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Genetic and metabolic engineering approaches for the production and delivery of L -asparaginases: An overview
Accepted Manuscript Genetic and metabolic engineering approaches for the production and delivery of L-asparaginases: an overview Jalaja Vidya, Syed Sajitha, Mrudula Vasudevan Ushasree, Raveendran Sindhu, Parameswaran Binod, Aravind Madhavan, Ashok Pandey PII: DOI: Reference:
S0960-8524(17)30711-3 http://dx.doi.org/10.1016/j.biortech.2017.05.057 BITE 18083
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
28 March 2017 2 May 2017 10 May 2017
Please cite this article as: Vidya, J., Sajitha, S., Ushasree, M.V., Sindhu, R., Binod, P., Madhavan, A., Pandey, A., Genetic and metabolic engineering approaches for the production and delivery of L-asparaginases: an overview, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.05.057
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Genetic and metabolic engineering approaches for the production and delivery of Lasparaginases: an overview Jalaja Vidya1*, Syed Sajitha1, Mrudula Vasudevan Ushasree1, Raveendran Sindhu1, Parameswaran Binod1, Aravind Madhavan1, 2 and Ashok Pandey1,3 1
Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram 695 019, Kerala, India 2
Rajiv Gandhi Centre for Biotechnology, Jagathy, Thiruvananthapuram – 695 014, India 3
Center of Innovative and Applied Bioprocessing, Sector 81, Mohali, Punjab, India
*Corresponding author. Tel 91-471-2515426 Fax 91-471-2491712 E-mail: [email protected] 1
Abstract L-asparaginase is one of the protein drugs for countering leukemia and lymphoma. A major challenge in the therapeutic potential of the enzyme is its immunogenicity, low-plasma half-life and glutaminase activity that are found to be the reasons for toxicities attributed to asparaginase therapy. For addressing these challenges, several research and developmental activities are going on throughout the world for an effective drug delivery for treatment of cancer. Hence there is an urgent need for the development of asparaginase with improved properties for efficient drug delivery. The strategies selected should be economically viable to ensure the availability of the drug at low cost. The current review addresses various strategies adopted for the production of asparaginase from different sources, approaches for increasing the therapeutic efficiency of the protein and new drug delivery systems for L-asparaginase. Key words: L-asparaginase II; recombinant production; protein stability; drug delivery
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1. Introduction Enzymes gained therapeutic importance about 40 years back, and they are using now as thrombolytic agents, digestive aids, debriding agents and even as oncolytic agents. In 1902, Dr. John Beard first discovered that enzymes could be used as a therapeutic agent against cancer. His discovery was one of the milestones in cancer therapy. Enzymes, if employed as therapeutic agents must be safe and effective in order to bring about the expected clinical advantage. The advent of protein engineering and recombinant technology can tackle all these problems to a certain extent, and these techniques marked the beginning of a new era in a therapeutic industry. The native properties of the enzymes can be altered for industrial needs in several ways, from the simple chemical modifications such as bio-conjugations to advanced engineering approaches based on protein structural information. The strategy adopted for the modification varies from protein to protein depending on the property, which is focused and includes alterations in thermal properties, half life, substrate affinity, immunogenicity, shelf life, etc. The climax of an engineered drug action depends on the way it is administered into the body and the extent to which it exists in the circulation. There comes the role of drug delivery systems for optimal action of the drug. Nowadays the therapeutic protein industry is focusing mainly on the drug design and novel delivery systems for the unsurpassed result in robust time. Protein therapeutic like enzymes which posses high specificity and potency, require targeted delivery at nanoscale levels for their optimal effectiveness. Hence within a decade, the medium for the drug delivery such as drug carriers and moieties for targeted delivery was rapidly evolved (Muzykantov, 2011). The present review addresses genetic and metabolic engineering strategies adopted for the improvement and delivery methods for anti-leukemic L- asparaginase. 3
2. Production of recombinant L-asparaginases Large scale production of bioactive compounds became a routine process worldwide, after the recognition of fermentation process. Of these bioactive compounds, proteins comprise a major class with wide applications in almost all the industries including food, feed, medicine, detergent, paper, fuel etc. The advent of recombinant DNA technology shed light into the production process and further modification of the proteins and peptides for industrial applications. The very first protein of pharmaceutical potential, produced was insulin and now more than 200 peptides and protein pharmaceutical are there in the FDA list (Demain and Vaishnav, 2009). By understanding the genetic makeup of the microbial systems, the recombinant DNA technology enabled the production of proteins and enzymes from diverse sources like plants and animals by microbial fermentation process for industrial purposes. L- asparaginase with comparable cytotoxicity was reported from Zymomonas mobilis (Einsfeldt et al., 2016). A synthetic mature gene with the N-terminal Histidine tag and an enterokinase cleavage site was cloned and expressed in E. coli. The codons were optimized to the codon usage of E. coli. The enzyme was expressed both extracellularly and intracellularly with a yield of 0.13IU/ml and 3.6 IU/ml respectively and the extracellular enzyme showed cytotoxic effect on leukemic cells. The enzyme from E. carotovora expressed in E. coli was produced by fed batch process in a laboratory scale fermentor and yielded 0.9g/l of soluble protein (Roth et al., 2013). Expression of recombinant L-asparaginase II from a thermotolerant E. coli isolated from camel manure showed anti-proliferative activity in leukemic cell lines (Muharram et al., 2014). A relatively thermostable enzyme from B. subtilis was cloned and expressed in B. subtilis 168 4
using the shuttle vector pMA5 and the purified enzyme showed low affinity towards L-glutamine (Jia et al., 2013). Secretion enables the downstream processing easier; hence most of the recombinant production is accompanied by cloning a secretory signal either the native signal or any other excellent signal sequence in frame with the asparaginase gene. Meena et al., 2015 reported ansA gene from marine actinobacteria Streptomyces griseus and a threefold increase in enzyme production was obtained after expression in E. coli M15. They also reported the optimized culture conditions for production of extracellular L-asparaginase by Box-Behnken design with a maximum yield of 56.78IU/ml. Efficient expression of an extracellular enzyme from E. coli in the culture medium was reported by Ghoshoon et al., 2015 . The full length gene with the signal sequence was cloned in E. coli and the L-asparaginase II mature protein was secreted in to the culture medium where the components were optimized by response surface methodology and a production of 17.386U/l was reported in optimized conditions. The role of L-asparaginase signal peptide in extracellular secretion of recombinant proteins was reported by Ismail et al., 2011 where the mutations on signal peptide were carried out for improving secretion of cyclodextrin glucanotransferase. Chityala et al., 2015 observed secreted expression of glutaminase free Lasparaginase of Pectobacterium carotovorum was achieved by cloning the gene in Bacillus subtilis. The ansB2 gene was cloned in pHT43 and transformed in Bacillus subtilis WB800N. Consecutive IPTG induction enhanced the enzyme production to 105 IU/ml with majority of the expressed protein localized to the extracellular medium. Feng et al., 2016 reported an improved enzyme production from Bacillus subtilis. Step by step increase in activity of L-asparaginase from Bacillus subtilis was achieved by combined 5
