review article gene delivery into plant cells for...

Review ArticleGene Delivery into Plant Cells for RecombinantProtein Production

Qiang Chen12 and Huafang Lai1

1Center for Infectious Disease and Vaccinology The Biodesign Institute Arizona State University 1001 S McAllister AvenueTempe AZ 85287 USA2School of Life Sciences Arizona State University Tempe AZ 85287 USA

Correspondence should be addressed to Qiang Chen shawnchenasuedu

Received 4 September 2014 Accepted 17 November 2014

Academic Editor Pedro H Oliveira

Copyright copy 2015 Q Chen and H Lai This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Recombinant proteins are primarily produced from cultures of mammalian insect and bacteria cells In recent years thedevelopment of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant proteinproduction with advantages in versatility speed cost scalability and safety over the current production paradigms In this paperwe review the recent progress in the methodology of agroinfiltration a solution to overcome the challenge of transgene deliveryinto plant cells for large-scale manufacturing of recombinant proteins General gene delivery methodologies in plants are firstsummarized followed by extensive discussion on the application and scalability of each agroinfiltrationmethod New developmentof a spray-based agroinfiltration and its application on field-grown plants is highlighted The discussion of agroinfiltration vectorsfocuses on their applications for producing complex and heteromultimeric proteins and is updated with the development ofbridge vectors Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in contextof their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes These newadvancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants facilitating theadoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications

1 Introduction

The approval of the first plant-derived therapeutic enzymefor Gaucherrsquos disease has demonstrated the promise of plant-based systems for recombinant protein (RP) production [1]In addition to the traditional advantages in cost scalabilityand safety over current bioreactor-based production platfo-rms progress in glycoengineering and expression vector dis-covery has also allowed plants to produce RPs with specificglycoforms to enhance functionality and at unprecedentedspeed to control potential pandemics and fight bioterrorism[2]

The traditional strategy of producing RPs in plants is tocreate stable transgenic lines of plantsThe target transgene isintegrated into the plant genome and the RP can be producedin successive generations [3] To eliminate the long timeframe of generating transgenic plants transient expressionsystems have been developed In this strategy the transgene is

not integrated into the plant genome but rather quicklydirects the production of the RP while residing transientlywithin the plant cell In addition to significantly shorteningthe production timeline this strategy also enhances RP accu-mulation level by eliminating the ldquoposition effectrdquo of variableexpression caused by the random integration of transgenewithin the genome [4] Besides its speed and high yieldthe transient expression system also offers the versatility forproducing personalized RPs such as therapeutics for patient-specific cancers and vaccines against viruses that have rapidantigenic drift andor multiple strains with unpredictableoutbreaks This flexibility also provides the ldquosurgerdquo capa-bility to rapidly produce recombinant counteragents in abioterrorism event Since no transgenic plant is created tran-sient expression also addresses regulatory issues and publicconcerns for genetically modified organisms (GMOs) Theseadvantages demonstrate the vast potential of transient expres-sion as a preferred method for RP production in plants

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 932161 10 pageshttpdxdoiorg1011552015932161

2 BioMed Research International

However scale-up of RP production by transient expressionposes a bigger challenge than transgenic plants because nogenetically stable seed bank is produced and scale-up is nolonger just a matter of increasing acreage to boost yield Toovercome this challenge a scalable transgene delivery meth-od must be developed for plant transient expression

2 Methods of Transgene Delivery

The method of choice for introducing transgenes into plantsdepends on the expression vector and the host plants Thesemethods include direct delivery by gene gun and indirectdelivery through using Agrobacterium tumefaciens or plantviruses [5]

21 Direct Delivery Methods DNA or RNA can be directlyintroduced into plant cells via a so-called microprojectilebombardmentmethod also known as a gene gun or biolisticsIn this method the transgene is coated onto microgold ortungsten particles and fired into plant cells ballistically [6]The advantage of this method resides in its versatility and abroad range of susceptible plants It can be used to delivertransgene to both nuclear and chloroplast genomes At leastin theory effective transgene delivery by biolistics is vectorindependent and can be applied to any plant host species [5]

22 Indirect GeneDeliveryMethods Indirect transgene deliv-ery exploits the ability of plant virus or certain pathogenicagrobacteria species (eg A tumefaciens) that naturallytransfer their genome (plant virus) or part of their tumorinducing plasmid (Ti plasmid) DNA (T-DNA) into plant cells(Agrobacterium) A transgene can enter plant cells as a by-product of viral infection if cloned into the full viral genomeInfection can be facilitated by rubbing plant tissue with atransgene carrying infectious viral particles or viral nucleicacids [3] However this method is only applicable to virusesor plant hosts that are susceptible to mechanical inoculationbut not to those that require specialized insects for viral trans-mission

Ti plasmids of Agrobacterium can be modified into deliv-ery vectors by replacing pathogenic genes in T-DNA withtransgenes transgene transfer from agrobacteria to plant cellsis accomplished through the natural interaction between Atumefaciens and its plant hosts [5] In contrast to biolisticsgene delivery by A tumefaciens requires the cloning of trans-gene into a modified Ti plasmid and is restricted to dicotyle-donous and a limited number of monocotyledonous plants[7] However delivery by Agrobacterium generally offersbetter efficiency transgene expression and inheritance thanbiolistics [5] It is speculated that Agrobacterium-based deliv-ery is advantageous because transgene copy numbers andintegration into the genome are better controlledThe coevo-lution ofAgrobacterium and its plant hostsmay favor the inte-gration of transgenes into genomic loci that are transcrip-tionally active which leads to its high level of expression[7] In transient expression biolistics often cause severetissue damage and effectively reduce the available biomass forRP production making indirect delivery by Agrobacterium

a preferredmethod As a result anAgrobacterium-based genedelivery via agroinfiltration has become the favorable genedelivery method for transient expression in plants [7 8]

3 Agroinfiltration for Expression ofRecombinant Proteins

On a per cell basis the yield of a RP is usually higher in tra-nsient expression than that in transgenic plants [4] Theelimination of the ldquoposition effectrdquo is responsible for this imp-rovement as the transgene is no longer randomly insertedinto genomic areas with variable transcriptional activity [9]However earlier gene delivery methods consisted of soakingleaf pieces in Agrobacterium culture in which only the celllayer on the edges may receive the transgene This limits theefficiency and scalability of transient systems

Agroinfiltration was invented to overcome this challengeBecause up to one-third of the leaf volume is intercellularspace it is possible to replace the air in these cavities witha suspension of Agrobacterium [1] Thus transgene-carryingagrobacteria are actively delivered into the intercellular spaceof the leaf tissue by agroinfiltration allowing for the effectiveaccess of agrobacteria to most leaf cells and making thetransfer of T-DNA a highly efficient event [8 10]

