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  • Methodology article
  • Open Access

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme

  • 1,
  • 1,
  • 1,
  • 1,
  • 2 and
  • 1Email author
BMC Biotechnology201818:20

https://doi.org/10.1186/s12896-018-0429-y

  • Received: 27 December 2017
  • Accepted: 15 March 2018
  • Published:

Abstract

Background

Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases.

Results

Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile.

Conclusions

We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

Keywords

  • Recombinant protein
  • Amebic cysteine protease
  • Expression and purification
  • Cytosolic oxidative folding
  • Proteolytic activity
  • Enzyme inhibition

Background

Proteases are hydrolytic enzymes that cleave the peptide bond that joins amino acids in proteins [1, 2]. Based on the cleavage site at the substrate, they are usually subdivided into exopeptidases and endopeptidases [1, 3]. However, according to their catalytic mechanism and the amino acid residue present at the active site, are so grouped in aspartic proteases, cysteine proteases, glutamic proteases, metalloproteases, asparagine proteases, serine proteases, threonine proteases, and proteases with mixed or unknown catalytic mechanism [4]. Because of their biochemical function play significant roles in several processes that are critical for cell life, such as degradation of potentially toxic misfolded or damaged polypeptides [5].

Worldwide, protozoan parasites infectious to humans represent a major threat to public health [6, 7]. Intracellular parasites, such as Toxoplasma, Leishmania, Plasmodium, and Trypanosoma, are among the most lethal [8]. Cryptosporidium, Entamoeba, and Giardia infect the gastrointestinal tract causing diarrhea, which is fatal if left untreated [9], while Trichomonas colonizes the epithelium of the urogenital tract producing inflammation in the cervix, vagina, and urethra [10]. These pathogens encode a variety of proteases involved in essentially all aspects of their biology, including (i) cell invasion, development, and migration, (ii) evasion of host immune system, and (iii) degradation of proteins for nutrition [11, 12]. Thus, these proteolytic enzymes have medical and pharmaceutical importance as they are valuable targets for designing novel or improved therapeutic compounds [3, 610, 1317].

Entamoeba histolytica (Eh), the causative agent of human amebiasis [18], has 86 genes encoding proteolytic enzymes: 50 cysteine proteases, 22 metalloproteases, 10 serine peptidases, and 4 aspartic proteases [19]. Of these, secreted cysteine proteases (CP) play significant roles in pathogenicity, i.e., proteolytic degradation of host extracellular matrix components [20, 21]. Different studies have shown that the high expression levels of three enzymes: EhCP1, EhCP2, and EhCP5, account for roughly 90% of the CP-specific activity [22, 23]. Interestingly, EhCP1 is unique to E. histolytica, since the orthologous gene is absent in E. dispar [22, 24], a morphologically undistinguishable but non-pathogenic ameba [25]. As an inactive precursor or zymogen, which comprises a pro-domain that blocks the active site, depends on the cleavage of the signal peptide and the limited proteolysis of the pro-domain to get its mature form (Additional file 1: Figure S1).

To date, several recombinant approaches have been undertaken to produce EhCP proteins using Escherichia coli cells as the bacterial system [24, 2629]. Although active enzymes were satisfactorily obtained, protein insolubility was the major challenge found, requiring an elaborate oxidative protein folding process to promote solubility. E. coli SHuffle Express, a mutant strain recently been developed and successfully proved to own an enhanced ability to correctly fold proteins with disulfide bonds in the cytoplasmic compartment [30]. Considering the foregoing, we took a chance on this bacterial system for soluble expression of recombinant EhCP proteins. Here, we present efficient recombinant production of fully soluble and active EhCP1 enzyme using E. coli SHuffle Express strain as the bacterial host.