approaches using combinations of different signal sequences and promoters and by random mutagenesis. Here eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for evaluation of highest secretion of the protein to the extracellular medium using HpaII promoter and the signal wapA proved to be the best. The activity was further improved by replacing the HpaII promoter with strong p43 promoter. This was then subjected to two rounds of error prone PCR reactions for random mutagenesis and the variants with enhanced activity was selected for N terminal deletion of 25 amino acid residues thereby increasing the activity to 100% that of wild enzyme. The enzyme activity reached 407.6 U/ml (2.5 g/l of ASN protein in 3l bioreactor under fed –batch process. Extracellular secretion of the periplasmic enzyme E. coli L-asparaginase II was achieved by expressing the protein as fusion tag consisting of the pelB signal sequence and various lengths of repeated aspartate residues (Kim et al., 2015). The recombinant enzyme fused with the pelB signal sequence and five aspartate tag was secreted efficiently into the culture medium. The role of secretory machinery of the host cell in extracellular production was proved by deletion of gspDE gene. Another approach for secretion of the recombinant Erwinia enzyme to the culture medium was investigated by Karamitros and Labrou (2014) and the enzyme from the extracellular medium was purified by simple and fast method using affinity adsorbent. The cloned gene from Erwinia chrysanthemi was expressed in seven different E. coli host strains to access the secretory efficiency of the hosts. The E. coli BL21 (DE3) rosetta strain showed the highest expression among all other strains selected and the protein from the culture broth was purified in single step by L-asp adsorbent column and the bound protein was eluted using L-aspartate. Huang et al., 2011 reported an L-asparaginase having dual application in food and leukemic treatment from
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fungi Rhizomucor miehei. The cloned enzyme expressed from E. coli showed a very low glutaminase activity and was a homodimer of 133kDa. Recombinant production of the L-asparaginase from diverse sources is presented in Table 1. 3. Approaches for increased production with desired quality The microbial production rate of proteins and bioactive compound widely depend on the growth factors or nutritional requirements and the environmental conditions which varies from organism to organism including pH, temperature, aeration, fermentation time etc. Hence depending upon the nature of microbe selected for the production, one can optimize the process parameters and the media components for better yield of the desired product in the fermentor. Large scale production of the product for industrial purpose can be obtained by fermentation process. Not all native proteins carry ideal properties for its application in industrial processes. The functional properties of each protein need to be characterized after purification process and further improvement in the characteristics was achieved primarily through protein engineering and host cell engineering for improved expression of recombinant proteins. The native properties of the enzymes can be altered for industrial needs by several ways, from the simple chemical modifications such as bio-conjugations to advanced engineering approaches based on protein structural information. The strategy adopted for the modification varies from protein to protein depending on the property, which is focused. A schematic view of the approaches for improving the stability of therapeutic protein was included in Fig. 1. The structural stability of the protein can be achieved by alteration of the protein primary structure through selected protein engineering tools like rational designs, random or site directed 7
mutagenesis, chimeric protein expression or as fusion proteins. By the mutagenesis, one can identify the favorable and unfavorable amino acid residues at particular position in a protein and this step-by-step replacement of residues results in gradual increase in the desired functional properties of protein.
3.1 Rational design Sudhir et al., 2016 developed glutaminase free L-asparaginase from Bacillus licheniformis through mutations in amino acid sequences for improving the half life and thermal properties. Four amino acid residues for mutagenesis were selected based on the sequence alignment with previously mutated asparaginases, hydrophobicity and electrostatic potential. The mutant D103V showed three fold better half life and thermal tolerance compared to the wild type protein. The mutant also exhibited increased substrate affinity with Km value of 0.42 mM and Vmax of 2778.9 mol min−1. Patel et al., 2009 developed an E. coli enzyme resistant to the lysosomal proteases by employing the tools like genetic algorithm and molecular dynamics. The cleavage start site of lysosomal protease asparagine endopeptidases (AEP) was identified as N24 and the mutant protein N24G showed the proteolytic resistance with a decreased enzyme activity. Later replacement of alanine at same position (N24A) resulted in an enzyme with higher activity due to unique hydrogen bonding network. For reducing the glutaminase activity, an additional mutation in the interface position 195 constructed a double mutant N24A R195S which is almost ideal for clinical purpose (Offman et al., 2016). Sannikova et al., 2016 modified recombinant enzyme from Wolinella succinogenes having an N terminal peptide which binds to heparin and amino acid substitutions V23Q and K24T to resist 8
trypsinolysis showed high therapeutic efficiency compared to the reference enzyme. Bansal et al., 2012 developed an L-asparaginase enzyme devoid of glutaminase activity by altering the substrate specificity by mutating active site amino acid residues in Pyrococcus furiosus. The mutant K274E showed improved enzymatic properties without any glutaminase activity. An identical result was observed by Verma et al. 2014 for E. coli where the amino acid substitutions in the residues in the subunit interfaces of the enzyme improved activity and stability of the engineered enzyme. Mehta et al., 2014 observed that the mutated variant of L-asparaginase II of E. coli W66Y and Y176F was more effective than the wild type enzyme against the ALL cells and showed a decreased glutaminase activity. The double mutant in the immune-dominant epitopes, K288S/Y176F exhibited reduced antigenicity and tenfold less immunogenicity compared to the wild type enzyme. Mutagenesis of residues involved in the catalytic triad and the substrate binding sites in L-asparaginase of Helicobactor pylori revealed the role of selected residues in glutaminase activity and cytotoxicity of the drug (Maggi et al., 2015). They showed that the double mutant M121C/T169M was unable to hydrolyze glutamine but have no cytotoxic effect. The mutant Q63E exhibited glutaminase activity with comparable cytotoxic effect as that of the wild type enzyme. Gervais and Foote, 2014 observed that deamidation of the Erwinia chrysanthemi enzyme ErA by mutating the residues N41D and N281D resulted in slightly improved specific activity in single and double mutants. N281D mutant showed low glutaminase activity but the thermal stability at elevated temperatures was reduced. An approach to improve the resistance towards the proteolysis, selected surface residues of E. coli enzyme were mutated to cysteine there by allowing the covalent linkage of the subunits to different lengths of maleimide-functionalized the polymers. The mutants A38C-T263C were expressed and were secreted as active protein (Ramirez Paz et al.,) 2016. In silico mutagenesis at 9
the substrate binding site of L-asparaginase II for reducing the affinity towards L-glutamine was done by replacing the amino acid Asp96 with alanine. The replacement showed a 40 % increased asparaginase activity and 30 % decrease in glutaminase activity. Replacement with residues other than alanine did not made any notable change in activities. Validation of this proposed model by site directed mutagenesis may produce the drug with fewer side effects suitable for the clinical purpose (Ln et al., 2011). 3.2 Directed evolution Directed evolution is a tool for protein engineering that mimics the process of natural evolution to evolve protein to user defined manner. This laboratory process functions on a molecular level and focus specific molecular properties. L-asparaginase genes from E. carotovora and E. chrysanthemi were subjected to staggered extension process and a library of variants was created by Kotzia and Labrou, 2009. A thermostable variant among the screened members were selected and identified which contained a single point mutation at position 133, where the negatively charged amino acid aspartate was replaced by alanine. Compared to the wild enzyme, the mutant showed better thermal properties like Tm and half life.
3.3 Random mutations Exploiting the limitations of the DNA repair systems and the polymerases, the mutations can be introduced randomly in a gene sequence in order to produce a cluster of variants for screening. Random mutant libraries were created by several methods which includes the use of error prone PCR, taking the advantage of the mutated strains (E. coli XL1red) or by chemical mutagens. Feng et al., 2016 reported that the random mutation of L-asparaginase II from B. subtilis produced variants with improved properties. Here secretion of recombinant L-asparaginase II 10
was achieved by p43 promoter and choice of wapA signal sequence. This variant form was the subject of error prone PCR for introducing random mutations and the mutant with enhanced activity was selected from mutants generated.