31 Application and Scalability of Agroinfiltration The sim-plest method of agroinfiltration is syringe infiltration In thismethod transgene-carryingAgrobacterium in the infiltrationmedium is injected into the leaf with a needleless syringe(Figure 1(a)) Syringe infiltration offers the flexibility of intro-ducing multiple transgene constructs into different areasallowing multiple assays to be performed on a single leaf [8]Thus it has been used for studying plant-pathogen interac-tions abiotic stresses gene functional analysis protein local-ization and protein-protein interactions [8] Syringe infiltra-tion also has numerous applications for RP production Forexample it can be used to quickly examine the expressionlevel of a RP under established conditions (Figure 1(b)) Iffurther optimization of expression is necessary its flexibilitypermits a quick assessment of various factorsrsquo effects on theyield expression kinetics and toxicity of the target proteinThese factors include concentrations of Agrobacterium cul-ture different expression vectors organelles favorable for RPaccumulation and the requirement for silencing suppressorsOnce optimized syringe infiltration can also be used to infil-trate several entire plants to rapidly obtain sufficient amounts(milligram level) of RPs for biochemical characterization andpreclinical functional studies as well as for developing puri-fication schemes Despite these utilities only a few plantspecies are naturally amenable to syringe infiltration and theprospect of its scalability is highly limited [11]

A scalable agroinfiltration technology will enhance thecompetitiveness of transient expression systems with the tra-ditional cell culture-based platforms for RP production Anagroinfiltration method using a vacuum chamber was devel-oped for such purposes [5] A prototype apparatus routinelyused in the laboratory is best illustrative of the vacuumchamber method First aerial parts of plants are submerged

BioMed Research International 3

(a) (b)

(c) (d)

Figure 1 Transgene delivery by agroinfiltration intoN benthamiana and lettuce plants Agrobacteria carrying the expression cassette of GFPor DsRed in geminiviral vectors were syringe-infiltrated into a N benthamiana leaf (a) and GFP or DsRed expression was observed 4 daysafter infiltration under UV light (b) Similarly A tumefaciens cells harboring the expression cassette of GFP in a geminiviral vector werevacuum infiltrated into a lettuce plant (c) and GFP expression was examined 4 days after infiltration (d) The yellow spot in (b) indicates theleaf area that was coinfiltrated with agrobacteria carrying the expression cassette of GFP and DsRed

into an Agrobacterium suspensionThe submerged plants arethen transferred into a desiccator that serves as the vacuumchamber A pump provides the vacuum that exposes the sub-merged plants to a negative atmospheric pressure and drawsthe air out of the interstitial space of the leaves Agroinfil-tration is achieved when the vacuum is released allowingagrobacteria in the medium to enter the intercellular spaceonce occupied by the air (Figure 1(c))

Vacuum infiltration can efficiently infiltrate plant speciesthat are unamenable to syringe infiltration effectively expan-ding the host range of agroinfiltration [12ndash14] Furthermorestudies demonstrated that vacuum infiltration resulted insimilar yield and temporal expression patterns for several RPscompared to that of syringe infiltrationThis suggests that theresults from the two agroinfiltration methods are mutuallytransferable the simple syringe infiltration can accuratelypredict the expression pattern of aRP in scale-up settingsNot

surprisingly research has shown the superiority of vacuuminfiltration in speed and robustness over syringe infiltrationFor example as the entire plant is subjected to agroinfiltrationwith vacuum the expression of the RP can be detected inall leaves of the entire plant (Figure 1(d)) Even at benchscale the time required for infiltrating a single 6-week-oldN benthamiana plant is significantly shortened 30 times byvacuum infiltration [15]

The scalability of vacuum infiltration has been examinedfor the production of RPs with biomedical applicationsTo test its scalability beyond the desiccator prototype wedesigned a vacuum chamber that is able to accommodate 16trays of plants per infiltration cycle Results indicated thatvacuum infiltration is highly scalable Specifically the accu-mulation level and the temporal expression patterns of virus-like particles (VLPs) of norovirus andmonoclonal antibodies(MAbs) against West Nile virus (WNV) were not affected


(a) (b)

Figure 2 N benthamiana plant growth (a) and agroinfiltration (b) at commercial production scale at Kentucky Bioprocessing LLC

regardless of whether they were produced under scale-upconditions or with the desiccator [16ndash19] This pilot-scalesystem has enabled us to produce the norovirus vaccine can-didate under current good manufacturing practice (cGMP)regulations which is sufficient both in quality and quantityfor a phase I human clinical trial [16 20 21] Biotechnol-ogy companies have further explored the scalability of thismethod For example a fully automated vacuum system wasdeveloped with the capability to agroinfiltrate up to 12 tonsof plant biomass per day allowing for the production of up to75 g ofMAb-based therapeutics per greenhouse lot (Figure 2)[1 15 22] This process can be further scaled up but itsrequirement of inverting plants grown in pots or trays mayimpose limitations on the ways the plants can be cultivatedand may confine the use of vacuum infiltration to high-valueRPs such as vaccines and therapeutics

For even larger scale RP production especially for low-cost but high-volume RPs it is desirable to develop new agro-infiltration technologies that allow gene delivery to wholeplants without using a vacuum Fortunately as only nontrans-genic plant material is used in transient expression biomasscan be generated in open fields by conventional agriculturalpractices without concerns for GMO This allows the explo-ration of a spray-based agroinfiltration method to delivertransgene into field-grown plants Initially approximately 2of leaf cells can receive and express the transgene throughspray agroinfiltration [1] New developments in this method-ology include the use of surfactant andor abrasives inthe Agrobacterium suspension to enhance transfection andnew Agrobacterium strains with super transfectivity [1 23]When improved spray agroinfiltration is combined with anexpression vector that can generate a replicon with cell-to-cell movement capability up to 90 of leaf cells can receivethe transgene and express the target protein at high levels of50 total soluble protein (TSP) [1] This provides a simpleand indefinitely scalable process of transgene delivery intofield-grown plants allowing transient expression on a largeagricultural scaleThe demonstration of large-scale agroinfil-tration under the US Food and Drug Administrationrsquos (FDA)cGMP guidelines supports the regulatory compliance of thistechnology and extends its application to manufacture RPswith human biomedical interests Collectively these studies

demonstrate that the value of vacuum and spray agroinfiltra-tion lies in their enormous scalability potential facilitatingthe adoption of plant transient expression-based systems forcommercial manufacturing of RPs

32 Vectors for Agroinfiltration Agroinfiltration is versatileand can be performed with any vectors as long as they canreplicate in A tumefaciens and initiate T-DNA transfer withthe help of virulent genes on chromosomes andor anotherplasmid [8]The earliest vectors used for agroinfiltrationweretranscriptional vectors such as pBIN19 or pCAMBIA drivenby nopaline synthase (pnos) or cauliflower mosaic virus(CaMV) 35S promoters (CaMV35S) While these transcrip-tional vectors are not as robust as later developed plant virus-based vectors they do have a broad host range and can beused in almost all plant species It is these vectors that demon-strated the superiority of transient expression in regard to thespeed and RP yield over the traditional protein expression intransgenic plants [20 24 25]

