World J Microbiol Biotechnol DOI 10.1007/s11274-015-1804-7

REVIEW

Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli Chunfang Ling • Junyan Zhang • Deqiu Lin Ailin Tao

•

Received: 25 January 2014 / Accepted: 11 January 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Papain-like cysteine proteases are widely expressed, fulfill specific functions in extracellular matrix turnover, antigen presentation and processing events, and may represent viable drug targets for major diseases. In depth and rigorous studies of the potential for these proteins to be targets for drug development require sufficient amounts of protease protein that can be used for both experimental and therapeutic purposes. Escherichia coli was widely used to express papain-like cysteine proteases, but most of those proteases are produced in insoluble inclusion bodies that need solubilizing, refolding, purifying and activating. Refolding is the most critical step in the process of generating active cysteine proteases and the current approaches to refolding include dialysis, dilution and chromatography. Purification is mainly achieved by various column chromatography. Finally, the attained refolded proteases are examined regarding their protease structures and activities. Keywords Papain-like cysteine proteases  Escherichia coli  Refolding  Purification  Activation C. Ling  D. Lin School of Life Science, South China Normal University, 55# Zhongshan Road West, Tianhe District, Guangzhou 510631, People’s Republic of China C. Ling  J. Zhang  D. Lin (&)  A. Tao (&) Guangdong Provincial Key Laboratory of Allergy & Clinical Immunology, The State Key Laboratory of Respiratory Disease, The Second Affiliated Hospital of Guangzhou Medical University, 250# Changgang Road East, Guangzhou 510260, Guangdong Province, People’s Republic of China e-mail: [email protected] A. Tao e-mail: [email protected]

Introduction One of the important protease families found in the prokaryotic, plant, and animal kingdoms is the cysteine protease family, which is involved in diverse aspects of the physiology and development of organisms (Dolinar et al. 1995; Dutta et al. 2010; Joo et al. 2007). Papain-like cysteine proteases, the most numerous subfamily of the cysteine protease class, have been identified as responsible for the key proteolytic activities in invasive, immune system related and degenerative disorders (Lecaille et al. 2002). For instance, cathepsin S regulates MHC class II dependent antigen presentation. Specific inhibition of cathepsin S can attenuate antibody response and, therefore, cathepsin S could be considered as a novel drug target for asthma and certain auto-immune diseases (Riese et al. 1998). Cathepsins are also involved in a variety of disease processes such as glomerulonephritis, arthritis and cancer metastasis (Smith and Gottesman 1989). Group 1 (DerF1 and DerP1) allergens are a contributing factor in atopic disease (perennial rhinitis, asthma, and atopic dermatitis) worldwide (Best et al. 2000; Yasuhara et al. 2001). In addition, parasite derived papain-like cysteine proteases are critical to the life cycle or pathogenicity of many parasites (Sajid and McKerrow 2002). Sufficient amounts of bioactive papain-like cysteine proteases would be useful for analysis of the relationship between enzymatic activity and pathogenesis as well as for understanding the development of therapeutic inhibitors. Expression of cysteine proteases at high expression rates has been accomplished using recombinant DNA technology. Among the heterologous recombinant DNA expressions systems, the E. coli expression system is the most convenient and frequently used, and plays a key role in producing useable amounts of genetically engineered

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proteins. However, expression of heterologous proteins in E. coli often leads to the formation of insoluble aggregates, termed ‘‘inclusion bodies’’ (IBs). Due to their refractile character, inclusion bodies are easily distinguished from other cell components (Jungbauer and Kaar 2007). However, it is challenging to convert these inactive and insoluble protein aggregates into soluble and correctly folded biologically active products, and then also remove the propeptide successfully. Many methods for recovering the bioactivities of recombinant proteins have been developed in recent years. In this review special emphasis is placed on the expression, refolding, purification and activation of cysteine protease (Fig. 1).