3.4 Epitope engineering The therapeutic protein can be engineered to reduce the immunogenicity there by increasing the retention time in circulation. The drug can evade the immune surveillance by one or several steps by avoid the binding to antigen presenting cell receptors, subsequent proteolysis to peptides for binding to the MHC class II molecules and pass up the binding to B and T cell receptors. Epitope mapping allows identifying and eliminating the T cell epitopes and Class II MHC agretopes and thereby reducing the immunogenicity to a greater extend. Thus a relatively less immunogenic version of the protein can be produced by detailed analysis of the epitopes and its bioengineering. Pokrovsky et al., 2016 studied the structural analysis of the bacterial enzyme from five different sources was done by bioinformatics tools and the epitopes were predicted using Discotope, ElliPro and EPSVR. Immunogenicity studies and comparative analysis of the epitopes provide an idea about the immunogenic part of the protein which can be further mutated to get a less immunogenic counterpart. In silico engineering of the L-asparaginase II of E. coli and Pectobacterium carotovorum for overcoming the limitations was attempted by Ramya and Pulicherla in 2015. Point mutations were introduced in the B cell and T cell epitopes to reduce the immunogenicity and the substrate affinity of the mutant variants were analyzed by docking studies. Molecular modeling tools were used to generate the mutated enzyme models and validated by molecular dynamics and simulations using Gromacs. Similarly T cell epitopes of E. coli L-asparaginase II was predicted 11
by screening the amino acid sequence using immune epitope database consensus prediction method and combinatorial saturation mutagenesis of the targeted sites produced a library of variants. An enzyme variant with eight amino acid substitutions in the predicted T cell epitopes resulted in a reduced T cell response and comparatively low antibody titer (Cantor et al., 2011). Enzyme from Rhizobium etli was proposed to be a therapeutic choice by its less immunogenicity based on epitope prediction compared to enzyme from E. coli and Erwinia. Further reduction in immunogenicity can be attributed by engineering the B cell epitope to move toward catalytically efficient enzyme with minimal immunogenicity (Huerta-Saquero et al. 2013). Structural modifications for property improvements for L-asparaginase are depicted in Supplementary Fig. S1.
3.5 Expression as fusion proteins The fusion of the target protein and the partner protein separately in correct molar ratios and production of a complex protein is a complex procedure for the industrial production and application of pharmaceutical drugs. A good way to solve this problem is by expression of the proteins as fusion proteins in any of the recombinant host, preferably E. coli. Genetic fusions were widely used to overcome the shorter half life of the therapeutic proteins. In order to address the problems related to in vivo stability and immunogenicity, the protein can be fused to albumin, Fc fragment of the antibody, transferring etc by genetic engineering tools. Other than these natural protein, the fusion of inert peptide repeat polymers were also there which have some added advantages like low production cost, ease of manipulation in E. coli system, efficient purification etc. (Strohl, 2015). The plasma half life can be improved by fusion of XTEN, an unstructured recombinant polypeptide (Schellenberger et al., 2009). By selectively 12
eliminating the hydrophobic amino acid content of the attached polypeptide, the immunogenicity can be reduced to a greater extent. A fusion construct of E. coli L-asparaginase and side chain Fv produced a fusion protein with similar kinetic properties like that of the native enzyme with better stability, longer half life and resistant to proteolysis (Guo et al., 2000a). The antibody part was fused to the N-terminus of the protein and was expressed as inclusion bodies and refolded to get the active fusion protein. Based on the three dimensional structural analysis it was proposed that the betterment in the properties of the fusion protein was attributed by the change in the electrostatic potential and steric hindrance (Guo et al., 2000b). 4. Advanced delivery systems for L-asparaginase delivery Among the biopharamaceuticals, protein drugs score the top most position and the advancement in recombinant DNA technology made the production, modification and delivery of these drugs much easier than the early days. The drug delivery route is equally important as the development process because this determines the rate of release of drug to the blood stream, dosage of drug to be administered, controllable level of drug etc. The therapeutic protein efficiency during the treatment depends mainly on the delivery systems, which offers enhanced pharmacokinetic properties which in turn increases the circulatory half life of the drug in patient’s body (Reichert, 2003). E. coli asparaginase is administered in the dose of 25000 U/m2 in adults and 2500-5000 U/m2 in case of children through intravenously or intramuscularly (Riccardi et al 1981, Ahlke E et al 1997). 4.1 RBC as a carrier for vascular drug delivery Targeted delivery of the drugs precisely in to the site of action was a challenging point in the development and application of the therapeutic drugs. Molecules which act as the drug carriers 13
solved the problem by improving the circulatory half life, controlling the timing of drug action, specificity of the target site etc. RBCs being a natural blood component, serve as a better choice as drug carriers for vascular drug delivery. Its non nuclear nature, increased life span and typical membrane structures make it an ideal natural transporter for drug loading and delivery. The entrapment of enzyme inside the erythrocytes help protection from immune system and proteases and asparagine can be continuously pumped into the RBC causing its hydrolysis. The half life of the enzyme will be equal to the half-life of erythrocytes (Domenech et al., 2011). The vehicle construction also considers the ABO and Rh blood group compatibility so that rejection must not happen (Agrawal et al 2013). Clinical trial of the RBC encapsulated asparaginase showed improved pharamacokinetics and delayed renal clearance (Agrawal et al, 2013). A novel encapsulation method of RBC incorporation of proteins with low molecular weight protamine in mice showed prolonged half life compared to the enzyme loaded RBC via hypotonic method (Kwon et al. 2009). Another approach to bypass the humoral immune response upon introduction of the engineered E. coli asparaginase which can bind RBC in situ was reported by Lorentz et al in 2015. Here the wild enzyme was conjugated to the ERY1 synthetic peptide with a 12 residue erythrocyte binding domain and linker sequence for conjugation and flanking arginine residue for reducing the pKa. The erythrocyte binding results in T cell deletion and by abolishing antigen-specific T cell help to B cells may effectively prevent the development of anti-drug antibodies. 4.2 Nanocarriers and nanoparticles The term nanocarriers and nanoparticles often used alone to mention the delivery method based on small molecules less than a micron. Nanocarriers are synthetic groups attached by covalent bonds and are of larger molar mass and are single-chain polymer–drug conjugates, polymer 14
colloids prepared by techniques such as emulsion polymerization, crosslinked nanogel matrices, dendrimers, and carbon nanotubes. Nanoparticles are comprise self-assemblies of smaller molecules, which are aggregated through intermolecular forces. This includes liposomes, polyplexes and other assemblies (Canelas et al., 2009). Drug delivery approaches for Lasparaginase is depicted in Fig. 2. 4.2.1 Liposomal delivery Liposomes are vesicles of phospholipids with an inner aqueous phase and made of one or more concentric lipid bilayers. The enclosed aqueous phase can carry the hydrophilic moieties and hydrophobic compounds can be entrapped in the lipid bilayer. Hence the liposomes act as a carrier of diverse group of compounds such as small drugs, proteins and nucleic acids. Asparaginases can also entrapped in liposomes without losing its activity and anti-cancer potentiality. Entrapment with the help of thin-layer hydration method has been successfully tried using soya bean lecithin and cholesterol. Cholesterol helps in retaining the stability of liposomal particle (Anindita and Venkatesh, 2012). Comparison of the Pharmacokinetics of the asparaginase encapsulated in large and small liposomes showed prolonged enzyme survival by small liposomal enzyme in circulation (Gaspar et al., 1996). More over liposomal encapsulation enhanced the antitumor activity and significantly reduced the production of anti asparaginase antibodies. 5. Conclusions Having a strong foundation of recombinant DNA technology and the protein engineering strategies, nowadays it became a trouble-free job to characterize the enzyme candidates from diverse sources and manipulate according to our interest. An overview o f the recent research 15
outcomes suggest that L-asparaginase with increased production and desired quality are tailored by availing one or other approach of protein engineering. Apart from the sources like E. coli and Erwinia, it opened the way for trial of L-asparaginase from any sources for identification of an alternate choice for chemotherapy against ALL. Combining the drug design and delivery systems, it became possible to create an ideal enzyme for therapeutic application. Acknowledgement We acknowledge CSIR and DBT for all the financial support for carrying out the research on Lasparaginase. One of the authors, Raveendran Sindhu, acknowledges Department of Biotechnology for financial support under DBT Bio-CARe scheme. Aravind Madhavan acknowledges Department of Biotechnology for financial support under DBT Research Associateship programme.