The robust replication andor transcription of plantviruses has led to the development of viral vectors for enhanc-ing the RP yield [25] Each type of plant virus offers its uniqueadvantages and limitations as an agroinfiltration vector andmay be useful for the production of a specific type of RPFor example double-stranded DNA plant viruses such asCaMV are useful only for producing small RPs because theyhave limited packaging capacity and can lose their genomefunctions when a fraction of their genomes are removed orsubstituted [25] In contrast single-stranded RNA viruses(eg tomato busy stunt virus (TBSV) tobacco mosaic virus(TMV) and potato virus X (PVX)) offer vectors for express-ing large RPs because they are more tolerant to large genesubstitutions and insertions [25] However these RNA-basedvectors have to be generated by an unscalable in vitro process

ldquoDeconstructedrdquo viral vectors represent a new generationof vectors that combine the robustness of full viral vectors andthe versatility of nonviral vectors The elimination of unnec-essary or unbeneficial genomic components during viral dec-onstruction significantly reduces the size of the repliconallowing the insertion of larger transgenes while maintainingthe robustness of viral replication and transcription Decon-structed RNA viral vectors can be delivered in the form of


DNA which will be transcribed and spliced into autonomousreplicons in plant cells [25] This not only effectively elim-inates the need for generating RNA vectors by an in vitroprocess but also allows for all deconstructed viral vectorsto be delivered by agroinfiltration Since agroinfiltration candeliver vectors to most of the cells on the entire plant[15] the viral systemic spreading function is no longerneeded This alleviates the concern for transgene loss duringsystemic spreading and allows the deletion of coat proteinto accommodate larger transgene insertion Agroinfiltrationalso broadens the range of plant species susceptible to viralvector delivery beyond the natural virus hosts and allows forthe delivery of vectors that are notmechanically transmissiblein natureTherefore the development and application of dec-onstructed viral vectors not only overcome the limitationsof full viral vectors but further enhance their versatility andtransgene expression in transient systems

The most commonly used deconstructed vectors rely ontheMagnICON system derived from TMV [26]This systemcan be used either in themodular or the fully assembled formdepending on the applicationThemodular system facilitatessimple cloning of the transgene because it is located in a sepa-rated plasmid of reasonable size Furthermore the transgenein the 31015840 module can be paired with a suite of 51015840 modulesthat contains different promoters andor targeting sequencesto various organelles As a result the modular MagnICONsystem provides the flexibility to test the expression of atransgenewith different promoters and in different organellesby simply mixing different A tumefaciens strains that carryvarious modules Thus it is best suited for the optimizationof transgene expression and small scale RP production Incontrast the fully assembled system sacrifices the flexibilityto gain robustness for industrial scale production It requiresonly one vector and one A tumefaciens culture for agroin-filtration greatly simplifying the upstream processing andreducing the overall cost of goodTheMagnICON system hasbeen tested at various production scales from a few plantsin a laboratory to 12 tons of plant materialday in industryCollectively they demonstrated that very high level accumu-lation of RPs (up to 5mg per g leaf freshweight (LFW)) can beachievedwithin 7ndash10 days after infiltration (dpi)They includeRPs of all sizes and complexity ranging from small subunitvaccines to large tetravalent antibodies [16 17 26ndash29]

In spite of the success the current MagnICON systemcannot produce RPs withmore than two heterosubunitsThisproblem is associated with the phenomenon called ldquocompet-ing repliconsrdquo as codelivery of viral vectors built on thesame viral backbone often results in early segregationand subsequent preferential amplification of only one of thevectors in a single cell [25] For example TMV and PVX areboth competing viruses but not with each other If the heavy(HC) and light chain (LC) genes of a MAb are both clonedinto the TMV or PVX vector only one of the chains will beproduced in a single cell and assembled MAb will not beproduced However if the HC and LC gene are built on theTMV and PVX backbone respectively both chains can beexpressed in the same cell permitting their proper assemblyinto a functional MAb [26] This allows the MagnICONsystem to produce MAbs However identifying additional

Rep

LB RB

LIR LIRSIRGOI

Figure 3 Geminiviral BeYDV vector for expression of recombinantproteins The left (LB pink triangle) and right border (RB pinktriangle) delineate the T-DNA construct that will be transferredinto plant cells by Agrobacterium Upon delivery into plant cellsexpression of Rep gene produces the Rep protein (brown cloud)that nicks the LIRs (red stars) in the T-DNA to release a single-stranded DNA molecule (between the two yellow arrows) ThisDNAmolecule recircularizes and is copied tomake double-strandedDNAs that can replicate by the rolling circle mechanism to producevery high copy numbers of DNA templates (circles) and in turnabundant mRNAs of gene of interest (GOI) for the translation of therecombinant protein Blue star SIR pink triangle LB and RB of theT-DNA red stars LIRs green arrow gene of interest brown cloudRep protein

viruses that are noncompeting with both TMV and PVXfor expressing proteins with three or more distinct subunitsis a very difficult if not impossible task [20 30] We havecircumvented this problem by developing a noncompetingvector system based on bean yellow dwarf virus (BeYDV)a monopartite virus in the Geminiviridae family [31] Uponinfection of plant cells very high copy numbers of BeYDVgenome are produced by rolling circle replication whichrequires only one single viral protein (replication associatedproteins (Rep)) (Figure 3) [31] In the first generation ofgeminiviral vectors the transgene and the Rep protein aresupplied in two separate modules [19 31] Coagroinfiltrationof the two modules resulted in high-level accumulation ofRPs which can be further increased by including a thirdmodule carrying a suppressor of gene silencing from TBSV(P19) [19] For example we showed that inclusion of the thirdP19 module increased the accumulation of hepatitis B coreantigen (HBcAg) in N benthamiana gt4-fold [21] Southernand Northern blot analyses indicated that codelivery of P19only marginally increased the replicon copy number butgreatly enhanced the accumulation ofHBcAg-specificmRNA[21]These results indicate that P19 indeed can increase targetmRNA and protein accumulation most likely by suppressingposttranscriptional silencing of the transgene We thenintegrated the transgene Rep and P19 modules into a singlevector system and demonstrated that the geminiviral systemis noncompeting and permits the efficient expression andassembly of MAbs [32] For large-scale manufacturing wedeveloped a single vector system that contains multiple repli-con cassettes with each encoding for a distinct protein Upon