Expression of recombinant papain-like cysteine protease in E. coli A variety of factors have been identified that significantly influence protein expression in E. coli among which codon usage, mRNA stability and RNA secondary structures have been the major concerns. It was found that exposure of the AUG initiation codon from the long-range intra-strand secondary structure at 50 -end of mRNA, significantly improved the translation of human interleukin 10 (huIL-10) and interferon a (huIFN-a) proteins and reached 10-fold greater expression than the wild-type gene, suggesting that this may be a strategy for the expression of human proteins in E. coli (Zhang et al. 2006). Biased codon usage can diminish heterologous protein production in E. coli. For example, the arginine codons AGG and AGA are particularly rare in E. coli. These rare codons have been shown to lower levels of protein expression and cause translational errors (Calderone et al. 1996). However, codon optimization has been shown to improve the expression levels of human cathepsin K genes and human malaria parasite Plasmodium falciparum Falcipain-2 in E. coli (Roy et al. 2012; Sarduy et al. 2012).

Cell Culture and Harvest Cell Disruption & Solubility Identification Inclusion Body Isolation & Solubilization Purification of Target Protein Target Protein Refolding & Testing Enzyme Activity Assay Experimental & Therapeutic Study

Fig. 1 Flow diagram of the production of recombinant papain-like cysteine proteases from inclusion bodies

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1a

Denatured and Reduced

Refolded

3

2

Misfolded

1b

Intermediate

3

Aggregated

Fig. 2 Simplified model of refolding reaction competes with misfolding and aggregation reactions during protein renaturation. 1 correct refolding pathway; 2 misfolding pathway; 3 aggregation pathway

In addition, cysteine protease sequences can affect protein expression and protein refolding from inclusion bodies. The cDNA sequence for papain-like cysteine proteases consist of three regions encoding pre (a signal sequence), pro (activation domain) and mature (contains a Cys–His– Asn catalytic triad) peptide (Rawlings and Salvesen 2013). The length of bioactive papain-like cysteine proteases are usually between 220 and 260 amino acids (Lecaille et al. 2002). Signal peptides are on average between 10 and 20 amino acids in length and are responsible for the translocation into the endoplasmic reticulum during ribosomal protein expression (Lecaille et al. 2002). However, the presequence often showed a cytotoxic effect resulting in suppressed cell growth (Chan and Fong 1988) and impaired protein synthesis (Vernet et al. 1989) when the pre-pro-form of protein was expressed in E. coli. Removal of the presequence of the cysteine protease is necessary when it is expressed in E. coli. However, the pro-peptide regulates the enzymatic activity of the recombinant protein and may function as an intramolecular chaperone in the refolding process (Dolinar et al. 1995; Takahashi et al. 2000; Yamamoto et al. 1999). Thus, recombinant proteins obtained from inclusion bodies of the house dust mite allergen DerF1 directly expressed in mature form in E. coli without the prosequence can not be refolded properly and show very low IgE binding ability (Takaomi et al. 2001). The N-terminal of recombinant cysteine proteases have usually been fused to purification tags such as hexahistidine (Chen and Sun 2012; Sijwali et al. 2001a, 2002) or glutathione-S transferase (GST; Kim et al. 2010; Lee et al. 2012). Expression vectors pET and pGEX are most commonly used to clone pro-enzymes cDNAs sequences. The E. coli strain BL21(DE3) are commonly used as expression hosts and are grown in Luria–Bertani medium contains 50–100 lg/ml ampicillin at 37 °C until OD600 0.6–0.8. The T7 RNA polymerase inducible system is widely used for the expression of papain-like cysteine proteases, such as cathepsin O (Velasco et al. 1994), cathepsin B (Chen and Sun 2012; Kuhelj et al. 1995), cathepsin L (Dolinar et al. 1995; Lee et al. 2012), cathepsin S (Kim et al. 2010; Kopitar et al. 1996; Tobbell et al. 2002) and cathepsin F (Santamaria et al. 1999). Protein expression is induced by addition of isopropyl-b-D-thiogalactoside (IPTG) at a final concentration variable from 0.1 to 2 mM for 3–5 h.

