FERMENTATION OF RECOMBINANT E. Coli
FERMENTATION OF RECOMBINANT E. coliTOP10F’/pPROEX HTa/BmSXP TO ACHIEVEHIGH YIELD OF BIOMASS ANDRECOMBINANT ANTIGENFOR DIAGNOSTIC APPLICATIONKHOO TENG KEWUNIVERSITI SAINS MALAYSIA2011
FERMENTASI E. coli REKOMBINANTOP10F’/pPROEX HTa/BmSXP BAGIMENDAPATKAN HASIL BIOJISIM DANANTIGEN REKOMBINAN YANG TINGGIUNTUK KEGUNAAN DIAGNOSISolehKHOO TENG KEWThesis yang diserahkan untukmemenuhi keperluan bagiIjazah Sarjana SainsJulai 2011
FERMENTATION OF RECOMBINANT E. coliTOP10F’/pPROEX HTa/BmSXP TO ACHIEVEHIGH YIELD OF BIOMASS ANDRECOMBINANT ANTIGENFOR DIAGNOSTIC APPLICATIONbyKHOO TENG KEWThesis submitted in fulfillment of the requirementsfor the Degree ofMaster of ScienceJuly 2011
ACKNOWLEDGEMENTSFirst and foremost, I thank my supervisor Dr. Amutha Santhanam, for hercontinuous support throughout this research project. Dr. Amutha was always there tolisten and to give advice. My frequent pestering with fermentation related questionshave all been adequately and patiently answered with a probe to think further. Shetaught me the effective means of expressing my ideas. She has also showed medifferent ways to approach a research problem and the need to be persistent toaccomplish any goal.My heartiest appreciation also goes all the way to Prof. Dr. Rahmah Noordin,my co-supervisor, whose encouragement, supervision and support from thepreliminary to the concluding level enabled me to develop an understanding of thediagnostic issues with lymphatic filariasis. Prof. Rahmah had contributed therecombinant E. coli TOP10F’/pPROEX HTa/BmSXP recombinant strain andpanLF RapidTM diagnostic kit which set up the base foundation that further madepossible the progressive continuation of this project. Her generosity in sharing herscientific knowledge, wisdom and experience in the molecular biology aspect of thestudy has allowed me to delve deeper in terms of understanding the objectives andworkflow of this project. Prof. Rahmah has also supervised me in the immunoassaypart of the work and granted me access to her lymphatic filariasis serum bank whichenabled the analytical study for the quality of the BmSXP recombinant antigenproduced. Together, we went through a lot of endearing efforts during the meticulousediting of this thesis, nevertheless, it is her motivation, along with her professionalhelp and guidance that has geared me up and given the polishing touch in this thesisii
write-up and presentation. I have truly explored the ideas, organization, requirementsand development of writing a good thesis under her wing of guidance.Also included in this long list, the helpful colleague, Pn. Norshahida Arifin,who never gets tired of my constant advice-seeking attitude and her generosity in thesharing of technical knowledge that have assisted me throughout the course. Hertremendous contributions of past projects experiments have paved a smoothbeginning that have brought my research project to light. Not forgot to mention, Dr.Surash Ramanathan from Centre for Drug Research, USM, who offered his expertiseand HPLC facility in quantifying the acetic acid concentration.I am also indebted to the cooperation and advices given by the lecturers ofINFORMM namely Prof. Asma, Prof. Rusli, Prof. Prabha, Prof. Phua, Dr. Chen andDr. Khoo. Thanks also to Pn. Sabariah, En. Zulkarnian, Pn. Nurulhasanah and En.Nyambar, also not forgetting the remarkable support from friends and colleaguesnamely En. Lee, Cik Tan and Pn. Yana. My hearty appreciation also goes to theadministrative department of INFORMM