Table 1 Examples of recombinant proteins produced in plants by agroinfiltration

Plant host Vector Biologic target Development stage ReferencesN benthamiana Nonviral vector Influenza A H5N1 HA VLP vaccine Phase III human trial [38 39]N benthamiana TMV NHL personalized vaccine Phase I human trial [37 40]N benthamiana CPMV BTV 4-component VLP vaccine Preclinical [30]N benthamiana (WT ΔXF) TMVPVX Tetravalent antibody WNV therapeutic Preclinical [28 41]N benthamiana TMV Cellulases for ethanol production Early development [1 42]N benthamiana TMVPVX Ebola immune complex-based vaccine Preclinical [27 43]N benthamiana TMV BeYDV CPMV HBcAg nonenveloped VLP vaccine Preclinical [19 44]Lettuce N benthamiana TMVPVX BeYDV Ebola therapeutics based on MAb Preclinical [18 32 34 45]Lettuce N benthamiana TMV BeYDV Norovirus NVCP VLP vaccine Preclinical [19 34 46]Lettuce N benthamiana TMVPVX BeYDV WNV therapeutics based on MAb Preclinical [17 28 34]Lettuce N benthamiana (WT ΔXF) TMV BeYDV WNV DIII vaccine Preclinical [31 34 47]HA hemagglutinin VLP virus-like particle NHL non-Hodgkinrsquos lymphoma BTV bluetongue virus WT wild-type ΔXF plants with double knockdown of120573-12-xylose and core 120572-13-fucose WNVWest Nile virus HBcAg hepatitis B core antigen MAb monoclonal antibody NVCP Norwalk virus capsid proteinDIII domain III of envelope protein

agroinfiltration into plant cells each cassette was shown toassemble into an independent replicon and produces highlevels of the proteinsubunit it codes for without competingwith the replication of other replicons or the production ofother proteins [32] This single vector system obviates theneed to generate several vector modules and manufacturemultiple inocula of A tumefaciens strains further reducingcapital and operational cost Recently a different geminiviralvector based on the mild strain of BeYDV-m has beendeveloped and has shown its robustness in expressing twovaccine candidates [33] These geminiviral systems may havebroader plant host ranges than theMagnICON system [18 1932 34] Overall the geminiviral replicon system overcomesthe difficulty of producing multiple heterosubunit proteins

Other examples of deconstructed viral vectors includesystems based on 51015840 and 31015840 untranslated regions (UTRs) ofcowpea mosaic virus (CPMV) RNA-2 and tobacco yellowdwarf Mastrevirus (TYDV) [35 36] One version of theCPMV-based vectors is replication independent and there-fore has great promise for agroinfiltration in plant hoststhat are not compatible with replication-dependent vectorsystems Excitingly the CPMV-based vector has allowed theexpression and assembly of Bluetongue VLPs inN benthami-ana that requires coexpression of four different proteincomponents [30] These plant-produced VLPs were shownto be immunogenic and provide protective immunity insheep against a challenge of a Bluetongue virus field isolatedemonstrating the utility of this vector in producing complexand heteromultimeric proteins [30]The TYDV-based vectorsystem represents ldquobridgerdquo vectors that allow the stable inher-itance of the transgene and a robust yet controlled transientexpression of a RP upon the induction with a specific chem-ical signal [36] Plants are allowed to accumulate biomassin the growth phase while the integrated transgene remainssilent and replicon amplification will be triggered upon ind-uction for RP production This type of bridge vector systemeffectively combines the strengths of both the stable and tran-sient expression systems and potentially offers a complete

platform for the rapid assessment of RP candidates and theirtransition to a large-scale commercial production

33 Plant Hosts for Agroinfiltration A prerequisite for a plantspecies to be successfully agroinfiltrated is its susceptibility toA tumefaciens infection Among susceptible plants howeverthe amenability of different species to agroinfiltration variessignificantly due to leaf structural differences in the cuticlethe density of stomata on the epidermis and the compactnessof mesophyll cells Due to technological improvements arapidly expanding spectrum of plant species is now amenablefor transgene delivery by agroinfiltration Since transientexpression systems for RP production do not generate trans-genic plants it does not have the risk of contaminating foodcrops or unintended transgene escape This further expandsplant hosts infected via agroinfiltration as an acceptable tech-nology to the public and regulatory agencies The choice of aparticular plant host for protein expression is determined byits compatibility with available expression vectors the natureof the target RP and the scale of production

331 Nicotiana Hosts Themost popular host plant for agro-infiltration is N benthamiana and related Nicotiana plantsincluding tobacco Besides being most amenable to agroin-filtration these plants can produce large amount of biomassrapidly and are prolific seed producers for the industrial scale-up of production [20] In addition they are permissive to thereplication of a variety of replicon-based vectors The FDAand other regulatory agencies are familiar with clinical trialmaterials from these plant hosts thus facilitating their accep-tance in regulation-compliant processes [29 37] As a resultnumerous RPs of various natures sizes and applications havebeen produced in Nicotiana hosts (Table 1) The examplesbelow demonstrate the advantages and versatility of utilizingthese plant hosts for agroinfiltration

In a clinical trialNicotiana host-produced biologics wereused to treat Non-Hodgkinrsquos lymphoma (NHL) NHL is


a group of blood cancers that is estimated to result inover 70800 new cases in the USA alone in 2014 In NHLeach malignant B cell clone expresses a unique cell surfaceimmunoglobulin (Ig) as the tumor-specific marker makingstandard treatments ineffective The variable nature of NHLcalls for patient-specific cancer treatments that require anexpression system with the flexibility to rapidly producepatient-specific vaccines One of these vaccines consists ofMAbs derived from each patientrsquos own tumor The cell cul-ture-based production platforms do not have the speed andflexibility to produce these personalized vaccines In con-trast plant production systems based on agroinfiltration canprovide the optimal platform to meet this demand Resultsshowed that 20 patient-specific MAbs were produced at highlevels inN benthamiana leaves within twoweeks of agroinfil-tration [29] The manufacturing process is robust requiringonly twoweeks forMAb-based vaccine expression and purifi-cation and less than 12 weeks from biopsy to vaccination [29]To test the safety and immunogenicity of the plant-expressedvaccine candidates a phase I human clinical trial was initiatedwith 12 patients Results indicated that the vaccine was well-tolerated without major side effects and 73 of the patientsdeveloped a tumor-specific immune response [37 40] Thisstudy demonstrated the rapidness and versatility of theagroinfiltration-based transient system in generating multi-ple patient-specific cancer vaccines and showcased the capac-ity of N benthamiana in producing vaccines that are safe toadminister and effective in the treatment NHL patients

Nicotiana plant hosts were also utilized for commercialscale enzyme production In the production of ethanol asa fuel extender large quantities of cellulase are needed tosaccharify cellulosic feedstocks For more than 30 years thehigh cost of cellulase from fungal fermentation has beena major impediment to the economic viability of cellulosicethanol programs [48] To reduce the cost of cellulase Nbenthamianawas used as a host to produce four cellulases forcell wall degradation via agroinfiltration [42] Results showedthat all four cellulases were expressed at high levels up to75TSP [42] Further analysis indicated that plant-producedcellulases are functional efficiently converting cellulose toglucose [42] Similar results were obtained between usingsyringe and spray agroinfiltration indicating the scalabilityof the upstream process [42] Importantly the necessity ofpurification and costs associated therewith are avoided inthe downstream processing as the cellulases can be simplypreserved at room temperature for up to four months indehydrated N benthamiana biomass as silage [42] Technoe-conomic analysis of a similar cellulase production systembased on transgenic N tabacum concludes that the plant-based system may offer a gt30 reduction in unit productioncosts and an 85 reduction in the required capital investmentcomparedwith the current fungal-based fermentation system[49]We speculate that the systembased on spray agroinfiltra-tion may result in a similar cost-saving benefit presenting asystem of cellulase production with unprecedented efficiencyand cost-effectiveness This process can find broad applica-tions for production of other cost-sensitive RPs

N benthamiana hosts also offer opportunities to produceRPs with enhanced functionalities (biobetters) For example