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32. Kishore V., Nishita KP., Manonmani HK. 2015. Cloning, expression and characterization of L-asparaginase from Pseudomonas fluorescens for large scale production in E. coli BL21. 3 Biotech. 5: 975-981. 33. Kotzia GA., Labrou NE. 2009. Engineering thermal stability of L-asparaginase by in vitro directed evolution. The FEBS J, 276: 1750-1761. 34. Kwon YM., Chung HS., Moon C., Yockman J., Park YJ., Gitlin SD., David AE., Yang VC. 2009. L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL). J Control. Rel. 139: 182–189. 35. Ln R., Doble M., Rekha V.P., Pulicherla KK. 2011. In silico engineering of Lasparaginase to have reduced glutaminase side activity for effective treatment of acute lymphoblastic leukemia. J Ped. Hematol. Oncol. 33: 617-621. 36. Lorentz KM., Kontos S., Diaceri G., Henry H., Hubbell JA. 2015. Engineered binding to erythrocytes induces immunological tolerance to E. coli asparaginase. Sci. Advan.1: e1500112. 37. Maggi M., Chiarelli L.R., Valentini G., Scotti C. 2015. Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants. PLoS One 10: e0117025. 38. Meena B., Anburajan L., Sathish T., Raghavan RV., Dharani G., Vinithkumar NV., Kirubagaran R. 2015. L-Asparaginase from Streptomyces griseus NIOT-VKMA29: optimization of process variables using factorial designs and molecular characterization of l-asparaginase gene. Sci. Rep. 24: 12404. 39. Mehta RK., Verma S., Pati R., Sengupta M., Khatua B., Jena RK., Sethy S., Kar SK., Mandal C., Roehm KH., Sonawane A. 2014. Mutations in subunit interface and b-cell 21
epitopes improve antileukemic activities of Escherichia coli Asparaginase-II evaluation of immunogenicity in mice. The J Biol. Chem. 289: 3555-3570. 40. Muharram MM., Abulhmd AT., Mounir M. 2014. Recombinant expression, purification of L-asparaginase-II from thermotolerant E. coli strain and evaluation of its antiproliferative activity. Afr. J Microbiol. Res. 8: 1610-1619. 41. Muzykantov VR. 2011.Targeted therapeutics and nanodevices for vascular drug delivery: quo vadis?. IUBMB Life, 63: 583-585. 42. Nguyen TC., Do TT., Nguyen THT., Quyen DT. 2014. Expression, purification and evaluation of recombinant L-asparaginase in methylotrophic yeast Pichia pastoris. J Vietnamese Environ. 6: 288-292. 43. Offman MN., Krol M., Patel N., Krishnan S., Liu J., Saha V., Bates PA. 2011. Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity. Blood, 117:1614-1621. 44. Oza VP., Parmar PP., Patel DH., Subramanian RB. 2011. Cloning, expression and characterization of L-asparaginase from Withania somnifera L. for large scale production. 3 Biotech. 1: 21–26. 45. Patel N., Krishnan S., Offman M.N., Krol M., Moss C.X., Leighton C., van Delft F.W., Holland M., Liu J., Alexander S., Dempsey C., Ariffin H., Essink M., Eden T.O., Watts C., Bates P.A., Saha V. 2009. A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase. The J Clini. Investig. 119:1964-1973.
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46. Pokrovsky VS., Kazanov MD., Dyakov IN., Pokrovskaya MV., Aleksandrova SS. 2016. Comparative immunogenicity and structural analysis of epitopes of different bacterial Lasparaginases. BMC Cancer, 16: 89. 47. Pourhossein M., Korbekandi,H. 2014. Cloning, expression, purification and characterisation of Erwinia carotovora L-asparaginase in Escherichia coli. Adv. Biomed. Res. 3: 82. 48. Ramirez-Paz J., Saxena M., Griebenow K. 2016. Optimized expression and purification of recombinant L-asparaginase II: Tetramer stabilization by site-specific covalent crosslinking of its subunits, The FASEB Journal, 30 (1 Supplement), lb171-lb171. 49. Ramya LN., Pulicherla KK. 2015. Studies on deimmunization of antileukaemic lasparaginase to have reduced clinical immunogenicity- an in silico approach. Pathol. Oncol. Res. 21: 909-920. 50. Reichert J.M. 2003. Trends in development and approval times for new therapeutics in the United States. Nat. Rev. Drug Dis. 2: 695–702. 51. Riccardi R., Holcenberg JS., Glaubiger DL., Wood JH., Poplack DG. 1981. LAsparaginase pharmacokinetics and asparagine levels in cerebrospinal fluid of rhesus monkeys and humans. Cancer Res. 41: 4554–4558. 52. Roth G., Nunes JES., Rosado LA., Bizarro CV., Volpato G., Nunes CP., Renard G., Basso LA., Santos DS., Chies JM. 2013. Recombinant Erwinia carotovora Lasparaginase II production in Escherichia coli fed-batch cultures. Braz. J Chemi. Engg. 30: 245 – 256.
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53. Sajitha S., Vidya J., Varsha K., Binod P., Pandey A. 2015. Cloning and expression of L -asparaginase from E. coli in eukaryotic expression system. Biochem. Engg. J. 102: 14– 17. 54. Sannikova EP., Bulushova NV., Cheperegin SE., Gubaydullin II., Chestukhina GG., Ryabichenko VV., Zalunin IA., Kotlova EK., Konstantinova GE., Kubasova TS., Shtil AA., Pokrovsky VS., Yarotsky SV., Efremov BD., Kozlov DG. 2016. The modified heparin-binding L-asparaginase of Wolinella succinogenes. Mol. Biotechnol. 58: 528– 539. 55. Schellenberger V., Wang CW., Geething NC., Spink BJ., Campbell A., To W., Scholle M.D., Yin Y., Yao Y., Bogin O., Cleland J.L., Silverman J., Stemmer W.P. 2009.A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27: 1186-1190. 56. Strohl WR. 2015. Fusion proteins for half-life extension of biologics as a strategy to make biobetters. Biodrugs 29: 215–239. 57. Sudhir AP., Agarwaal VV., Dave BR., Patel DH., Subramanian RB. 2016. Enhanced catalysis of l-asparaginase from Bacillus licheniformis by a rational redesign. 58. Upadhyay AK., Singh A., Mukherjee KJ., Panda AK. 2014. Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein. Front. Microbiol. 5: 486. 59. Verma S., Mehta RK., Maiti P., Röhm KH., Sonawane A. 2014. Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II. Biochimica et Biophysica Acta, 1844: 1219–1230. 24
60. Vidya J., Vasudevan UM., Soccol CR., Pandey A. 2011. Cloning, functional expression and characterization of l-asparaginase ii from E. coli MTCC 739. Food Technol. Biotechnol. 49: 286–290.
61. Vidya J., Vasudevan UM., Pandey A., 2013. Effect of surface charge alteration on stability of L-aspaarginase II from Escherichia sp. Enz. Microb. Technol. 56: 15 – 19. 62. Wink PL., Bogdawa HM., Renard G., Chies J.M., Basso LA., Santos DS. 2014. Comparison between Two Erwinia carotovora L-Asparaginase II constructions: cloning, heterologous expression, purification, and kinetic characterization. J Microbial Biochemical Technol. 2: 13-19. 63. Yano S., Minato R., Thongsanit J., TachikiT., Wakayama M. 2008. Over-expression of type I L-asparaginase of Bacillus subtilis in Escherichia coli, rapid purification and characterization of recombinant type I L-asparaginase. Ann. Microbiol. 58: 711-716.