World J Microbiol Biotechnol Table 1 Papain-like cysteine proteases expressed in E. coli Protease

Plasmid

Solubility

Refolding

Purification

Yield (mg/ l)

References

Cathepsin V

pET-32/28

Soluble

–

Ni–NTA superflow column

0.5

Novinec et al. (2012)

Cathepsin L

pGEX-4T-1

Soluble

–

Glutathione-Sepharose 4B column

–

Lee et al. (2012)

Cathepsin L

pMD56.1

Inclusion body

Dialysis

Sephacryl S-200 column

0.145

Dolinar et al. (1995)

Cathepsin S

pET3a

Inclusion body

Two-step dilution

Sephacryl S-200 column

0.2

Kopitar et al. (1996)

Cathepsin S

PT73.3

Inclusion body

Dilution

Thiopropyl-Sepharose column

–

Tobbell et al. (2002)

Cathepsin S

pGEX-4T-1

Soluble

–

Glutathione-Sepharose 4B column Ni–NTA column

–

Kim et al. (2010)

Falcipain-2

pRSET-B

Inclusion body

Dilution

–

Sijwali et al. (2002)

Falcipain-2

pBAD24

Inclusion body

Dilution

Ni2?-chelating Sepharose FF

50

Sarduy et al. (2012)

Falcipain-3

pQE-30

Inclusion body

Dilution

Ni–NTA column

–

Sijwali et al. (2001b)

Cathepsin O

pETH7

Inclusion body

Dilution

Electrophoresis

–

Velasco et al. (1994)

Cathepsin F

pGEX-3X

Soluble

–

Glutathione-Sepharose 4B column

–

Santamaria et al. (1999)

Cathepsin K

pQE-30

Inclusion body

Dilution

Ni–NTA agarose column

35

Hwang and Chung (2002)

Cathepsin B

pET3a

Inclusion body

Dialysis

Fast flow Sepharose S column

3

Kuhelj et al. (1995)

Cathepsin B

pET32a

Soluble

–

Ni–NTA column

–

L. Chen and Sun (2012)

Der f 1

pGEMEX-1

Inclusion body

Two-step gel filtration chromatography

–

70

Takahashi et al. (2000)

Der p 1 Cathepsin F-like

pKN172 pT7-7

Inclusion body Inclusion body

Dialysis Dialysis

Superdex SX200 column Immunoaffinity resin

– –

Asturias et al. (2009) Joo et al. (2007)

Cathepsin F-like

pET-22b

Inclusion body

Dilution

SP-Sepharose FF column

0.3

Miyaji et al. (2010)

Vinckepain-2

pQE-30

Inclusion body

Dilution

Ni–NTA column

–

Singh et al. (2002)

Berghepain 2

pRAET-A

Inclusion body

Dilution

Ni–NTA column

–

Singh et al. (2007)

Papain

pET30 EK/ LIC

Inclusion body

Dilution

Ni–NTA column and Sephacryl S-200 column

400

Choudhury et al. (2009)

Vivapain-2

pQE30

Inclusion body

Dilution

Ni–NTA column and Q-Sepharose column

–

Na et al. (2004)

Vivapain-3

pQE30

Inclusion body

Dilution

Ni–NTA column and Q-Sepharose column

–

Na et al. (2004)

Chabaupain-1

pQE30-RK6

Soluble

–

Ni–NTA column and Sephadex G-25 column

–

Caldeira et al. (2009)

Chabaupain-2

pQE30-RK6

Inclusion body

Dilution

Ni–NTA column

–

Caldeira et al. (2009)

However, most of the papain-like cysteine proteases are expressed in inclusion bodies. Changing cell culture conditions such as lower temperature (Schein and Noteborn 1988) or suboptimal pH (Kopetzki et al. 1989) can result in decelerated cell growth and result in the production of soluble recombinant proteins. Therefore, when papain-like cysteine protease chabaupain-1 of Plasmodium chabaudi was expressed at 17 °C, protein was produced in a soluble form (Caldeira et al. 2009).