namely, En. Irwan, Cik. Noroslinda, En.Azam, En. Azzizi, Cik Kammini and Pn. Asma. Last but not least, to all the scientificofficers and scientific assistants, my fellow comrades who fought the same battle andmy family members, whom direct or indirect involvement and endless support aswell as guidance that led to the completion of this dissertation.This research project was funded by a short term grant from Universiti SainsMalaysia, project number 304/CIPPM/638108. The fermentation and downstreamprocessing facilities were funded by Prof. Rahmah’s research grant from EuropeanCommission, project number 304/CIPPM/650394. The tenure throughout mypostgraduate studies was also covered by the prestigious USM Fellowship.iii
TABLE OF CONTENTSAcknowledgementiiTable of ContentsivList of TablesxList of FiguresxiiiList of Abbreviations and SymbolsxviiAbstrakxxiiAbstractxxvCHAPTER I - INTRODUCTION1.1Introduction to filariasis11.1.1Lymphatic filariasis11.1.2Wuchereria bancrofti21.1.3Transmission and life cycle51.1.4Clinical manifestation61.1.5Diagnosis of lymphatic filariasis81.1.6Elimination of filariasis111.1.7panLF Rapid 12E. coli nt protein production in E. coli181.2.3Small scale fermentation using shake flask culture221.2.4Large scale fermentation using bioreactor231.2.5Mode of fermentation271.2.5.1Batch fermentation27iv
1.2.5.2Fed-Batch fermentation281.2.5.3Continuous fermentation291.2.6Challenges in fermentation of recombinant E. coli301.2.6.1Secretion of acetic acid301.2.6.2Improving efficiency311.2.7Downstream processing341.2.7.1Cell disruption341.2.7.2Affinity chromatography381.2.8Quality of the recovered target protein421.2.8.1Western blot analysis421.2.8.2ELISA assays431.3Statement of problem431.4Objectives of the study441.5Writing style45CHAPTER II - MATERIALS AND METHODS2.1Common methodology462.1.1Material weighing462.1.2pH determination462.1.3Optical density determination462.1.4Sterilization462.2Bacterial strain472.3Growth and maintenance of bacterial strain512.4Experimental layout512.5Small scale studies in shake flasks55v
2.5.1Varying the initial inoculum volume552.5.2Varying the culture medium572.5.3Varying the pH582.5.4Varying the agitation rate582.5.5Varying the initial glucose concentration582.5.6Varying the inducer concentration592.5.7Varying the induction time592.5.8Varying the post-induction temperature602.6Batch and fed-batch fermentations up scaling602.6.1Preparation of inoculum for bioreactor612.6.2Fermentation process set-up632.6.3Fermentation parameter632.7Batch fermentation642.7.1Varying the seed inoculum642.7.2Varying the media64Fed-batch fermentation652.82.8.1Varying the mode of feeding method652.8.2Varying the initial inoculum volume682.8.3Varying the induction parameter682.9Analytical methods692.9.1Dry cell weight estimation692.9.2Viable cell concentration estimation692.9.3Residual glucose concentration quantification702.9.4Acetic acid concentration quantification702.9.5Plasmid stability70vi
2.10Protein purification protocol712.10.1Sample preparation for protein recovery2.10.2Small-scale Fast Performance of Liquid Chromatography (FPLC) proteinpurification - Manual spun column method2.10.37172Large-Scale Fast Performance of Liquid Chromatography (FPLC) proteinpurification – Automated AKTA prime system732.10.3.1 Preparation of the AKTA Prime machine and HisTrap column 732.112.10.3.2 Binding, washing and elution of histidine-tagged protein732.10.3.3 Washing the HisTrap column and AKTATMprime machine74Optimization of protein purification752.11.1Varying the imidazole concentration in lysis buffer752.11.2Varying the imidazole concentration in wash buffer762.11.3Varying the volume of washing buffer762.11.4Varying the salt concentration in buffers solution772.12Protein analysis772.12.1Bio-Rad protein assay reagent (Bradford assay)772.12.2SDS-PAGE782.12.3Western blot802.12.4ELISA82CHAPTER III – RESULTS AND DISCUSSION: IMPROVEMENT OFFERMENTATION CONDITION AND INDUCTION STRATEGY3.1Overview843.1.1Inoculum volume843.1.2Culture medium85vii
