GPIs are involved in the pathogenicity of protozoan parasites. In this report, we determine if GPIs are responsible for inhibition or induction of host cell apoptosis. We show here that T. gondii GPIs fail to block apoptosis that was triggered in human-derived cells via extrinsic or intrinsic apoptotic pathways. Furthermore, characteristics of apoptosis, e.g. caspase-3/7 activity, phosphatidylserine exposition at the cell surface or DNA strand breaks, were not observed in the presence of T. gondii GPIs. These results indicate that T. gondii GPIs are not involved in survival or in apoptosis of host cells. This absence of effect on apoptosis could be a feature common to GPIs of other parasites.     

Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii

Membrane microdomains or rafts, sterol- and sphingolipid-rich microdomains in the plasma membrane have been studied extensively in mammalian cells. Recently, rafts were found to mediate virulence in a variety of parasites, including Toxoplasma gondii. However, it has been difficult to examine a two-dimensional distribution of lipid molecules at a nanometer scale. We tried to determine the distribution of glycosphingolipids GM1 and GM3, putative raft components in the T. gondii cell membrane in this study, using a rapid-frozen and freeze-fractured immuno-electron microscopy method. This method physically stabilized molecules in situ, to minimize the probability of artefactual disruption. Labeling of GM3, but not GM1, was observed in the exoplasmic (or luminal), but not the cytoplasmic, leaflet of the inner membrane complex (IMC) in T. gondii infected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of the T. gondii plasma membrane. In contrast to HFF-1, T. gondii infected in mouse fibroblast (MF), labelings of both GM1 and GM3 were detected in the IMC luminal leaflet, although GM1′s gold labeling density was very low. The same freeze-fracture EM method showed that both GM1 and GM3 were expressed in the exoplasmic leaflet of the MF plasma membrane. However, labeling of only GM3, but not GM1, was detected in the exoplasmic leaflet of the HFF-1 plasma membrane. These results suggest that GM1 or GM3, localized in the IMC, is obtained from the plasma membranes of infected host mammalian cells. Furthermore, the localization of microdomains or rafts in the luminal leaflets of the intracellular confined space IMC organelle of T. gondii suggests a novel characteristic of rafts.     

Analysis of recombinant protein toxicity in <Emphasis Type="Italic">E. coli</Emphasis> through a phage λ-based genetic screening system

The aspartic protease from the human immunodeficiency virus type 1 (HIV-1) is highly toxic to E. coli, thus impairing its yield in production processes. Proteolytic cleavage of essential cellular proteins is probably a major contributor to the bacteriocidal effect but this has not been proven. Through an adapted high-throughput λ-based screening system, we have analyzed a set of HIV-1 protease mutants with distinguishable catalytic properties and we show that inactive enzymes are as toxic to E. coli cells as the wild-type enzyme. Together with additional data from directed molecular evolution approaches, these results indicate that the toxicity of the viral protease is not linked to its proteolytic activity. Our study also reveals that the λ-based screening system is a robust new tool for the genetic analysis of highly toxic recombinant products in E. coli.     

Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to micronemal protein protease 1 activity during invasion

Host cell entry by the Apicomplexa is associated with the sequential secretion of invasion factors from specialized apical organelles. Secretion of micronemal proteins (MICs) complexes by Toxoplasma gondii facilitates parasite gliding motility, host cell attachment and entry, as well as egress from infected cells. The shedding of MICs during these steps is mediated by micronemal protein proteases MPP1, MPP2 and MPP3. The constitutive activity of MPP1 leads to the cleavage of transmembrane MICs and is linked to the surface rhomboid protease 4 (ROM4) and possibly to rhomboid protease 5 (ROM5). To determine their importance and respective contribution to MPP1 activity, in this study ROM4 and ROM5 genes were abrogated using Cre‐recombinase and CRISPR‐Cas9 nuclease, respectively, and shown to be dispensable for parasite survival. Parasites lacking ROM4 predominantly engage in twirling motility and exhibit enhanced attachment and impaired invasion, whereas intracellular growth and egress is not affected. The substrates MIC2 and MIC6 are not cleaved in rom4‐ko parasites, in contrast, intramembrane cleavage of AMA1 is reduced but not completely abolished. Shedding of MICs and invasion are not altered in the absence of ROM5; however, this protease responsible for the residual cleavage of AMA1 is able to cleave other AMA family members and exhibits a detectable contribution to invasion in the absence of ROM4.     

Protease excretion during pineapple micropropagation in temporary immersion bioreactors

Summary Biotechnology has become an important tool to produce plant secondary metabolites and proteases are among them. Although pineapple plants have been found to produce proteases, most of the biotechnological investigations on this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore we decided to modify this phase, looking for an increase of protease excretion. Three independent experiments were performed to evaluate the effects of culture duration, and levels of gibberellic acid (GA) and 6-benzyladenine (BA). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. As happens in investigations focused on protease production, the specific protease activity was the most important indicator recorded here. It maximized at 21 d of culture. Moreover, GA (4.2 μM) increased specific activity in the culture medium while BA produced a negative effect. Results shown here demonstrate that conditions adquate for propagation purposes (15-d pre-elongation phase; 2.8 μM GA; 2.2 μM BA) are not necessarily adequate for protease excretion.