Solubilization of inclusion bodies A standard protocol for the solubilization of inclusion bodies starts with cells that are harvested by centrifugation, resuspended in lysis buffer, and then mechanically broken by French Press or sonication. Insoluble and soluble fractions are separated by centrifugation. The inclusion body fraction is then washed several times in alkaline buffer (e.g. 2 % Triton x-100, 1–10 mM EDTA, 50–100 mM Tris–

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World J Microbiol Biotechnol Table 2 Classification of small molecule additives Classification

Type of additive

Effect on protein–protein interaction

Effect on proteinstability

References

Denaturant

Urea

Reduce

Reduce

Tsumoto et al. (2003)

Folding enhancer

Trehalose

Enhance

Enhance

Chen et al. (2003); Coutard et al. (2012)

Reduce

Neutral

Coutard et al. (2012); Sijwali et al. (2001a)

Neutral

Enhance

Clark et al. (1999); Raman et al. (1996)

Guanidine HCl Glucose Sucrose Glycerol Glycine Proline PEG PNIPAAma Aggregation suppressor

L-Arginine

Tween 20 NDSBb201 NDSB 256 Triton X-100 Polyethylene glycol KCl Oxido-shuffling reagents

GSH/GSSGc Cysteine/cystine Cysteamine/cystamine DTT/ODTTd

a

Poly N-isopropylacrylamide

b

Non detergent sulfobetaine

c

Reduced and oxidized glutathione

d

Reduced and oxidized dithiothreitol

HCl, pH 8.0) and solubilized in alkaline buffer containing high concentrations of chaotropic agents such as urea (8 M) or guanidinium hydrochloride (6–7 M). Inclusion body proteins often contain disulfide bonds that reduce their solubility, therefore, low molecular weight thiol reagents such as dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol or sodium thiosulfate are added to allow for the reduction of disulfide bonds by thiol disulfide exchange. The solution is then stirred at room temperature for 1–3 h to solubilize proteins, clarified by centrifugation at 10,000g for 30 min, and the supernatant subsequently filtered using a 0.45 lm PVDF Millipore membrane (Alessio et al. 1999; Hwang and Chung 2002).

Purification of recombinant proteases Purification of recombinant proteases is best performed using various column chromatography steps, but this must be optimized for each individual protease. Chromatography

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is used for the removal of denaturants, most host proteins and the separation of folding intermediates. Proteins have been purified based on (1) their surface properties, i.e., ion exchange chromatography (IEC); (2) their net charge, i.e., size exclusion chromatography (SEC); (3) their high-affinity ligands, i.e., immobilized metal affinity chromatography (IMAC; Bro¨mme 2001). IEC has been widely used to purification recombinant papain-like cysteine protease (Table 1), such as cathepsin B (Kuhelj et al. 1995), Falcipain-2 (Sijwali et al. 2002), vinckepain-2 (Singh et al. 2002), cathepsin F-like cysteine protease domain (Miyaji et al. 2010) and cruzipain (Eakin et al. 1992). Using IEC column as a refolding reactor, purification and refolding can be achieved simultaneously. In addition, dual gradient IEC has been established to maximize the refolding yields (Li et al. 2002). IMAC has opened up a new prospect for efficient purification and refolding of proteases equipped with engineered polyhistidine tags in one step. The polyhistidine tags with immobilized metal ions can form high-affinity

World J Microbiol Biotechnol

complexes even in the presence of high concentrations of denaturants (Li et al. 2004). Using a silica-based matrix instead of agarose-based material for IMAC to refold hydrophobic proteins contained multiple disulfide bonds can significantly improve proteins refolding yields (Sharapova et al. 2011). One of the limitations of IMAC is that the intended protein use can be affected by metal ion carryover or by the fact that the target protein carries a tag that may require further tag removal and cleanup of the final product.