Results

First, using pQE30 as the parental vector, the recombinant pQEhCP1 plasmid (Additional file 1: Figure S2) was successfully constructed by subcloning the gene fragment encoding the pro-mature EhCP1 protein under the control of the T5/lacO (promoter-operator) sequence. Competent E. coli SHuffle Express cells were efficiently transformed with pQEhCP1 and recombinant protein was easily induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). After bacterial culturing for 18 h at 30 °C, recombinant EhCP1 protein tagged with a hexahistidine sequence at the N-terminus was readily purified by two consecutive chromatographic protocols: nickel-affinity and gel permeation. Analytical expression assays revealed proper gelatinase activity (Additional file 1: Figure S3). Furthermore, quantitative analysis of the expression and purification process showed a reasonable outcome in terms protein purity and protease activity (Table 1).
Table 1

Purification of recombinant EhCP1 (summary)

Purification

Step

Total Protein

(mg) b

Total Activity (units) c

Specific Activity (units/mg) c

Recovery Yield

(%) d

Enzyme Purity

(%) e

Crude lysate a

69.75

9918.5

142.2

100.0

2.4

Nickel-affinity

0.81

4731.2

5841.0

47.7

97.0

Gel permeation

0.65

3915.0

6023.0

39.5

100.0

aObtained from five cell pellets, each from a 200-mL batch (total: 1 L of bacterial culture). bProtein concentration determined by Bradford assay. cProtease activity measured by the chromogenic assay. dRelative amount of total activity at each step as compared to the first. eRelative amount of specific activity at each step as compared to the last

Protease activity of recombinant EhCP1 was effectively measured using two synthetic peptides as substrates, Z-Arg-Arg-pNA (chromogenic) and Z-Arg-Arg-AMC (fluorogenic). While considering the pH value of 6.5 as standard (as maximal activity was found within 5.5 and 7.5 [24]), the assay temperature was simply decided from preliminary measurements (using the initial rate as a test indicator): 37 °C for chromogenic and 22 ± 1 °C for fluorogenic. Since the precise amide bond at the substrate was efficiently recognized and hydrolyzed by enzymatic action, each released product (p-nitroaniline, pNA; 7-amino-4-methylcoumarine, AMC) was correspondingly monitored and used to calculate the protease activity. Depicted in Fig. 1, the enzyme concentration positively affects the rate of reaction, i.e., an increase in the recombinant EhCP1 amount improves the protease activity. Likewise, the substrate concentration stimulates the rate of reaction, and thus the protease activity (Fig. 2). Thoroughly examining the latter, using enzyme at 0.05 mg/mL (for chromogenic assay) or 0.005 mg/mL (for fluorogenic assay), we obtained sound kinetic parameters for the hydrolysis of both substrates: Z-Arg-Arg-pNA (Km = 57.1 μM; Vmax = 29.3 units; SA = 5856.6 units/mg) and Z-Arg-Arg-AMC (Km = 6.7 μM; Vmax = 19 FU/min; SA = 38,088 FU/min/mg).
Fig. 1
Fig. 1

Effect of the enzyme concentration on the rate of reaction. a Hydrolysis of Z-Arg-Arg-pNA by recombinant EhCP1 (0–4.65 μg). b Hydrolysis of Z-Arg-Arg-AMC by recombinant EhCP1 (0–0.93 μg). Data represent the mean ± S.E.M. (bars) of 2–3 independent experiments

Fig. 2
Fig. 2

Effect of the substrate concentration on the rate of reaction. a Hydrolysis of Z-Arg-Arg-pNA (0–200 μM) by recombinant EhCP1 (0.05 mg/mL). b Hydrolysis of Z-Arg-Arg-AMC (0–10 μM) by recombinant EhCP1 (0.005 mg/mL). Data represent the mean ± S.E.M. (bars) of 3 independent experiments

Proven so far, recombinant EhCP1 exhibits specific catalytic abilities, proceeds effectively on the substrate, and follows an apparent Michaelis-Menten kinetics. Finally, to assert its value as a therapeutic target, the effect of L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine (E-64) on the protease activity was further evaluated. E-64 is a well-known CP-specific inhibitor that functions as an effective active-site titrant [31]. Displayed in Fig. 3, the sigmoidal curve indicates that inhibition of protease activity follows a typical dose-response profile. Also, using the enzyme concentration as before (for kinetic parameters), we found the IC50 at 1 μM.
Fig. 3
Fig. 3