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Fig. 1
26
Fig. 2
27
Figure captions
Fig. 1. Approaches for property improvement of protein drugs Fig. 2 Drug delivery approaches for L-asparaginase Supplementary Fig. 1 Structural modification for property improvement of L-asparaginase
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Table 1: Recombinant production of L-asparaginases Source
Expression host/
Activity
Reference
stable at 50oC even
Vidya et al., 2011
Features
E. coli MTCC 739
E. coli BL21DE3
after 30 minutes.
P. carotovorum
E. coli BL21DE3,
3411.43U/L/h
MTCC 1428
Batch and fed batch
No glutaminase
reactor
activity
E. coli BL21DE3
Melting temperature
E. coli K12
Gosawmi et al., 2014
Upadhyay et al., 2014
64 oC, specific activity 190IU/Mg E. coli
Pichia pink
2.5IU/ml
Sajitha et al., 2015
S. griseus
E. coli M15
123 IU/Ml
Meena et al., 2015
Z. mobilis
E. coli, N-terminal
Extracellular activity
Ensfeldt et al., 2016
His tag, entereokinase
0.13IU/ml
cleavage site
Intracellular activity 3.6IU/ml
E. carotovora
E. coli, fed batch
0.9g/l
Roth et al., 2013
fermentor E. coli
E. coli
E. coli
E. coli, extra cellular
Muharram, 2014 17,386U/L
Ghoshoon et al., 2015
Extra cellular
Chityala et al., 2015
production by Response Surface Methodology P. carotovorum
B. subtilis
production of glutaminase free 29
enzyme B. subtilis
B. subtilis , Fed-batch
407.6IU/mL
Feng et al., 2016
E. coli BL21DE3
Extracellular
Karamitros and
Rosetta
production
Labrou, 2014
process in 3L bioreactor E. chrysanthemi
17800IU/L Rhizomucor miehei
E. coli
Low glutaminase
Huang et al., 2014
activity, specific activity of 1,985 U/mg P.aeuroginosa
E .coli BL21DE3
93.4IU/mL
Dalfard et al., 2016
pLysS P.flourescence
E. coli DE3
270.122 U/mL
Sinha and Singh, 2016
E.chrysanthemi
P. pastoris
Extracellular
Nguyen et al., 2014
production of glycosylated protein Specific activity 6.25IU/mg E. carotovora NCYC
Specific activity 430
Pourhossein and
IU/mg
Korbekandi, 2014
E. coli BL21DE3,
Specific activities of
Wink et al., 2011
E. coli C41 DE3
mature and full length
E. coli
1526 E. chrysanthemi
protein 208 and 237U/Mg E. coli
E. coli, pelB leader
Extracellular
and five aspartate tag
secretion,
Kim et al., 2015
activity 40.8U/mL W. sonmifera
E. coli BL21DE3
specific activity of 17.3 IU/mg 30
Oza et al., 2011
E. coli top10
E. coli DH5α, B.
AQ1 xylanase
subtilis DB104
promoter and shuttle
Helianti et al., 2012
vector pSKE194 Intracellular and extracellular expression S. cereviseae
P. pastoris
AOX1 promoter,
Ferrara et al., 2006
enzyme secreted to periplasmic space Yield in 2l fermentor 85,600 U/L Erwinia carotovora
E. coli BL21DE3
Glutaminase free
Subsp. Atroseptica
enzyme
Scri 1043
Batch fermentor
Goswami et al., 2015
24.57U/ml Fed batch 96.78 U/ml E. coli IBB13
E. coli E13, E. coli
63-117% more
E20
activity than the
Cornes et al., 2002
parent strain E. coli BL21 P. fluorescence B. subtilis
Staphylococcus sp. OJ82
Specific activity
Kishore et al., 2015
0.94IU/mg E. coli Rosseta
Specific activity
GamiB
21.7U/mg
E. coli BL21DE3
60% activity retention in 2M NaCl
31
Yano et al., 2008
Han et al., 2014
HIGHLIGHTS
Overview of different strategies adopted for production of recombinant L-asparaginase. Discusses approaches for increasing the therapeutic efficiency of L-asparaginase. Discusses new drug delivery systems of L-asparaginase.
32
S0960-8524(17)30711-3 http://dx.doi.org/10.1016/j.biortech.2017.05.057 BITE 18083
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
28 March 2017 2 May 2017 10 May 2017
Please cite this article as: Vidya, J., Sajitha, S., Ushasree, M.V., Sindhu, R., Binod, P., Madhavan, A., Pandey, A., Genetic and metabolic engineering approaches for the production and delivery of L-asparaginases: an overview, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.05.057
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Genetic and metabolic engineering approaches for the production and delivery of Lasparaginases: an overview Jalaja Vidya1*, Syed Sajitha1, Mrudula Vasudevan Ushasree1, Raveendran Sindhu1, Parameswaran Binod1, Aravind Madhavan1, 2 and Ashok Pandey1,3 1
Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram 695 019, Kerala, India 2
Rajiv Gandhi Centre for Biotechnology, Jagathy, Thiruvananthapuram – 695 014, India 3
Center of Innovative and Applied Bioprocessing, Sector 81, Mohali, Punjab, India
*Corresponding author. Tel 91-471-2515426 Fax 91-471-2491712 E-mail: [email protected] 1
Abstract L-asparaginase is one of the protein drugs for countering leukemia and lymphoma. A major challenge in the therapeutic potential of the enzyme is its immunogenicity, low-plasma half-life and glutaminase activity that are found to be the reasons for toxicities attributed to asparaginase therapy. For addressing these challenges, several research and developmental activities are going on throughout the world for an effective drug delivery for treatment of cancer. Hence there is an urgent need for the development of asparaginase with improved properties for efficient drug delivery. The strategies selected should be economically viable to ensure the availability of the drug at low cost. The current review addresses various strategies adopted for the production of asparaginase from different sources, approaches for increasing the therapeutic efficiency of the protein and new drug delivery systems for L-asparaginase. Key words: L-asparaginase II; recombinant production; protein stability; drug delivery
2
1. Introduction Enzymes gained therapeutic importance about 40 years back, and they are using now as thrombolytic agents, digestive aids, debriding agents and even as oncolytic agents. In 1902, Dr. John Beard first discovered that enzymes could be used as a therapeutic agent against cancer. His discovery was one of the milestones in cancer therapy. Enzymes, if employed as therapeutic agents must be safe and effective in order to bring about the expected clinical advantage. The advent of protein engineering and recombinant technology can tackle all these problems to a certain extent, and these techniques marked the beginning of a new era in a therapeutic industry. The native properties of the enzymes can be altered for industrial needs in several ways, from the simple chemical modifications such as bio-conjugations to advanced engineering approaches based on protein structural information. The strategy adopted for the modification varies from protein to protein depending on the property, which is focused and includes alterations in thermal properties, half life, substrate affinity, immunogenicity, shelf life, etc. The climax of an engineered drug action depends on the way it is administered into the body and the extent to which it exists in the circulation. There comes the role of drug delivery systems for optimal action of the drug. Nowadays the therapeutic protein industry is focusing mainly on the drug design and novel delivery systems for the unsurpassed result in robust time. Protein therapeutic like enzymes which posses high specificity and potency, require targeted delivery at nanoscale levels for their optimal effectiveness. Hence within a decade, the medium for the drug delivery such as drug carriers and moieties for targeted delivery was rapidly evolved (Muzykantov, 2011). The present review addresses genetic and metabolic engineering strategies adopted for the improvement and delivery methods for anti-leukemic L- asparaginase. 3