N benthamiana with ldquohumanizedrdquo glycosylation pathwayshave been developed to enhance the safety and efficacy ofplant-producedMAbs [50]The difference inN-glycosylationbetween plant and mammalian-produced MAbs may alterthe stability andor efficacy of plant-producedMAbs or causepotential adverse effects through immune complex forma-tion To overcome this challenge a double knockdown (ΔXF)N benthamiana line was created to suppress the productionof the two plant-specific glycans 120573-12-xylose and core 120572-13-fucose [51] Results indicated that anti-EbolaMAbs producedin theΔXFplant line had no plant-specificN-glycans but con-tained the highly homogenous (90) mammalian glycoformGnGn [52] The lack of fucose and the high homogeneity ofplant-derived MAbs have led to their higher affinity to theFc receptor (FcgRIII) and their enhanced potency againstEbola virus over the mammalian cell-produced MAbs [52]The superior potency of plant-produced MAbs was furtherdemonstrated in a challenge study with nonhuman primatesin which plant-produced MAbs were far more protectiveagainst a lethal Ebola challenge than those produced inmam-malian cells [45] In a remarkable and exciting developmentthese plant-made MAbs were recently used to treat twoAmerican Ebola patients and showed promising results [53]Similarly our studies showed that ΔXF plant-derived anti-WNVMAbs displayed enhanced viral neutralization in com-parison to their mammalian counterparts [28] These newNbenthamianahosts are being applied to produce biobetter RPsbeyond the realm of MAbs

332 Non-Nicotiana Hosts Certain RPs require a non-Nico-tiana plant host for their optimal expression Fortunatelyimprovements in technologies have allowed the applicationof agroinfiltration to many plant species beyond Nicotianaplants including lettuce tomato alfalfa petunia potatocotton grapevine switchgrass radish pea lupine flax citruslentil sunflower and Arabidopsis [11] Agroinfiltration meth-ods have also been applied to woody trees including aspenpoplar birch eucalyptus pines and spruces [13] In additionto leaf tissue petals of tobacco petunia Antirrhinum majusGerbera jamesonii several species of Dendrobium flowersand the fruits of tomatoes and strawberries have also beensuccessfully agroinfiltrated with transgene constructs [54]

Among these options lettuce is a prime example to demo-nstrate the special utility of non-Nicotiana hosts in producingRPs Despite the aforementioned advantages of Nicotianahosts they do produce unusually higher levels of phenolicsand alkaloids than other plant species These compoundsfoul purification resins and are difficult to remove from thetarget RP in downstream processing adding to productionresources and costs [55 56]This is especially problematic forRPs with pharmaceutical applications as they need to be freeof these plant compounds to meet regulations of the FDAThus there is a need to identify plant hosts that produce lowerlevels of phenolics and alkaloids yet retain the robustness ofRP production Lettuce (Lactuca sativa) is already cultivatedcommercially in large scales and its yield and speed ofbiomass generation can easily match those of Nicotianaplants Lettuce produces negligible quantities of phenolics


and alkaloids and thus would overcome the challenge oftheir removal during downstream processing Agroinfiltra-tion with nonviral vectors indicated that lettuce expresses avariety of functional RPs albeit the expression levels were low[57 58] To further demonstrate the potential of lettuce as ahost for RP production we explored the use of deconstructedviral vectors to express pharmaceutical proteins in lettuce(Table 1) We first examined the expression of a VLP vaccinecandidate for norovirus based on the capsid protein ofNorwalk virus (NVCP) with geminiviral vectors NVCP wasexpressed at levels which are comparable with that in Nbenthamiana at the highest expression levels ever reportedfor a vaccine in lettuce [34] Furthermore lettuce-producedNVCP efficiently assembled intoVLPswith a diameter typicalof native NVCP VLPs [21 34] Moreover these VLPs arefully functional and can induce potent immune response inmice [21]These studies demonstrated that lettuce is as robustas Nicotiana plants for RP production with agroinfiltrationBeyond that this study also demonstrated the superiorityof lettuce over Nicotiana plants for expressing VLPs Owingto their structural resemblance to native viruses but lackinginfectious viral genomes VLPs have been shown to havetremendous potential in immunogenicity multivalency andsafety as vaccines against many diseases [20] However theporous and dynamic nature of theVLP structure alsomakes itsusceptible to trap contaminant molecules inside As a resultit is a very difficult task to remove plant secondary metabo-lites from the feedstream of Nicotiana plant-produced VLPs[16] If not resolved this problem will diminish the vastpotential of VLPs and plant transient expression technologyDue to the low level of secondarymetabolites in lettuce tissueNVCP VLPs can be purified to high purity from lettuceextracts without the extra need for elimination of phenolicsand alkaloids [34]

The advantage of lettuce has also been showcased for theproduction of MAbs another group of proteins with highpharmaceutical relevance MAbs can be efficiently producedin several plant hosts For example MAbs against Ebola virusandWNVcanbe expressed at very high levels inN benthami-ana with MagnICON and geminiviral vectors [17 32] Theseplant-produced MAbs are fully functional in protecting ani-mals from lethal challenge of viral infections [17] In additionto the native MAbs large MAb variants such as tetravalentantibodies and recombinant immune complexes have beensuccessfully produced in N benthamiana [27 28 41 43]The most popular and efficient method of purifying MAbsis Protein A affinity chromatography As a RP itself ProteinA is costly and can be easily damaged by a cleaning reagentUnfortunately phenolics and alkaloids in N benthamianafeedstream foul Protein A resins and are hard to removefrom the target MAb Consequently extra purification stepsare required to remove these secondary metabolites from thefeedstream before loading to Protein A resin [17] Moreoverfrequent cleaning of resins with harsh reagents is necessary toprevent fouling [59]These extra measures complicate down-stream processing shorten the life span of protein A andadd extra capital and operational cost for MAb productionOur results indicate that the ammonium sulfate precipitationstep for MAb purification which is partially responsible for

removing secondarymetabolites from tobacco extract can bebypassed due to the negligible amounts of plant compoundsin the lettuce feedstream [18 34] Thus lettuce extracts con-taining MAbs can be directly loaded onto the Protein Acolumn avoiding resin fouling concerns [18 34] As a resultthe life cycle of Protein A resin is prolonged and the overallproduction cost of MAbs is reduced

Our success in producing functional VLPs and MAbswith commercially produced lettuce demonstrated anotheradvantage of lettuce as a host for large-scale agroinfiltrationSince agricultural and food industries have already estab-lished the infrastructure and technology needed for large-scale lettuce growing and processing they can be rapidlyadapted for the production of RPsThis suggests that biomassproduction could be subcontracted to existing commercialgrowers This will forego the need for capital investment ofpurpose-built biomass facilities but allows access to poten-tially unlimited quantities of inexpensive plant material forlarge-scale manufacturing of RPs Besides lettuce other plantspecies are being explored as hosts for agroinfiltration toallow for the production of RPs with unique properties