Refolding of recombinant proteases Refolding of purified proteins obtained from inclusion bodies is still a challenging and limiting process affected by several factors including the denaturant, small molecule additives, refolding buffer pH and protein concentration. Protein refolding is initiated by the removal of the denaturant allowing intramolecular interaction and the formation of a folded structure. The process of protein refolding is not a single reaction and is complicated by other non-desirable reactions, such as mis-folding and aggregation that result in the formation of inactive proteins. Intermediate protein structures, which are unstable and less soluble, can also readily misfold and form aggregates during the refolding process. It is therefore important to facilitate folding of the intermediate protein structures into more stable native structures by optimizing the refolding buffer and selecting proper refolding strategies to obtain maximum yields of native protein. Screening tests were performed in a systematic micro-dilution format. In briefly, the denatured protein was often diluted with refolding buffer in 100-fold and evaluated proteins refolding efficiencies by assaying refolding reaction for hydrolysis of synthetic substrate. The screening tests have been successfully to evaluating the efficiency of refolding buffers of Falcipain-2 (Sijwali et al. 2001a, 2002), Falcipain-3 (Sijwali et al. 2001b) and cathepsin S (Tobbell et al. 2002). ‘‘Reverse screening’’ using partial unfolding native proteins and analyzing the recovery of them by reverse-phase high performance chromatography, it may become a new approach to assess the efficiency of refolding additives (Ejima et al. 2006). Both nonspecific (hydrophobic) interactions of predominantly unfolded polypeptide chains and incorrect interactions of partially formed folding intermediates can result in unproductive aggregation (Fig. 2). Consequently, the formation of aggregates predominates at high concentrations of denatured protein. Since aggregation is driven by protein concentration, once optimal refolding conditions are applied, the yield of correctly refolded protein could be solely determined by protein concentration.

Protein refolding by dialysis Dialysis to remove high concentrations of denaturant can be accomplished by buffer exchange using ultrafiltration membranes (Clark 2001). Using step-wise dialysis, the inactive protein passes through progressively decreasing concentrations of denaturant (Vallejo and Rinas 2004). However, if the folding or stability of each domain is different in multi-domain proteins, equilibration at higher concentrations of denaturant may result in the folding of the most stable domains (Tsumoto et al. 2003). Therefore, the hybrid protein (DerP1 and DerP2) contained multidomain could refold well by step-wise dialysis (Asturias et al. 2009). In one step dialysis, the inclusion body proteins will fold into the intermediate and native structures as the concentration of denaturant is decreased. For example, when denatured cathepsin L-like cysteine protease dialyzed against refolding buffer contained 1 M urea, it can re-form and convert to the native structure successfully (Ahn et al. 2007). In addition, using highly hydrophilic materials such as cellulose acetate, which are more compatible with unfolded protein molecules, can minimize protein binding to the membranes and significantly improve renaturation yields (West et al. 1998).

Protein refolding by dilution The most commonly used method in small-scale refolding studies is dilution of the solubilized protein directly into renaturation buffer. Either rapid or slow dilution is the two common methods. Coutard et al. (2012) suggested that after taking into account the bimodal distribution of proteins with various isoelectric points (pI), that an acidic refolding buffer should be used for alkaline proteins and an alkaline buffer used for acidic proteins. Most papain-like cysteine proteases are acidic proteins, and should refold in alkaline refolding buffer. For many proteins, refolding requires excessive reaction volumes since renaturation yields decrease above a limiting concentration of about 50 lg/ml (Rudolph and Lilie 1996). Small molecules additives are usually added to the refolding solvent to facilitate protein refolding in vitro. According to Tsumoto et al. (2003), the additives can be classified and are summarized here in Table 2. A folding enhancer improves protein–protein interactions in principle, while an aggregation suppressor reduces side chain interactions. In order to improving protein refolding efficiency, the types and quantities of additives should be optimized for any given protein. Folding enhancer as glycerol (30 % w/v) or sucrose (20 % w/v) were often used to improve refolding efficiency of papain-like cysteine proteases (Na et al. 2004; Sarduy et al. 2012; Sijwali et al.