3.1.3Nutrient feeding strategy883.1.4IPTG induction strategy903.23.1.4.1IPTG induction concentration913.1.4.2IPTG induction time923.1.4.3Continuous IPTG induction93Small scale studies in shake flask culture943.2.1Varying the initial inoculum volume943.2.2Varying the medium963.2.3Varying the pH1013.2.4Varying the agitation rate1033.2.5Varying the initial glucose concentration1053.2.6Varying the inducer concentration1073.2.7Varying the induction time1103.2.8Varying the post-induction temperature1123.2.9Batch culture under optimized parameters1153.3Batch fermentation1163.3.1Varying the initial glucose concentration1163.3.2Varying the seed inoculum preparation1173.3.3Varying the media120Fed-batch fermentation1213.43.4.1Feeding strategies1213.4.2Specific growth rate (µ)1253.4.2.1Feed source without additional supplementation3.4.2.2Feed source with additional 5% yeast extract supplementation 1293.4.3Varying the induction parameterviii125131
3.4.3.1Varying the IPTG concentration1323.4.3.2Varying the induction time1343.4.3.3Improvement of induction strategy1363.4.4Monitoring of acetic acid accumulation1413.4.5Monitoring of plasmid stability146CHAPTER IV - RESULTS AND DISCUSSION: OPTIMIZATION OFPROTEIN PURIFICATION4.1Overview1494.2Imidazole concentration in lysis buffer1514.3Imidazole concentration in wash buffer1534.4Volume of wash buffer and salt concentration in wash buffer1554.5Purity of the recovered BmSXP recombinant protein1574.6Quality of the recovered BmSXP recombinant antigen1614.6.1Western blot analysis1614.6.2ELISA167CHAPTER V - SUMMARY173References183Appendix A – Media and Solutions Protocol200Appendix B – Determination of Fermentation Kinetic Parameters209Appendix C – Preparation of Calibration Graph210Appendix D – Cost Savings Calculation215List of Publications217ix
LIST OF TABLESPagesTable 1.1Advantages and disadvantages of protein production at21different compartments of E. coliTable 2.1Medium composition for small scale studies in shake flasks56Table 2.2Medium composition for batch and fed-batch fermentations62up scalingTable 2.3Feed solution composition67Table 2.4Kinetic parameter values applied during the fed-batch67fermentationTable 2.5Recipes for polyacrylamide resolving and stacking for 2 small79gels (0.75 mm thick gel)Table 3.1Effect of different initial inoculum volume on growthcharacteristicsofrecombinant95E. coliTOP10F’/pPROEX HTa/BmSXPTable 3.2Effect of different medium on growth characteristics of97recombinant E. coli TOP10F’/pPROEX HTa/BmSXPTable 3.3Effect of different initial pH on growth characteristics of102recombinant E. coli TOP10F’/pPROEX HTa/BmSXPTable 3.4Effect of different agitation rate on viability of recombinant104E. coli TOP10F’/pPROEX HTa/BmSXPTable 3.5Effect of different initial glucose concentration on growthcharacteristicsofrecombinantTOP10F’/pPROEX HTa/BmSXPxE. coli106