2. Production of recombinant L-asparaginases Large scale production of bioactive compounds became a routine process worldwide, after the recognition of fermentation process. Of these bioactive compounds, proteins comprise a major class with wide applications in almost all the industries including food, feed, medicine, detergent, paper, fuel etc. The advent of recombinant DNA technology shed light into the production process and further modification of the proteins and peptides for industrial applications. The very first protein of pharmaceutical potential, produced was insulin and now more than 200 peptides and protein pharmaceutical are there in the FDA list (Demain and Vaishnav, 2009). By understanding the genetic makeup of the microbial systems, the recombinant DNA technology enabled the production of proteins and enzymes from diverse sources like plants and animals by microbial fermentation process for industrial purposes. L- asparaginase with comparable cytotoxicity was reported from Zymomonas mobilis (Einsfeldt et al., 2016). A synthetic mature gene with the N-terminal Histidine tag and an enterokinase cleavage site was cloned and expressed in E. coli. The codons were optimized to the codon usage of E. coli. The enzyme was expressed both extracellularly and intracellularly with a yield of 0.13IU/ml and 3.6 IU/ml respectively and the extracellular enzyme showed cytotoxic effect on leukemic cells. The enzyme from E. carotovora expressed in E. coli was produced by fed batch process in a laboratory scale fermentor and yielded 0.9g/l of soluble protein (Roth et al., 2013). Expression of recombinant L-asparaginase II from a thermotolerant E. coli isolated from camel manure showed anti-proliferative activity in leukemic cell lines (Muharram et al., 2014). A relatively thermostable enzyme from B. subtilis was cloned and expressed in B. subtilis 168 4
using the shuttle vector pMA5 and the purified enzyme showed low affinity towards L-glutamine (Jia et al., 2013). Secretion enables the downstream processing easier; hence most of the recombinant production is accompanied by cloning a secretory signal either the native signal or any other excellent signal sequence in frame with the asparaginase gene. Meena et al., 2015 reported ansA gene from marine actinobacteria Streptomyces griseus and a threefold increase in enzyme production was obtained after expression in E. coli M15. They also reported the optimized culture conditions for production of extracellular L-asparaginase by Box-Behnken design with a maximum yield of 56.78IU/ml. Efficient expression of an extracellular enzyme from E. coli in the culture medium was reported by Ghoshoon et al., 2015 . The full length gene with the signal sequence was cloned in E. coli and the L-asparaginase II mature protein was secreted in to the culture medium where the components were optimized by response surface methodology and a production of 17.386U/l was reported in optimized conditions. The role of L-asparaginase signal peptide in extracellular secretion of recombinant proteins was reported by Ismail et al., 2011 where the mutations on signal peptide were carried out for improving secretion of cyclodextrin glucanotransferase. Chityala et al., 2015 observed secreted expression of glutaminase free Lasparaginase of Pectobacterium carotovorum was achieved by cloning the gene in Bacillus subtilis. The ansB2 gene was cloned in pHT43 and transformed in Bacillus subtilis WB800N. Consecutive IPTG induction enhanced the enzyme production to 105 IU/ml with majority of the expressed protein localized to the extracellular medium. Feng et al., 2016 reported an improved enzyme production from Bacillus subtilis. Step by step increase in activity of L-asparaginase from Bacillus subtilis was achieved by combined 5
approaches using combinations of different signal sequences and promoters and by random mutagenesis. Here eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for evaluation of highest secretion of the protein to the extracellular medium using HpaII promoter and the signal wapA proved to be the best. The activity was further improved by replacing the HpaII promoter with strong p43 promoter. This was then subjected to two rounds of error prone PCR reactions for random mutagenesis and the variants with enhanced activity was selected for N terminal deletion of 25 amino acid residues thereby increasing the activity to 100% that of wild enzyme. The enzyme activity reached 407.6 U/ml (2.5 g/l of ASN protein in 3l bioreactor under fed –batch process. Extracellular secretion of the periplasmic enzyme E. coli L-asparaginase II was achieved by expressing the protein as fusion tag consisting of the pelB signal sequence and various lengths of repeated aspartate residues (Kim et al., 2015). The recombinant enzyme fused with the pelB signal sequence and five aspartate tag was secreted efficiently into the culture medium. The role of secretory machinery of the host cell in extracellular production was proved by deletion of gspDE gene. Another approach for secretion of the recombinant Erwinia enzyme to the culture medium was investigated by Karamitros and Labrou (2014) and the enzyme from the extracellular medium was purified by simple and fast method using affinity adsorbent. The cloned gene from Erwinia chrysanthemi was expressed in seven different E. coli host strains to access the secretory efficiency of the hosts. The E. coli BL21 (DE3) rosetta strain showed the highest expression among all other strains selected and the protein from the culture broth was purified in single step by L-asp adsorbent column and the bound protein was eluted using L-aspartate. Huang et al., 2011 reported an L-asparaginase having dual application in food and leukemic treatment from
6
fungi Rhizomucor miehei. The cloned enzyme expressed from E. coli showed a very low glutaminase activity and was a homodimer of 133kDa. Recombinant production of the L-asparaginase from diverse sources is presented in Table 1. 3. Approaches for increased production with desired quality The microbial production rate of proteins and bioactive compound widely depend on the growth factors or nutritional requirements and the environmental conditions which varies from organism to organism including pH, temperature, aeration, fermentation time etc. Hence depending upon the nature of microbe selected for the production, one can optimize the process parameters and the media components for better yield of the desired product in the fermentor. Large scale production of the product for industrial purpose can be obtained by fermentation process. Not all native proteins carry ideal properties for its application in industrial processes. The functional properties of each protein need to be characterized after purification process and further improvement in the characteristics was achieved primarily through protein engineering and host cell engineering for improved expression of recombinant proteins. The native properties of the enzymes can be altered for industrial needs by several ways, from the simple chemical modifications such as bio-conjugations to advanced engineering approaches based on protein structural information. The strategy adopted for the modification varies from protein to protein depending on the property, which is focused. A schematic view of the approaches for improving the stability of therapeutic protein was included in Fig. 1. The structural stability of the protein can be achieved by alteration of the protein primary structure through selected protein engineering tools like rational designs, random or site directed 7
mutagenesis, chimeric protein expression or as fusion proteins. By the mutagenesis, one can identify the favorable and unfavorable amino acid residues at particular position in a protein and this step-by-step replacement of residues results in gradual increase in the desired functional properties of protein.