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2001a). PEG 3350 and PEG 8000 were found to be more effective than PEG 1000 in promoting protein refolding (Cleland et al. 1992). L-arginine added to the refolding buffer at concentrations in the range of 0.4–0.8 M (Clark et al. 1999) decreased aggregate formation during the refolding process. The formation of disulfide bonds in proteins requires a sufficiently oxidizing environment (Raines 1997). The common methods used to promote the formation of disulfide bonds during refolding are (1) air oxidation when in the presence of trace amounts of metal ions, (2) the use of mixed disulfides, and (3) the oxido-shuffling system (Clark et al. 1999). The oxido-shuffling system utilizes low molecular weight thiols in reduced and oxidized form to promote the formation of disulfide bonds and has been the most widely applied oxidation method. Among those thiols, reduced glutathione and oxidized glutathione are the most commonly used pair to promote papain-like cysteine proteases to form disulfide bonds. For most proteins which containing several disulfide bonds (e.g. RNase A), maximum disulfide bond formation is observed in the presence of 1–3 mM low molecular weight reduced thiol and at approximately one-fifth to one-tenth of the concentration of the respective oxidized form. The yields and rates of oxidative refolding are strongly dependent on the low mol. wt [–S–S–]/[–SH]. (Rudolph and Lilie 1996; Wetlaufer et al. 1987).

step. However, chromatographic speed and small sample volume are the two limiting factors that restrict the applicability of SEC in large scale processes (Jungbauer and Kaar 2007).

Secondary structures of refolded proteins Circular dichroism (CD) determines the secondary structure (b-sheet and/or a-helix) of refolded proteins by measuring its spectrum (Vincentelli et al. 2004). However, this process is limited when the amount of protein available is not sufficient or when NDSB is present in the refolding buffer. Dynamic light scattering can be used to assess the conditions of protein aggregation after the refolding process (Vincentelli et al. 2004). A valid folding criterion can be determined by X-ray crystallogenesis (protein folding and dispersion homogeneity) because only correctly folded proteins with an even aggregation state yield well-ordered crystals (Vincentelli et al. 2004). The protein structure can also be analyzed by NMR spectroscopy (Werner et al. 1994). In addition, reversed phase chromatography monitors the folding status and effectively separates intermediate states, including correctly as well as incorrectly formed disulfide bonds, during the refolding process (Wu et al. 1998).

Solvent-exchange by size exclusion chromatography

Cysteine proteases activation and enzyme activity examination

Size exclusion chromatography (SEC) is an effective way to refold denatured proteins in vitro. The SEC column separates the unfolded protein from the denaturant due to their large difference in size. The two key factors affecting the successful SEC refolding process are the loading of the protein onto the column in the presence of a denaturant solution and the change in protein size that occurs as the protein renatures during elution with the refolding buffer (Li et al. 2004). The DerF1 protein was successfully renatured by a two-step gel filtration column and resulted in a yield of 70 mg of pro-DerF1 from 1 l of culture (Takahashi et al. 2000). Choosing a solvent plug of 8 M urea as a ‘‘chaperon’’ to escort the denatured protein into the SEC, reduced the ability of the denatured protein to form aggregates when encountering a refolding buffer without denaturant (Liu and Chang 2003). Higher protein application concentrations and sample volumes and lower flow rates can decrease refolding yields. Linear decrease in the urea concentration during SEC was very effective in enhancing refolding yields at relatively high initial protein concentration (Gu et al. 2001). SEC may potentially provide not only good refolding but also purification in one

The pro-region can be removed auto-catalytically by incubating the precursor proteins in pH 4.0–6.0 acetate buffer (Table 3). Autocatalytic activation can be facilitated in the presence of negatively charged glycosaminoglycans with dextran sulfate being most commonly used (Bromme et al. 2004). However, not all proteases, such as cathepsin C, can be activated auto-catalytically. Human recombinant cathepsin C can be activated by Cathepsins L and S but not by autocatalytic processing (Dahl et al. 2001). Pepsin is often added to the incubation buffer for hetero-catalysis. Cathepsin L and cathepsin S could be facilitated by pepsin for hetero-catalysis, the maximum proteolytic activity of them were not change, but slightly reduced the time to reach the maximum activity (Kramer et al. 2007). In addition, the mature cathepsin K can be used as seed to induce the activation of procathepsin K (McQueney et al. 1997). Autocatalytic as well as pepsin activation lead to a partial degradation of the recombinant enzyme, thus the optimal time point for a balance between minimal degradation and maximal activation must be determined. Autocatalytic processing or pepsin activation can be terminated by raising the pH to 5.5.