Table 3.6Effect of different seed inoculum preparative methods and119inoculum age on growth characteristics of recombinant E. coliTOP10F’/pPROEX HTa/BmSXPTable 3.7Growth performance of recombinant E. coli in three different124feeding strategies of fed-batch fermentationTable 3.8Growth performance of recombinant E. coli in exponential127feeding strategy at varying specific growth rate (µ) duringfed-batch fermentationTable exponential feeding strategy at 0.20 h 1 µ during fed-batchfermentation at various harvesting timeTable 3.10Effect of different yeast extract supplementation 30E. coliTOP10F’/pPROEX HTa/BmSXPTable 3.11Growth performance of recombinant E. coli in exponential133feeding strategy at varying IPTG concentration duringfed-batch fermentationTable 3.12Growth performance of recombinant E. coli in exponential135feeding strategy at varying IPTG induction time duringfed-batch fermentationTable 3.13Growth performance of recombinant E. coli in exponential139feeding strategy at varying IPTG induction strategy duringfed-batch fermentationTable 3.14Plasmid stability performance of recombinant E. coli inoptimized exponential feeding rate of µset 0.20 h-1xi147
Table 3.15Paired sample t-test statistical analysis to assess the plasmid147stability performance of recombinant E. coliTable 4.1Comparison of BmSXP recombinant protein recovery154concentrations at five different imidazole increasing gradientconcentrations in wash bufferTable 4.2OD values of positive serum samples employed for evaluation169of sensitivity of ELISA using sera of patients infected withW. bancrofti infection (bancroftian filariasis)Table 4.3OD values of positive serum samples employed for evaluation170of sensitivity of ELISA using sera of patients infected withB. malayi infection (brugian filariasis)Table 4.4OD values of negative serum samples employed for171evaluation of specificity of ELISA using sera of patientsinfected with other parasitic helminthes and protozoaTable 4.5OD values of negative serum samples employed for172evaluation of specificity of ELISA using sera of healthyindividualsTable 4.6Result for the t-test analysis of positive and negative serumxii172
LIST OF FIGURESPagesFigure 1.1Lymphatic filariasis endemic areas3Figure 1.2Relative sizes of mf developmental stages that occur within4compatible mosquito hosts: Cyclodevelopmental transmissionFigure 1.3The life cycle of W. bancrofti7Figure 1.4Elephantiasis (lymphoedema) of lower limb7Figure 1.5panLF Rapid rapid immunochromatographic diagnostic kit14for the detection of both bancroftian and brugian filariasisinfectionFigure 1.6Strategies for the production of recombinant proteins in20E. coliFigure 1.7Schematic diagram of a stirred-tank reactor (STR)24Figure 1.8Instrument setup for fed-batch fermentation26Figure 1.9Chemical structures of histidine and imidazole41Figure 2.1Map of BmSXP recombinant plasmid48Figure 2.2Gene sequence map of pBmSXP (BmSXP gene located at49124-585 bp)Figure 2.3A Upstream (fermentation) flowchart of the experimental layout53Figure 2.3B Downstream (purification) flowchart of the experimental54layoutFigure 3.1ThegrowthprofileofrecombinantE. coliTOP10F’/pPROEX HTa/BmSXP in LB medium, inoculatedwith various volume of initial inoculumxiii95
Figure 3.2Effect of different medium on total cellular protein production99of recombinant E. coli TOP10F’/pPROEX HTa/BmSXPbased on various culture harvesting timeFigure 3.3Effect of different IPTG concentrations on growth profile of108recombinant E. coli TOP10F’/pPROEX HTa/BmSXP andexpression of BmSXP recombinant proteinFigure 3.4Effect of different induction times on growth profile of111recombinant E. coli TOP10F’/pPROEX HTa/BmSXP andBmSXP recombinant proteinFigure 3.5Effect of different post-induction temperatures on growthprofileof113E. colirecombinantTOP10F’/pPROEX HTa/BmSXP and BmSXP recombinantprotein expressionFigure 3.6SDS-PAGE analysis of the pooled fractions collected114(fractions 3-10) from 37oC and 30oC post-inductiontemperature cell lysate after small-scale FPLC proteinpurificationFigure 3.7ThegrowthprofileofTOP10F’/pPROEX HTa/BmSXPrecombinantduringE. colicultivationinmodified TB medium inoculated with 10% v/v workingvolume concentrated seed inoculum used as whole with aninoculum age of 8-hxiv119