4 Conclusions

Thedevelopment of deconstructed viral vectors has reinvigo-rated the field of plant-made RPs and provided a productionplatformwith superior protein yield speed scalability versa-tility safety and cost-saving benefits A recent breakthroughin plant glycoengineering allows plants to produce RPs withtailor-made N-glycans and expands the utility of plants indeveloping biobetters with superior functional and safetyprofilesThe lack of a scalable technology to deliver transgeneinto plant cells was one of the remaining hurdles for the com-mercial application of plant transient systems As discussedin this review this challenge has been overcome effectively byvarious agroinfiltration technologies We believe that furtheroptimization of agroinfiltration technologies will expeditethe acceptance of plant transient expression systems for thecommercial production of a broad range of RPs

Abbreviations

RP Recombinant proteinGMO Genetically modified organismcGMP current good manufacturing practiceTSP Total soluble proteinLFW Leaf fresh weightdpi Days postinfiltrationBeYDV Bean yellow dwarf virusTMV Tobacco mosaic virusPVX Potato virus XTBSV Tomato busy stunt virusVLP Virus-like particleNHL Non-Hodgkinrsquos lymphomaWNV West Nile virusBTV Bluetongue virusMAb Monoclonal antibody


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank members of the Chen Laboratory for theircontributions to the results describedThe authors also ackn-owledge the critical reading of the paper by J CaspermeyerProjects performed by our laboratory in this review weresupported in part by NIH-NIAID Grants U01 AI075549 andR33AI101329 to Q Chen

References

[1] Y Y Gleba D Tuse and A Giritch ldquoPlant viral vectors fordelivery by Agrobacteriumrdquo in Plant Viral Vectors K Palmerand Y Gleba Eds vol 375 of Current Topics in Microbiologyand Immunology pp 155ndash192 Springer Berlin Germany 2014

[2] M-A DrsquoAoust M M-J Couture N Charland et al ldquoThe pro-duction of hemagglutinin-based virus-like particles in plants arapid efficient and safe response to pandemic influenzardquo PlantBiotechnology Journal vol 8 no 5 pp 607ndash619 2010

[3] H M Davies ldquoCommercialization of whole-plant systemsfor biomanufacturing of protein products evolution andprospectsrdquo Plant Biotechnology Journal vol 8 no 8 pp 845ndash861 2010

[4] T V Komarova S Baschieri M Donini C Marusic E Ben-venuto and Y L Dorokhov ldquoTransient expression systems forplant-derived biopharmaceuticalsrdquo Expert Review of Vaccinesvol 9 no 8 pp 859ndash876 2010

[5] A L Rivera M Gomez-Lim F Fernandez and A M LoskeldquoPhysical methods for genetic plant transformationrdquo Physics ofLife Reviews vol 9 no 3 pp 308ndash345 2012

[6] J C Sanford ldquoBiolistic plant transformationrdquo Physiologia Plan-tarum vol 79 no 1 pp 206ndash209 1990

[7] H Klee R Horsch and S Rogers ldquoAgrobacterium-mediatedplant transformation and its further applications to plantbiologyrdquo Annual Review of Plant Physiology vol 38 pp 467ndash486 1987

[8] Z Vaghchhipawala C M Rojas M Senthil-Kumar and K SMysore ldquoAgroinoculation and agroinfiltration simple tools forcomplex gene function analysesrdquoMethods inMolecular Biologyvol 678 pp 65ndash76 2011

[9] Q Chen ldquoExpression and manufacture of pharmaceuticalproteins in genetically engineered horticultural plantsrdquo inTransgenic Horticultural Crops Challenges and Opportunities-Essays by Experts BMou andR Scorza Eds pp 83ndash124 Tayloramp Francis Boca Raton Fla USA 2011

[10] N Grimsley B Hohn T Hohn and R Walden ldquoAgroinfectionan alternative route for viral infection of plants by using the Tiplasmidrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 83 no 10 pp 3282ndash3286 1986

[11] TWroblewski A Tomczak and RMichelmore ldquoOptimizationof Agrobacterium-mediated transient assays of gene expressionin lettuce tomato andArabidopsisrdquo Plant Biotechnology Journalvol 3 no 2 pp 259ndash273 2005

[12] Q Chen M Dent J Hurtado et al ldquoTransient protein expres-sion by agroinfiltration in lettucerdquo inRecombinant Proteins fromPlants R Menassa J Macdonald and I Kolotilin Eds 2014

[13] N Takata and M E Eriksson ldquoA simple and efficient transienttransformation for hybrid aspen (Populus tremula times P tremu-loides)rdquo Plant Methods vol 8 no 1 article 30 2012

[14] X Chen R Equi H Baxter et al ldquoA high-throughput transientgene expression system for switchgrass (Panicum virgatum L)seedlingsrdquo Biotechnology for Biofuels vol 3 article 9 2010

[15] K Leuzinger M Dent J Hurtado et al ldquoEfficient agroinfiltra-tion of plants for high-level transient expression of recombinantproteinsrdquo Journal of Visualized Experiments no 77 Article IDe50521 2013

[16] H Lai and Q Chen ldquoBioprocessing of plant-derived virus-likeparticles of Norwalk virus capsid protein under current GoodManufacture Practice regulationsrdquoPlantCell Reports vol 31 no3 pp 573ndash584 2012

[17] H Lai M Engle A Fuchs et al ldquoMonoclonal antibody pro-duced in plants efficiently treats West Nile virus infection inmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 6 pp 2419ndash2424 2010

[18] J He H Lai C Brock and Q Chen ldquoA novel system forrapid and cost-effective production of detection and diagnosticreagents ofWestNile virus in plantsrdquo Journal of Biomedicine andBiotechnology vol 2012 Article ID 106783 10 pages 2012

[19] Z Huang Q Chen B Hjelm C Arntzen and H Mason ldquoADNA replicon system for rapid high-level production of virus-like particles in plantsrdquo Biotechnology and Bioengineering vol103 no 4 pp 706ndash714 2009

[20] Q Chen and H Lai ldquoPlant-derived virus-like particles as vac-cinesrdquo Human Vaccines and Immunotherapeutics vol 9 no 1pp 26ndash49 2013

[21] Q Chen ldquoVirus-like particle vaccines for norovirus gastroen-teritisrdquo in Molecular Vaccines M Giese Ed pp 153ndash181Springer Vienna Austria 2013

[22] G P Pogue F Vojdani K E Palmer et al ldquoProduction of phar-maceutical-grade recombinant aprotinin and a monoclonalantibody product using plant-based transient expression sys-temsrdquo Plant Biotechnology Journal vol 8 no 5 pp 638ndash6542010

[23] P Roemer L Bortesi D Tiede A Giritch and Y GlebaldquoAgrobacterium for transient transfection of whole plantsrdquoPatent application WO2013149726 A1 E P Office Ed 2013

[24] M C Canizares L Nicholson and G P Lomonossoff ldquoUse ofviral vectors for vaccine production in plantsrdquo Immunology andCell Biology vol 83 no 3 pp 263ndash270 2005

[25] C Lico Q Chen and L Santi ldquoViral vectors for production ofrecombinant proteins in plantsrdquo Journal of Cellular Physiologyvol 216 no 2 pp 366ndash377 2008