3.1 Rational design Sudhir et al., 2016 developed glutaminase free L-asparaginase from Bacillus licheniformis through mutations in amino acid sequences for improving the half life and thermal properties. Four amino acid residues for mutagenesis were selected based on the sequence alignment with previously mutated asparaginases, hydrophobicity and electrostatic potential. The mutant D103V showed three fold better half life and thermal tolerance compared to the wild type protein. The mutant also exhibited increased substrate affinity with Km value of 0.42 mM and Vmax of 2778.9 mol min−1. Patel et al., 2009 developed an E. coli enzyme resistant to the lysosomal proteases by employing the tools like genetic algorithm and molecular dynamics. The cleavage start site of lysosomal protease asparagine endopeptidases (AEP) was identified as N24 and the mutant protein N24G showed the proteolytic resistance with a decreased enzyme activity. Later replacement of alanine at same position (N24A) resulted in an enzyme with higher activity due to unique hydrogen bonding network. For reducing the glutaminase activity, an additional mutation in the interface position 195 constructed a double mutant N24A R195S which is almost ideal for clinical purpose (Offman et al., 2016). Sannikova et al., 2016 modified recombinant enzyme from Wolinella succinogenes having an N terminal peptide which binds to heparin and amino acid substitutions V23Q and K24T to resist 8
trypsinolysis showed high therapeutic efficiency compared to the reference enzyme. Bansal et al., 2012 developed an L-asparaginase enzyme devoid of glutaminase activity by altering the substrate specificity by mutating active site amino acid residues in Pyrococcus furiosus. The mutant K274E showed improved enzymatic properties without any glutaminase activity. An identical result was observed by Verma et al. 2014 for E. coli where the amino acid substitutions in the residues in the subunit interfaces of the enzyme improved activity and stability of the engineered enzyme. Mehta et al., 2014 observed that the mutated variant of L-asparaginase II of E. coli W66Y and Y176F was more effective than the wild type enzyme against the ALL cells and showed a decreased glutaminase activity. The double mutant in the immune-dominant epitopes, K288S/Y176F exhibited reduced antigenicity and tenfold less immunogenicity compared to the wild type enzyme. Mutagenesis of residues involved in the catalytic triad and the substrate binding sites in L-asparaginase of Helicobactor pylori revealed the role of selected residues in glutaminase activity and cytotoxicity of the drug (Maggi et al., 2015). They showed that the double mutant M121C/T169M was unable to hydrolyze glutamine but have no cytotoxic effect. The mutant Q63E exhibited glutaminase activity with comparable cytotoxic effect as that of the wild type enzyme. Gervais and Foote, 2014 observed that deamidation of the Erwinia chrysanthemi enzyme ErA by mutating the residues N41D and N281D resulted in slightly improved specific activity in single and double mutants. N281D mutant showed low glutaminase activity but the thermal stability at elevated temperatures was reduced. An approach to improve the resistance towards the proteolysis, selected surface residues of E. coli enzyme were mutated to cysteine there by allowing the covalent linkage of the subunits to different lengths of maleimide-functionalized the polymers. The mutants A38C-T263C were expressed and were secreted as active protein (Ramirez Paz et al.,) 2016. In silico mutagenesis at 9
the substrate binding site of L-asparaginase II for reducing the affinity towards L-glutamine was done by replacing the amino acid Asp96 with alanine. The replacement showed a 40 % increased asparaginase activity and 30 % decrease in glutaminase activity. Replacement with residues other than alanine did not made any notable change in activities. Validation of this proposed model by site directed mutagenesis may produce the drug with fewer side effects suitable for the clinical purpose (Ln et al., 2011). 3.2 Directed evolution Directed evolution is a tool for protein engineering that mimics the process of natural evolution to evolve protein to user defined manner. This laboratory process functions on a molecular level and focus specific molecular properties. L-asparaginase genes from E. carotovora and E. chrysanthemi were subjected to staggered extension process and a library of variants was created by Kotzia and Labrou, 2009. A thermostable variant among the screened members were selected and identified which contained a single point mutation at position 133, where the negatively charged amino acid aspartate was replaced by alanine. Compared to the wild enzyme, the mutant showed better thermal properties like Tm and half life.
3.3 Random mutations Exploiting the limitations of the DNA repair systems and the polymerases, the mutations can be introduced randomly in a gene sequence in order to produce a cluster of variants for screening. Random mutant libraries were created by several methods which includes the use of error prone PCR, taking the advantage of the mutated strains (E. coli XL1red) or by chemical mutagens. Feng et al., 2016 reported that the random mutation of L-asparaginase II from B. subtilis produced variants with improved properties. Here secretion of recombinant L-asparaginase II 10
was achieved by p43 promoter and choice of wapA signal sequence. This variant form was the subject of error prone PCR for introducing random mutations and the mutant with enhanced activity was selected from mutants generated.
3.4 Epitope engineering The therapeutic protein can be engineered to reduce the immunogenicity there by increasing the retention time in circulation. The drug can evade the immune surveillance by one or several steps by avoid the binding to antigen presenting cell receptors, subsequent proteolysis to peptides for binding to the MHC class II molecules and pass up the binding to B and T cell receptors. Epitope mapping allows identifying and eliminating the T cell epitopes and Class II MHC agretopes and thereby reducing the immunogenicity to a greater extend. Thus a relatively less immunogenic version of the protein can be produced by detailed analysis of the epitopes and its bioengineering. Pokrovsky et al., 2016 studied the structural analysis of the bacterial enzyme from five different sources was done by bioinformatics tools and the epitopes were predicted using Discotope, ElliPro and EPSVR. Immunogenicity studies and comparative analysis of the epitopes provide an idea about the immunogenic part of the protein which can be further mutated to get a less immunogenic counterpart. In silico engineering of the L-asparaginase II of E. coli and Pectobacterium carotovorum for overcoming the limitations was attempted by Ramya and Pulicherla in 2015. Point mutations were introduced in the B cell and T cell epitopes to reduce the immunogenicity and the substrate affinity of the mutant variants were analyzed by docking studies. Molecular modeling tools were used to generate the mutated enzyme models and validated by molecular dynamics and simulations using Gromacs. Similarly T cell epitopes of E. coli L-asparaginase II was predicted 11
by screening the amino acid sequence using immune epitope database consensus prediction method and combinatorial saturation mutagenesis of the targeted sites produced a library of variants. An enzyme variant with eight amino acid substitutions in the predicted T cell epitopes resulted in a reduced T cell response and comparatively low antibody titer (Cantor et al., 2011). Enzyme from Rhizobium etli was proposed to be a therapeutic choice by its less immunogenicity based on epitope prediction compared to enzyme from E. coli and Erwinia. Further reduction in immunogenicity can be attributed by engineering the B cell epitope to move toward catalytically efficient enzyme with minimal immunogenicity (Huerta-Saquero et al. 2013). Structural modifications for property improvements for L-asparaginase are depicted in Supplementary Fig. S1.
3.5 Expression as fusion proteins The fusion of the target protein and the partner protein separately in correct molar ratios and production of a complex protein is a complex procedure for the industrial production and application of pharmaceutical drugs. A good way to solve this problem is by expression of the proteins as fusion proteins in any of the recombinant host, preferably E. coli. Genetic fusions were widely used to overcome the shorter half life of the therapeutic proteins. In order to address the problems related to in vivo stability and immunogenicity, the protein can be fused to albumin, Fc fragment of the antibody, transferring etc by genetic engineering tools. Other than these natural protein, the fusion of inert peptide repeat polymers were also there which have some added advantages like low production cost, ease of manipulation in E. coli system, efficient purification etc. (Strohl, 2015). The plasma half life can be improved by fusion of XTEN, an unstructured recombinant polypeptide (Schellenberger et al., 2009). By selectively 12