Effect of the E-64 concentration on the protease activity of recombinant EhCP1. The enzyme and inhibitor (0–6 μM) interact for 15 min before the assay. Hydrolysis of 200 μM Z-Arg-Arg-pNA (a) and 10 μM Z-Arg-Arg-AMC (b) by recombinant EhCP1 (as in Fig. 2). Data represent the mean ± S.E.M. (bars) of 3 independent experiments

Discussion

We used E. coli SHuffle Express cells harboring a recombinant plasmid to produce EhCP1 protein. Along with the mature domain, the recombinant enzyme comprises the pro-domain, which is necessary for preserving its inactive state and apparently required for proper folding [28]. As compared to those reported producing an active thioredoxin-tagged protein (Trx-EhCP1) [24]: insoluble material (about 10 mg/L) that requires optimal refolding conditions to get reliable enzymatic activity, our approach represents progress in the recombinant production of soluble and active EhCP1 protein. Even so, since a limited yield of pure protein was found (0.65 mg/L), we acknowledge the need for further studies to improve production levels. Despite the apparent dissimilarity between the calculated Km values for two synthetic peptides used as substrates, Z-Arg-Arg-pNA and Z-Arg-Arg-AMC, which was attributed to the chemical nature of the leaving group (pNA or AMC) [32], the observed value for hydrolysis of Z-Arg-Arg-AMC (6.7 μM) closely resembles those reported using other recombinant enzymes, such as Trx-EhCP1 (2 μM) [24] and EhCP5 (7.29 μM) [33]. Also, as a protease sensitive to E-64 (a CP-specific inhibitor), it exhibits an IC50 value (1 μM) comparable to that established for recombinant EhCP5 (6 μM) [33]. Altogether, it is fair to presume suitability of the recombinant EhCP1 as a therapeutic target.

Conclusions

Search for new or improved compounds with therapeutic activity requires the identification and validation of targets (i.e., proteins with the potential of being blocked, inhibited, modulated or regulated, when interacting with a drug) [34]. The availability of genomic data has notably contributed towards distinguishing putative targets [35, 36]. Since isolation and purification of proteins from their source is often a challenging task, the recombinant production using an efficient expression system represents a promising approach to get suitable material (i.e., pure and functional proteins) [36, 37].

In this study, we report the recombinant production of EhCP1 using E. coli SHuffle Express strain as the bacterial host. We present a simple protocol for the expression and purification of the recombinant protein as a soluble and active enzyme. Through the analysis of protease activity and enzymatic inhibition, we confirm its reliability as a therapeutic target. Finally, we propose an approachable bacterial cell system for the recombinant production of other amebic proteins, particularly those with a need for proper oxidative folding.

Methods

Materials

DNA amplification reagents and purification kits were acquired from Qiagen (Germantown, MD, USA). Bacterial culture medium components were obtained from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). Protein analysis and purification reagents were supplied by Bio-Rad Laboratories (Hercules, CA, USA), GE Healthcare Bio-Sciences (Pittsburgh, PA, USA), and Qiagen. Endonucleases and other enzymes were available from New England Biolabs (Ipswich, MA, USA). Unless otherwise mentioned, additional reagents were provided by Sigma-Aldrich (St. Louis, MO, USA). All materials were biochemical or biotechnological research grade.

Bacterial strain and plasmids

The Escherichia coli strains and plasmids used in this study are listed in Table 2. Bacterial cultures were grown in LB medium (10 g/L tryptone; 5 g/L yeast extract; 10 g/L NaCl) supplemented with ampicillin (0.15 mg/mL). Xl1-Blue MRF’ was used as the host for molecular cloning, while SHuffle Express for protein expression.
Table 2

E. coli strains and plasmids

Strains or Plasmids

Relevant Genotype or Features

Source

Strains

XL1-Blue MRF′

Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacI q ZΔM15 Tn10 (Tet R )]

Stratagene a

SHuffle Express

fhuA2 [lon] ompT ahpC gal λatt::pNEB3-r1-cDsbC (Spec R , lacI q ) ΔtrxB sulA11 R(mcr-73::miniTn10–Tet S )2 [dcm] R(zgb-210::Tn10 –Tet S ) endA1 Δgor ∆(mcrC-mrr)114::IS10