Figure 3.8ThegrowthprofileofrecombinantE. coli122TOP10F’/pPROEX HTa/BmSXP during cultivation in ated seed inoculum used as whole with an inoculumage of 8-hFigure 3.9Comparison of cell mass in three different feeding strategies124of fed-batch fermentationFigure 3.10Comparison of cell mass in different feeding rate (µ 0.10,1280.15, 0.20, 0.25, and 0.30 h-1) and feeding strategy(µ 0.20 h-1 supplemented with 5.0% yeast extract) offed-batch fermentationFigure 3.11Comparison of cell mass in three different induction timings135(bacterial phase) of fed-batch fermentationFigure 3.12Comparison of cell mass in four different induction strategies139of fed-batch fermentationFigure 3.13Profile of acetate production in different feeding rate142(µset 0.10, 0.15, 0.20, 0.25, and 0.30 h-1)Figure 3.14Profile of acetate production in optimized feeding rate of142µset 0.20 h-1Figure 3.15Fed-batch fermentation MFCS/Win data of recombinant145E. coli cultured in optimized exponential feeding rate ofµset 0.20 h-1Figure 4.1Comparison of BmSXP recombinant protein recoveryconcentrations at four different imidazole concentrations inlysis bufferxv152
Figure 4.2Comparison of BmSXP recombinant protein concentrations at156two different salt concentrations in wash buffer andtwo different volumes of wash bufferFigure 4.3Chromatogram output of the purification at 30 mM imidazole156concentration in wash buffer and 10 CVFigure 4.4Chromatogram output of the elution step showing the elution158of the target protein in concurrent with the fractions collectionFigure 4.5SDS-PAGE analysis of the pooled fractions collected159(fractions 5-30) from performing laboratory scale proteinpurification with 20, 30, 40, 45 and 50 mM imidazole in washbuffer, with 10 CV and 300 mM salt concentration in washbuffer set as constantsFigure 4.6Western blot analysis of the recovered BmSXP recombinant163antigen using patients’ serum samples diagnosed withW. bancrofti infectionFigure 4.7Western blot analysis of the recovered BmSXP recombinant164antigen using patients’ serum samples diagnosed withB. malayi infectionFigure 4.8Western blot analysis of the recovered BmSXP recombinant165antigen using patients’ serum samples diagnosed with ca,Toxocariasis)Figure 4.9Western blot analysis of the recovered BmSXP recombinantantigen using healthy individuals’ serum samplesxvi166
LIST OF ABBREVIATIONS AND benzthiazoline-6-sulphonic acid)4Aluminium chloride hexahydrate5Air-lift fermenter6Beta7Brugia malayi8Base pair9Bovine serum albumin10Celsius11Calcium chloride dihydrate12Colony forming units13Centimeter14Cobalt (II) chloride hexahydrate15Cut off valueCOV16Central Processing UnitCPU17Carbon-source18Copper (II) chloride dihytrate19Column volumeCV20DaltonDa21Digital Control UnitDCU22Diurnal subperiodicDSP23Dry cell weightABTSAlCl3.6H2OALFβB. .2H2ODCWxvii