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World J Microbiol Biotechnol Table 3 Activation of papain-like cysteine proteases Enzyme

Activation buffer

Enzyme substrate

References

Cathepsin V

pH 4.0, 5 mM DTT

Za-Phe-Arg-AMCb (5 lM)

Novinec et al. (2012)

Cathepsin S

pH 4.5, 0.75 M disodium hydrogen phosphate/potassium dihydrogen phosphate, 2.5 mM DTT, 25 lg/ml dextran sulphate

Z-Phe-Val-Arg-AMC, Z-Phe-ArgAMC

Kopitar et al. (1996)

Cathepsin S

pH 4.5, 25 mM NaH2PO4, 0.5 M NaCl, 5 mM EDTA, 5 mM DTT

Z-Val-Val-Arg-AMC

Tobbell et al. (2002)

Cathepsin K

pH 4.0, 0.2 M sodium acetate, 5 mM DTT, 5 mM EDTA

Z-Phe-Arg-AMC (0.5 mM), GlyPro-Arg-pNA (0.5 mM)

Hwang and Chung (2002)

Cathepsin B

pH 3.5, porcine pepsin (1/100)

Z-Phe-Arg-AMC, Z-Arg-ArgAMC

Kuhelj et al. (1995)

Cathepsin C

pH 4.5, 20 mM citric acid, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, cathepsin L and S

Gly-Phe-pNA, Gly-Arg-pNA

Dahl et al. (2001)

Falcipain-2

pH 5.5, 100 mM sodium acetate, 10 mM DTT

Z-Leu-Arg-AMC, Z-Phe-ArgAMC

Sarduy et al. (2012); Sijwali et al. (2002)

Falcipain-3

pH 5.5, 100 mM sodium acetate, 5 mM DTT

Z-Phe-Arg-AMC (50 lM), Z-LeuArg-AMC (50 lM)

Sijwali et al. (2001b)

Der f 1

pH 4.0, 100 mM acetate buffer

N-suc-Leu-Val-Tyr-AMC, BocVal-Leu-Lys-AMC, Suc-AlaPro-Ala-AMC

Takahashi et al. (2000)

Vinckepain-2

pH 5.5, 5 mM DTT

Z-Leu-Arg-AMC

Singh et al. (2002)

Cathepsin F-like cysteine protease

pH 4.5, 20 mM sodium acetate, 1 mM EDTA, 5 mM DTT, porcine pepsin (1/10)

Tos-Gly-Pro-Lys-pNAc

Miyaji et al. (2010)

Cruzipain

pH 5.5, 0.1 M sodium acetate, 5 mM DTT

Z-Phe-Arg-AMC, benzoyl-ProPhe-Arg-pNA

Eakin et al. (1992)

Papain

pH 5.0, 0.1 M sodium acetate, 2 mM EDTA

Azocasein

Choudhury et al. (2009)

Vivapain-2

pH 5.5, 5 mM DTT

Z-Leu-Arg-AMC

Na et al. (2004)

Vivapain-3

pH 5.5, 5 mM DTT

Z-Leu-Arg-AMC

Na et al. (2004)

Berghepain-2

pH 5.5, 5 mM DTT

Z-Leu-Arg-AMC (50 lM), Z-PheArg-AMC (50 lM)