[26] A Giritch S Marillonnet C Engler et al ldquoRapid high-yieldexpression of full-size IgG antibodies in plants coinfectedwith noncompeting viral vectorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no40 pp 14701ndash14706 2006

[27] W Phoolcharoen S H Bhoo H Lai et al ldquoExpression of animmunogenic Ebola immune complex in Nicotiana benthami-anardquo Plant Biotechnology Journal vol 9 no 7 pp 807ndash816 2011

[28] H Lai J He J Hurtado et al ldquoStructural and functional charac-terization of an anti-West Nile virus monoclonal antibody andits single-chain variant produced in glycoengineered plantsrdquoPlant Biotechnology Journal vol 12 no 8 pp 1098ndash1107 2014

[29] M Bendandi S Marillonnet R Kandzia et al ldquoRapid high-yield production in plants of individualized idiotype vaccinesfor non-Hodgkinrsquos lymphomardquo Annals of Oncology vol 21 no12 pp 2420ndash2427 2010


[30] E C Thuenemann A E Meyers J Verwey E P Rybickiand G P Lomonossoff ldquoA method for rapid production ofheteromultimeric protein complexes in plants assembly ofprotective bluetongue virus-like particlesrdquo Plant BiotechnologyJournal vol 11 no 7 pp 839ndash846 2013

[31] QChen JHeW Phoolcharoen andH SMason ldquoGeminiviralvectors based on bean yellow dwarf virus for production ofvaccine antigens and monoclonal antibodies in plantsrdquoHumanVaccines vol 7 no 3 pp 331ndash338 2011

[32] Z HuangW Phoolcharoen H Lai et al ldquoHigh-level rapid pro-duction of full-size monoclonal antibodies in plants by a single-vectorDNAreplicon systemrdquoBiotechnology andBioengineeringvol 106 no 1 pp 9ndash17 2010

[33] G L Regnard R P Halley-Stott F L Tanzer I I Hitzeroth andE P Rybicki ldquoHigh level protein expression in plants throughthe use of a novel autonomously replicating geminivirus shuttlevectorrdquoPlant Biotechnology Journal vol 8 no 1 pp 38ndash46 2010

[34] H Lai J He M Engle M S Diamond and Q Chen ldquoRobustproduction of virus-like particles and monoclonal antibodieswith geminiviral replicon vectors in lettucerdquo Plant Biotechnol-ogy Journal vol 10 no 1 pp 95ndash104 2012

[35] F Sainsbury M Sack J Stadlmann H Quendler R Fischerand G P Lomonossoff ldquoRapid transient production in plantsby replicating and non-replicating vectors yields high qualityfunctional anti-HIV antibodyrdquo PLoS ONE vol 5 no 11 ArticleID e13976 2010

[36] BDugdale C LMortimerMKato TA James RMHardingand J L Dale ldquoIn plant activation an inducible hyperexpres-sion platform for recombinant protein production in plantsrdquoThe Plant Cell vol 25 no 7 pp 2429ndash2443 2013

[37] A A McCormick S Reddy S J Reinl et al ldquoPlant-producedidiotype vaccines for the treatment of non-Hodgkinrsquos lym-phoma safety and immunogenicity in a phase I clinical studyrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 105 no 29 pp 10131ndash10136 2008

[38] C Potera ldquoVaccine manufacturing gets boost from tobaccoplants Canada-basedmedicago opens US Facility to exploit itsinfluenza vaccine productionmethodrdquoGenetic Engineering andBiotechnology News vol 32 no 6 pp 8ndash10 2012

[39] Medicago ldquoClinical trials update avian flu vaccinerdquo GeneticEngineering amp Biotechnology News vol 31 no 14 p 61 2011

[40] IconGenetics ldquoIcon Genetics successfully completes Phase Iclinical study with personalized vaccines for treatment of non-Hodgkinrsquos lymphomardquo 2014 httpwwwicongeneticscomhtml5975htm

[41] J He H Lai M Engle et al ldquoGeneration and analysis of novelplant-derived antibody-based therapeutic molecules againstWestNile virusrdquoPLoSONE vol 9 no 3 Article ID e93541 2014

[42] S Hahn A Giritch and Y Gleba ldquoProduction storage and useof cell wall-degrading enzymesrdquo Patent Application E P OfficeEd 2013

[43] W Phoolcharoen J M Dye J Kilbourne et al ldquoA nonrepli-cating subunit vaccine protects mice against lethal Ebola viruschallengerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 51 pp 20695ndash207002011

[44] F Sainsbury and G P Lomonossoff ldquoExtremely high-level andrapid transient protein production in plants without the use ofviral replicationrdquo Plant Physiology vol 148 no 3 pp 1212ndash12182008

[45] G G Olinger Jr J Pettitt D Kim et al ldquoDelayed treatment ofEbola virus infectionwith plant-derivedmonoclonal antibodies

provides protection in rhesus macaquesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 109 no 44 pp 18030ndash18035 2012

[46] L Santi L Batchelor Z Huang et al ldquoAn efficient plant viralexpression system generating orally immunogenic Norwalkvirus-like particlesrdquoVaccine vol 26 no 15 pp 1846ndash1854 2008

[47] J He L Peng H Lai J Hurtado J Stahnke and Q ChenldquoA plant-produced antigen elicits potent immune responsesagainst west nile virus in micerdquo BioMed Research Internationalvol 2014 Article ID 952865 10 pages 2014

[48] D Klein-Marcuschamer P Oleskowicz-Popiel B A Simmonsand H W Blanch ldquoThe challenge of enzyme cost in theproduction of lignocellulosic biofuelsrdquo Biotechnology and Bio-engineering vol 109 no 4 pp 1083ndash1087 2012

[49] D Tuse T Tu and KMcDonald ldquoManufacturing economics ofplant-made biologics case studies in therapeutic and industrialenzymesrdquo BioMed Research International vol 2014 Article ID256135 16 pages 2014

[50] Q Chen and H Lai ldquoPlant-derived monoclonal antibodiesas human biologics for infectious disease and cancerrdquo inPlant-derived Pharmaceuticals Principles and Applications forDeveloping Countries K L Hefferon Ed pp 42ndash75 CABICryodon UK 2014

[51] R Strasser J Stadlmann M Schahs et al ldquoGeneration ofglyco-engineered Nicotiana benthamiana for the productionof monoclonal antibodies with a homogeneous human-like N-glycan structurerdquo Plant Biotechnology Journal vol 6 no 4 pp392ndash402 2008

[52] L Zeitlin J Pettitt C Scully et al ldquoEnhanced potency of afucose-free monoclonal antibody being developed as an Ebolavirus immunoprotectantrdquo Proceedings of the National Academyof Sciences of the United States of America vol 108 no 51 pp20690ndash20694 2011

[53] E C Hayden and S Reardon ldquoShould experimental drugs beused in the Ebola outbreakrdquo Nature 2014

[54] R A L Van Der Hoorn F Laurent R Roth and P J GM De Wit ldquoAgroinfiltration is a versatile tool that facilitatescomparative analyses of Avr9Cf-9-induced and Avr4Cf-4-induced necrosisrdquoMolecular Plant-Microbe Interactions vol 13no 4 pp 439ndash446 2000