New England Biolabs

Plasmids

pQE30

Lactose regulation, ColE1 origin, AmpR

Qiagen

pQEhCP1

pQE30-based, recombinant EhCP1

This study

aAgilent Technologies (Santa Clara, CA, USA)

Construction of pQEhCP1

The plasmid expressing recombinant EhCP1 was obtained by subcloning the gene fragment encoding the pro-mature polypeptide (UniProt Q01957, Ile14 to Leu315) into the commercially available vector pQE30. Molecular cloning was performed according to standard protocols [38]. The gene fragment was amplified by PCR from genomic DNA of E. histolytica (HM1:IMSS strain) using the synthetic primers PROEHCP1F (5′-gcg gat cca ttg att tca ata cat ggg ttg cca ata ac-3′) and PROEHCP1R (5′-cga agc ttt cag aga tat tca aca cca gtt gga taa ag-3′), which were designed to include the BamHI and HindIII restriction sites at the 5′ and 3′ ends, respectively. After endonucleolytic digestion, the PCR product was inserted into pQE30. The recombinant plasmid (pQEhCP1) was extracted from transformed XL1-MRF’ cells and characterized by an astringent endonucleolytic analysis. The authenticity of the insert was confirmed by DNA sequencing.

Expression of recombinant EhCP1

The recombinant EhCP1 protein was expressed in the cytosolic compartment of SHuffle Express cells harboring the pQEhCP1 plasmid. A fresh culture of transformed cells was sub-cultured (1:100) and grown at 37 °C for 2 h with shaking (300 rpm). Gene expression was induced by addition of IPTG to a final concentration of 0.5 mM. Expression was allowed by further growing at 30 °C for 18 h with shaking. Bacterial cells were harvested by centrifugation at 9300 x g, 10 °C for 10 min. Five cell pellets, each from a 200-mL batch, were obtained and preserved at − 20 °C.

Bacterial protein extracts were prepared by cell lysis under native conditions. Each pellet was suspended in 5 mL of TT-L buffer (1% Triton X-100; 100 mM Tris-HCl, pH 8.0; supplemented with 0.2 mg/mL lysozyme) and thoroughly mixed by rocking for 5 min. Cell lysis was achieved by sonication, using a typical procedure (10 cycles: 30 s ON, 30 s OFF; on ice bath), followed by rocking for 10 min. The cellular debris was removed by centrifugation at 9300 x g for 15 min at 10 °C. The supernatant (soluble fraction) was further cleared by centrifugation at 16000 x g, 10 °C for 15 min. Protein concentration was determined by conducting a Bradford assay [39], using BSA as the standard. Protease activity was estimated by conducting a chromogenic assay [40], using Z-Arg-Arg-pNA as the substrate.

Purification of recombinant EhCP1

Under native conditions, the recombinant EhCP1 protein was purified from the cleared soluble fractions (CSF) of bacterial extracts by two consecutive standard chromatographic protocols: nickel-affinity and gel permeation. Initially diluted with one volume of BW buffer (300 mM NaCl; 100 mM Tris-HCl, pH 8.0; 20 mM imidazole, pH 8.0), CSF was then gradually loaded to a column containing nickel-nitrilotriacetic (Ni-NTA) agarose. After extensive washing, the bound protein was eluted with E buffer (300 mM NaCl; 100 mM Tris-HCl, pH 8.0; 250 mM imidazole, pH 8.0). Fractions containing a significant concentration of pure protein (> 95%) were pooled and slowly loaded to a PD-10 column (i.e., Sephadex® G-25). Recombinant protein was eluted using 20 mM Tris–HCl buffer (pH 8.0). The complete purification process was monitored by SDS-PAGE [41] and gelatin zymography [42] analyses. The concentration and protease activity of recombinant EhCP1 were measured as before.