Singh et al. (2007)

a

Benzyloxycarbonyl

b

7-amido-4-methylcoumarin

c

p-Nitroanilide

Enzyme activity examination are often performed in an acetate or phosphate buffer, between pH 5 and 6, containing 1–2.5 mM EDTA and reducing agents (e.g., 1–2.5 mM DTT or 5–10 mM L-cysteine) in order to be fully active (Bromme et al. 2004). The buffer, DTT and EDTA concentrations vary between protocols, and their effect on the enzyme activities was in controversy. Some researchs deemed that these factors don’t have a significant effect on the enzyme activities (Bromme et al. 2004; Joo et al. 2007; Prasad et al. 2012). However, some other literatures showed different perspectives. DTT was found to enhance enzyme activities of knowpains (Prasad et al. 2012), Falcipain-2 (Sarduy et al. 2012), DerF1 and DerP1 (Takai et al. 2002) in a dose-dependent manner. Different pH buffers also showed

significant effects on cathepsin F-like cysteine protease (Joo et al. 2007), knowpains (Prasad et al. 2012), Tr-cp 14 (Asp et al. 2004) and Falcipain-2 (Sarduy et al. 2012) activities; the maximum activities often obtained in acidic pH. The concentration of cysteine protease used in enzyme activity assays is between 0.1 and 10 nM (Bromme et al. 2004). The cysteine proteinase activity assay is best monitored by synthetic fluorogenic substrates such as aminomethylcoumarin (AMC). The cathepsins with endopeptidase activity can be followed by their Z-Phe-Arg-AMC cleaving activity (e.g., cathepsins B, F, K, L, O, S and V; Bro¨mme 2001). Cathepsins with aminopeptidase activity are measured with other substrates such as Ala–Ala-AMC (cathepsin C) or Arg-AMC (cathepsin H; Bro¨mme 2001).

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Cysteine protease inhibitors provide a useful tool to study protease activity. Compounds synthesized included a wide range of peptide aldehydes, methyl nitriles and ketones as reversibly acting inhibitors and halomethyl ketones, diazomethanes, acyloxymethyl ketones, O-acylhydroxamates, and epoxysuccinyl derivatives as irreversible inhibitors (Lecaille et al. 2002). Peptide aldehydes possess low steric hindrance to nucleophilic addition and highly electron-deficient, as a result they tend to be reactive and undergo nucleophilic addition with thiols and hydration with water (Yamashita and Dodds 2000). Peptide methyl ketones possessing a range of substituents at the a position of methyl ketones (Marquis et al. 1999). Peptide aldehydes and peptide methyl ketones belong to transitionstrate analogues (Demuth 1990). Peptide halomethyl ketones, peptide diazomethyl ketones and epoxides are alkylators of the free thiol of the active site cysteine (Yamashita and Dodds 2000). Acyloxymethyl ketone interacted with the active site of the cysteine protease, and the ketone was attacked by the active site cysteine forming a thiohemiketal in an irreversible step (Yamashita and Dodds 2000). Natural eposysuccinyl peptide derivatives possess trans-dicarboxylic acid structural feature, and show strongly and irreversibly inhibitory activities against cathepsin B, cathepsin L and papain (Leung-Toung et al. 2002). E-64 is a selective but broad-specificity irreversible inhibitor for all mammalian cathepsins with the exception of cathepsin X (Bromme et al. 2004).

Conclusion This review provides an overview of the protocols for generating active papain-like cysteine proteases in E. coli expression systems, including protein expression, refolding, purification, activation and activity assays. E. coli based expression is clearly advantageous if large amounts of inactive proteins are needed and with a minimal investment in time and costs. However, how to effectively generate large amounts of bioactive protein from the aggregated inclusion bodies has still not been satisfactorily achieved. Refolding in vitro is the critical procedure required to produce active papain-like cysteine protease and recently, various procedures have become available for achieving inclusion body protein refolding in vitro. The proper method for any particular protein needs to be determined on a case-by-case basis to allow for correct folding of each recombinant protein with high yields. In the future, we will be benefit from fully understanding how proteins refold on columns and the role of additives in inhibiting protein aggregation. This will facilitate the choosing of proper ionic strength and ion type. Other desirable goals would be to increase the final protein

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concentration refolding is performed under as well as minimize the costs and amount of chemicals needed. Currently, improving the production of active papain-like cysteine proteases for industrial applications is still a challenge. Acknowledgments This work was supported by Great Project (2011ZX08011-005) from the Major Program of National Science and Technology of China and the National Natural Science Foundation of China (81373128).

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