[55] A C A Roque C R Lowe and M A Taipa ldquoAntibodiesand genetically engineered related molecules production andpurificationrdquo Biotechnology Progress vol 20 no 3 pp 639ndash6542004

[56] C Hey and C Zhang ldquoProcess development for antibodypurification from tobacco by protein a affinity chromatographyrdquoChemical Engineering and Technology vol 35 no 1 pp 142ndash1482012

[57] V Negrouk G Eisner H-I Lee K Han D Taylor and HC Wong ldquoHighly efficient transient expression of functionalrecombinant antibodies in lettucerdquo Plant Science vol 169 no2 pp 433ndash438 2005

[58] J Li M Chen X-W Liu H-C Zhang F F Shen and G PWang ldquoTransient expression of an active human interferon-beta in lettucerdquo Scientia Horticulturae vol 112 no 3 pp 258ndash265 2007

[59] Q Chen ldquoExpression and purification of pharmaceutical pro-teins in plantsrdquo Biological Engineering vol 1 no 4 pp 291ndash3212008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


However scale-up of RP production by transient expressionposes a bigger challenge than transgenic plants because nogenetically stable seed bank is produced and scale-up is nolonger just a matter of increasing acreage to boost yield Toovercome this challenge a scalable transgene delivery meth-od must be developed for plant transient expression

2 Methods of Transgene Delivery

The method of choice for introducing transgenes into plantsdepends on the expression vector and the host plants Thesemethods include direct delivery by gene gun and indirectdelivery through using Agrobacterium tumefaciens or plantviruses [5]

21 Direct Delivery Methods DNA or RNA can be directlyintroduced into plant cells via a so-called microprojectilebombardmentmethod also known as a gene gun or biolisticsIn this method the transgene is coated onto microgold ortungsten particles and fired into plant cells ballistically [6]The advantage of this method resides in its versatility and abroad range of susceptible plants It can be used to delivertransgene to both nuclear and chloroplast genomes At leastin theory effective transgene delivery by biolistics is vectorindependent and can be applied to any plant host species [5]

22 Indirect GeneDeliveryMethods Indirect transgene deliv-ery exploits the ability of plant virus or certain pathogenicagrobacteria species (eg A tumefaciens) that naturallytransfer their genome (plant virus) or part of their tumorinducing plasmid (Ti plasmid) DNA (T-DNA) into plant cells(Agrobacterium) A transgene can enter plant cells as a by-product of viral infection if cloned into the full viral genomeInfection can be facilitated by rubbing plant tissue with atransgene carrying infectious viral particles or viral nucleicacids [3] However this method is only applicable to virusesor plant hosts that are susceptible to mechanical inoculationbut not to those that require specialized insects for viral trans-mission

Ti plasmids of Agrobacterium can be modified into deliv-ery vectors by replacing pathogenic genes in T-DNA withtransgenes transgene transfer from agrobacteria to plant cellsis accomplished through the natural interaction between Atumefaciens and its plant hosts [5] In contrast to biolisticsgene delivery by A tumefaciens requires the cloning of trans-gene into a modified Ti plasmid and is restricted to dicotyle-donous and a limited number of monocotyledonous plants[7] However delivery by Agrobacterium generally offersbetter efficiency transgene expression and inheritance thanbiolistics [5] It is speculated that Agrobacterium-based deliv-ery is advantageous because transgene copy numbers andintegration into the genome are better controlledThe coevo-lution ofAgrobacterium and its plant hostsmay favor the inte-gration of transgenes into genomic loci that are transcrip-tionally active which leads to its high level of expression[7] In transient expression biolistics often cause severetissue damage and effectively reduce the available biomass forRP production making indirect delivery by Agrobacterium

a preferredmethod As a result anAgrobacterium-based genedelivery via agroinfiltration has become the favorable genedelivery method for transient expression in plants [7 8]

3 Agroinfiltration for Expression ofRecombinant Proteins

On a per cell basis the yield of a RP is usually higher in tra-nsient expression than that in transgenic plants [4] Theelimination of the ldquoposition effectrdquo is responsible for this imp-rovement as the transgene is no longer randomly insertedinto genomic areas with variable transcriptional activity [9]However earlier gene delivery methods consisted of soakingleaf pieces in Agrobacterium culture in which only the celllayer on the edges may receive the transgene This limits theefficiency and scalability of transient systems

Agroinfiltration was invented to overcome this challengeBecause up to one-third of the leaf volume is intercellularspace it is possible to replace the air in these cavities witha suspension of Agrobacterium [1] Thus transgene-carryingagrobacteria are actively delivered into the intercellular spaceof the leaf tissue by agroinfiltration allowing for the effectiveaccess of agrobacteria to most leaf cells and making thetransfer of T-DNA a highly efficient event [8 10]

31 Application and Scalability of Agroinfiltration The sim-plest method of agroinfiltration is syringe infiltration In thismethod transgene-carryingAgrobacterium in the infiltrationmedium is injected into the leaf with a needleless syringe(Figure 1(a)) Syringe infiltration offers the flexibility of intro-ducing multiple transgene constructs into different areasallowing multiple assays to be performed on a single leaf [8]Thus it has been used for studying plant-pathogen interac-tions abiotic stresses gene functional analysis protein local-ization and protein-protein interactions [8] Syringe infiltra-tion also has numerous applications for RP production Forexample it can be used to quickly examine the expressionlevel of a RP under established conditions (Figure 1(b)) Iffurther optimization of expression is necessary its flexibilitypermits a quick assessment of various factorsrsquo effects on theyield expression kinetics and toxicity of the target proteinThese factors include concentrations of Agrobacterium cul-ture different expression vectors organelles favorable for RPaccumulation and the requirement for silencing suppressorsOnce optimized syringe infiltration can also be used to infil-trate several entire plants to rapidly obtain sufficient amounts(milligram level) of RPs for biochemical characterization andpreclinical functional studies as well as for developing puri-fication schemes Despite these utilities only a few plantspecies are naturally amenable to syringe infiltration and theprospect of its scalability is highly limited [11]

A scalable agroinfiltration technology will enhance thecompetitiveness of transient expression systems with the tra-ditional cell culture-based platforms for RP production Anagroinfiltration method using a vacuum chamber was devel-oped for such purposes [5] A prototype apparatus routinelyused in the laboratory is best illustrative of the vacuumchamber method First aerial parts of plants are submerged


(a) (b)

(c) (d)







(a) (b)












Rep

LB RB

LIR LIRSIRGOI
























4 Conclusions


Abbreviations





Acknowledgments


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)







(a) (b)












Rep

LB RB

LIR LIRSIRGOI
























4 Conclusions


Abbreviations





Acknowledgments


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)












Rep

LB RB

LIR LIRSIRGOI
























4 Conclusions


Abbreviations





Acknowledgments


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Rep

LB RB

LIR LIRSIRGOI
























4 Conclusions


Abbreviations





Acknowledgments


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















4 Conclusions


Abbreviations





Acknowledgments


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article gene delivery into plant cells for...

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