Protease assays using synthetic substrates

Enzymatic hydrolysis of Z-Arg-Arg-pNA (chromogenic) or Z-Arg-Arg-AMC (fluorogenic) was used to assess the protease activity of recombinant EhCP1. The released product, p-nitroaniline (pNA) or 7-amino-4-methylcoumarine (AMC), was properly measured as a function of time to measure the proteolytic activity. Unless otherwise mentioned, standard assays (0.1 mL) were as follows:

(i) Chromogenic. Conducted at 37 °C, the enzyme (5 μg; i.e., 0.05 mg/mL) was initially activated for 10 min in citrate-phosphate buffer (50 mM; pH 6.5) containing 5 mM dithiothreitol (DTT). The reaction was started by adding the substrate (100 μM) to the assay mix. The linear increment of absorbance at 415 nm (A415) was determined from a 10-min kinetic analysis. Specific activity (SA) was expressed as units/mg (one unit is equal to 0.001 of A415 per min).

(ii) Fluorogenic. Performed at 22 ± 1 °C, the enzyme (0.5 μg; i.e., 0.005 mg/mL) was initially activated for 10 min in citrate-phosphate buffer (50 mM; pH 6.5) containing 5 mM DTT. The reaction was started by adding the substrate (5 μM) to the assay mix. The linear increment of fluorescence was determined from a 5-min kinetic analysis. Fluorescence was monitored at excitation and emission wavelengths of 355 and 460 nm. SA was expressed as FU/min/mg.

Inhibition assay

The effect of E-64 on the protease activity of recombinant EhCP1 was determined using different concentrations of the inhibitor (0 to 6 μM). The enzyme was initially incubated with the inhibitor for 15 min in reaction buffer without DTT. Then, this activator was added to the enzyme/inhibitor solution and after 10 min of additional incubation, the residual protease activity was measured as above, with a minor modification: 200 μM Z-Arg-Arg-pNA or 10 μM Z-Arg-Arg-AMC were used as the substrate.

Enzyme and inhibition parameters

Km values were determined by linear least squares regression fitting of reciprocal values of the initial velocity against the substrate concentration according to the Lineweaver-Burk equation. IC50 values were established by non-linear least squares regression of the protease activity against the logarithm of the inhibitor concentration according to a dose-dependent variable-slope fitting curve. Both parameters were calculated using GraphPad Prism v. 4.0 for Windows (GraphPad Software, San Diego, CA, https://www.graphpad.com).

Abbreviations

AMC: 

7-Amino-4-Methylcoumarine

CP: 

Cysteine Protease(s)

E-64: 

L-Trans-3-Carboxyoxiran-2-Carbonyl-L-Leucylagmatine

Eh

Entamoeba histolytica

EhCP1: 

Cysteine Protease 1 of Entamoeba histolytica

pNA: 

p-Nitroaniline

Z-Arg-Arg-AMC: 

Benzyloxycarbonyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin

Z-Arg-Arg-pNA: 

Benzyloxycarbonyl-L-arginyl-L-arginine-p-nitroanilide

Declarations

Acknowledgments

Authors thank Luciano Aldaba, Helios Gallego and Gabriela Monroy for the excellent technical assistance during the initial stages of the study.

Funding

This work was supported in part by grants from the Mexican Council for Science and Technology (CONACyT; CB-2010/01–155714 and SSA/IMSS/ISSSTE-2011/01–161544) and the Autonomous University of Baja California (UABC; CPI/300/2/N/112/1). The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the paper, and in the decision to publish the results.

Availability of data and materials

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Authors’ contributions

EJ-K performed experiments and analyzed data; REM designed experiments, analyzed data and contributed materials; PLAM and SGM-L analyzed data and contributed materials; AIR analyzed data and contributed analysis tools; MAR conceived and supervised the study, contributed reagents/materials/analysis tools and wrote the paper. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not Applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Calzada Universidad 14418, Parque Industrial Internacional, 22390 Tijuana, BCN, México
(2)
Centro de Graduados e Investigación en Química, Instituto Tecnológico de Tijuana, Boulevard Industrial S/N, Mesa de Otay, 22510 Tijuana, BCN, México

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Copyright

© The Author(s). 2018

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