eliminating the hydrophobic amino acid content of the attached polypeptide, the immunogenicity can be reduced to a greater extent. A fusion construct of E. coli L-asparaginase and side chain Fv produced a fusion protein with similar kinetic properties like that of the native enzyme with better stability, longer half life and resistant to proteolysis (Guo et al., 2000a). The antibody part was fused to the N-terminus of the protein and was expressed as inclusion bodies and refolded to get the active fusion protein. Based on the three dimensional structural analysis it was proposed that the betterment in the properties of the fusion protein was attributed by the change in the electrostatic potential and steric hindrance (Guo et al., 2000b). 4. Advanced delivery systems for L-asparaginase delivery Among the biopharamaceuticals, protein drugs score the top most position and the advancement in recombinant DNA technology made the production, modification and delivery of these drugs much easier than the early days. The drug delivery route is equally important as the development process because this determines the rate of release of drug to the blood stream, dosage of drug to be administered, controllable level of drug etc. The therapeutic protein efficiency during the treatment depends mainly on the delivery systems, which offers enhanced pharmacokinetic properties which in turn increases the circulatory half life of the drug in patient’s body (Reichert, 2003). E. coli asparaginase is administered in the dose of 25000 U/m2 in adults and 2500-5000 U/m2 in case of children through intravenously or intramuscularly (Riccardi et al 1981, Ahlke E et al 1997). 4.1 RBC as a carrier for vascular drug delivery Targeted delivery of the drugs precisely in to the site of action was a challenging point in the development and application of the therapeutic drugs. Molecules which act as the drug carriers 13
solved the problem by improving the circulatory half life, controlling the timing of drug action, specificity of the target site etc. RBCs being a natural blood component, serve as a better choice as drug carriers for vascular drug delivery. Its non nuclear nature, increased life span and typical membrane structures make it an ideal natural transporter for drug loading and delivery. The entrapment of enzyme inside the erythrocytes help protection from immune system and proteases and asparagine can be continuously pumped into the RBC causing its hydrolysis. The half life of the enzyme will be equal to the half-life of erythrocytes (Domenech et al., 2011). The vehicle construction also considers the ABO and Rh blood group compatibility so that rejection must not happen (Agrawal et al 2013). Clinical trial of the RBC encapsulated asparaginase showed improved pharamacokinetics and delayed renal clearance (Agrawal et al, 2013). A novel encapsulation method of RBC incorporation of proteins with low molecular weight protamine in mice showed prolonged half life compared to the enzyme loaded RBC via hypotonic method (Kwon et al. 2009). Another approach to bypass the humoral immune response upon introduction of the engineered E. coli asparaginase which can bind RBC in situ was reported by Lorentz et al in 2015. Here the wild enzyme was conjugated to the ERY1 synthetic peptide with a 12 residue erythrocyte binding domain and linker sequence for conjugation and flanking arginine residue for reducing the pKa. The erythrocyte binding results in T cell deletion and by abolishing antigen-specific T cell help to B cells may effectively prevent the development of anti-drug antibodies. 4.2 Nanocarriers and nanoparticles The term nanocarriers and nanoparticles often used alone to mention the delivery method based on small molecules less than a micron. Nanocarriers are synthetic groups attached by covalent bonds and are of larger molar mass and are single-chain polymer–drug conjugates, polymer 14
colloids prepared by techniques such as emulsion polymerization, crosslinked nanogel matrices, dendrimers, and carbon nanotubes. Nanoparticles are comprise self-assemblies of smaller molecules, which are aggregated through intermolecular forces. This includes liposomes, polyplexes and other assemblies (Canelas et al., 2009). Drug delivery approaches for Lasparaginase is depicted in Fig. 2. 4.2.1 Liposomal delivery Liposomes are vesicles of phospholipids with an inner aqueous phase and made of one or more concentric lipid bilayers. The enclosed aqueous phase can carry the hydrophilic moieties and hydrophobic compounds can be entrapped in the lipid bilayer. Hence the liposomes act as a carrier of diverse group of compounds such as small drugs, proteins and nucleic acids. Asparaginases can also entrapped in liposomes without losing its activity and anti-cancer potentiality. Entrapment with the help of thin-layer hydration method has been successfully tried using soya bean lecithin and cholesterol. Cholesterol helps in retaining the stability of liposomal particle (Anindita and Venkatesh, 2012). Comparison of the Pharmacokinetics of the asparaginase encapsulated in large and small liposomes showed prolonged enzyme survival by small liposomal enzyme in circulation (Gaspar et al., 1996). More over liposomal encapsulation enhanced the antitumor activity and significantly reduced the production of anti asparaginase antibodies. 5. Conclusions Having a strong foundation of recombinant DNA technology and the protein engineering strategies, nowadays it became a trouble-free job to characterize the enzyme candidates from diverse sources and manipulate according to our interest. An overview o f the recent research 15
outcomes suggest that L-asparaginase with increased production and desired quality are tailored by availing one or other approach of protein engineering. Apart from the sources like E. coli and Erwinia, it opened the way for trial of L-asparaginase from any sources for identification of an alternate choice for chemotherapy against ALL. Combining the drug design and delivery systems, it became possible to create an ideal enzyme for therapeutic application. Acknowledgement We acknowledge CSIR and DBT for all the financial support for carrying out the research on Lasparaginase. One of the authors, Raveendran Sindhu, acknowledges Department of Biotechnology for financial support under DBT Bio-CARe scheme. Aravind Madhavan acknowledges Department of Biotechnology for financial support under DBT Research Associateship programme.
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Fig. 1
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Fig. 2
27
Figure captions
Fig. 1. Approaches for property improvement of protein drugs Fig. 2 Drug delivery approaches for L-asparaginase Supplementary Fig. 1 Structural modification for property improvement of L-asparaginase
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Table 1: Recombinant production of L-asparaginases Source
Expression host/
Activity
Reference
stable at 50oC even
Vidya et al., 2011
Features
E. coli MTCC 739
E. coli BL21DE3
after 30 minutes.
P. carotovorum
E. coli BL21DE3,
3411.43U/L/h
MTCC 1428
Batch and fed batch
No glutaminase
reactor
activity
E. coli BL21DE3
Melting temperature
E. coli K12
Gosawmi et al., 2014
Upadhyay et al., 2014
64 oC, specific activity 190IU/Mg E. coli
Pichia pink
2.5IU/ml
Sajitha et al., 2015
S. griseus
E. coli M15
123 IU/Ml
Meena et al., 2015
Z. mobilis
E. coli, N-terminal
Extracellular activity
Ensfeldt et al., 2016
His tag, entereokinase
0.13IU/ml
cleavage site
Intracellular activity 3.6IU/ml
E. carotovora
E. coli, fed batch
0.9g/l
Roth et al., 2013
fermentor E. coli
E. coli
E. coli
E. coli, extra cellular
Muharram, 2014 17,386U/L
Ghoshoon et al., 2015
Extra cellular
Chityala et al., 2015
production by Response Surface Methodology P. carotovorum
B. subtilis
production of glutaminase free 29
enzyme B. subtilis
B. subtilis , Fed-batch
407.6IU/mL
Feng et al., 2016
E. coli BL21DE3
Extracellular
Karamitros and
Rosetta
production
Labrou, 2014
process in 3L bioreactor E. chrysanthemi
17800IU/L Rhizomucor miehei
E. coli
Low glutaminase
Huang et al., 2014
activity, specific activity of 1,985 U/mg P.aeuroginosa
E .coli BL21DE3
93.4IU/mL
Dalfard et al., 2016
pLysS P.flourescence
E. coli DE3
270.122 U/mL
Sinha and Singh, 2016
E.chrysanthemi
P. pastoris
Extracellular
Nguyen et al., 2014
production of glycosylated protein Specific activity 6.25IU/mg E. carotovora NCYC
Specific activity 430
Pourhossein and
IU/mg
Korbekandi, 2014
E. coli BL21DE3,
Specific activities of
Wink et al., 2011
E. coli C41 DE3
mature and full length
E. coli
1526 E. chrysanthemi
protein 208 and 237U/Mg E. coli
E. coli, pelB leader
Extracellular
and five aspartate tag
secretion,
Kim et al., 2015
activity 40.8U/mL W. sonmifera
E. coli BL21DE3
specific activity of 17.3 IU/mg 30
Oza et al., 2011
E. coli top10
E. coli DH5α, B.
AQ1 xylanase
subtilis DB104
promoter and shuttle
Helianti et al., 2012
vector pSKE194 Intracellular and extracellular expression S. cereviseae
P. pastoris
AOX1 promoter,
Ferrara et al., 2006
enzyme secreted to periplasmic space Yield in 2l fermentor 85,600 U/L Erwinia carotovora
E. coli BL21DE3
Glutaminase free
Subsp. Atroseptica
enzyme
Scri 1043
Batch fermentor
Goswami et al., 2015
24.57U/ml Fed batch 96.78 U/ml E. coli IBB13
E. coli E13, E. coli
63-117% more
E20
activity than the
Cornes et al., 2002
parent strain E. coli BL21 P. fluorescence B. subtilis
Staphylococcus sp. OJ82
Specific activity
Kishore et al., 2015
0.94IU/mg E. coli Rosseta
Specific activity
GamiB
21.7U/mg
E. coli BL21DE3
60% activity retention in 2M NaCl
31
Yano et al., 2008
Han et al., 2014
HIGHLIGHTS
Overview of different strategies adopted for production of recombinant L-asparaginase. Discusses approaches for increasing the therapeutic efficiency of L-asparaginase. Discusses new drug delivery systems of L-asparaginase.
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