24Double-distilled waterddH2O25Dissolved oxygen26Escherichia coli27For example28Enzyme-linked immunosorbent assayELISA29Iron (II) sulphateFeSO430Fast performance liquid chromatographyFPLC31Gravityg32Gramg33Global Alliance to Eliminate Lymphatic FilariasisGAELF34Global Programme to Eliminate Lymphatic FilariasisGPELF35Hour36Sulphuric acidH2SO437Boric acidH3BO338High cell density cultureHCDC39Histidine40High performance liquid chromatography41Horseradish peroxidase42Hertz43Immunoglobulin E44Immobilized metal affinity chromatography45Institute for Research in Molecular Medicine46Isopropyl β-D-1-thiogalactopyranoside47kilo Dalton48diPotassium hydrogen phosphateDOE. viii
49Potassium dihydrogen B53Lymphatic filariasisLF54Specific growth rateµ55Maximum specific growth rate56Molar57Multiple cloning sites58Millimolar59Mass drug administration60Microfilariae61Multi fermenter control system62Microgramµg63Milligrammg64Miligram per gram dry cell weightmg.g DCW-165Magnesium sulphate lilitermL69Micrometerµm70Millimetermm71Manganese sulphate72Megapascal73Malaysian ix
74Nocturnal periodicNP75Nocturnal subperiodicNSP76Sodium chlorideNaCl77Sodium hydrogen phosphateNaH2PO478Sodium molybdate dihydrateNa2MoO4.2H2O79Ammonium chlorideNH4Cl80Ammonium sulphate(NH4)2SO481Nickel82Nickel ions83Nickel- nitrilotriacetic acid84Nanometer85Nitrogen-source86Optical density87Open reading frame88ProductP89Percentage%90Phosphate buffered saline91Personal computerPC92Pagepg93Pounds per square inchpsi94Rotations per minute95Super broth96Single-distilled water97Sodium dodecyl sulphate polyacrylamide gelNiNi2 xSDS-PAGE
98Stirred-tank reactorSTR99Terrific broth100Tris-buffered saline101Tris-buffered saline Tween 20102Tricarboxylic acidTCA103Total cell proteinsTCP104Universiti Sains Malaysia105Volt106Volume per volume per minute107Wuchereria bancrofti108World Health Organization109Timesx110BiomassX111Yield coefficient of product from substrateTBTBSTBS-TUSMVvvmW. bancroftiWHOY P/S(Product yield)112Yield coefficient of product from biomassY P/X(Overall specific productivity)113Yield coefficient of biomass from substrateY X/S(Biomass yield)114Zinc sulphate heptahydrateZnSO4.7H2Oxxi
FERMENTASI E. coli REKOMBINANTOP10F’/pPROEX HTa/BmSXP BAGI MENDAPATKAN HASILBIOJISIM DAN ANTIGEN REKOMBINAN YANG TINGGIUNTUK KEGUNAAN DIAGNOSISABSTRAKpanLF Rapid merupakan satu ujian pantas pengesanan antibodi IgG4berdasarkan pada pengesanan antibodi anti-filarial IgG4 yang bertindak balas denganantigen rekombinan B. malayi, BmR1 dan BmSXP. Kit diagnostik ini adalah sangatberguna untuk pengesanan limfatik filariasis (LF), terutamanya dalam membantuWHO dalam aktiviti sertifikasi dan pengawasan pasca-pemberian ubat secarabesar-besaran selari dengan usaha Program Penghapusan LF Sedunia atau ‘GlobalProgramme to Eliminate Lymphatic Filariasis’. Pengeluaran kit ujian ini telahmenerima permintaan yang ketara di pasaran, maka peningkatan penghasilan ke skalabesar dan peningkatan efisiensi penulenan adalah perlu untuk meningkatkan kadarpengeluaran dan juga mengurangkan kos pengeluaran secara besar-besaran. Dalamkajian ini hasil BmSXP antigen rekombinan telah dimaksimumkan melaluipenghasilan biomass yang tinggi dengan menggunakan kultur sekelompok di dalambioreaktor, dan tahap pemulihan protein sasaran ini telah dioptimumkan rekombinan(TOP10F’/pPROEX HTa/BmSXP) pada awalnya telah dioptimumkan dalamfermentasi berskala kecil dengan menggunakan kelalang goncang di mana iamenghasilkan 4.2 g.L-1 dan 0.576 mg.g DCW-1 antigen rekombinan BmSXP. Prosesxxii
penaikkan-skala kemudian dijalankan dengan menggunakan kaedah fermentasikultur sekelompok di mana sel ditumbuhkan di dalam media kaldu Terrifc brothterubahsuai dan glukosa disuapkan secara eksponen pada kadar yang terkawalmenggunakan ‘Multifermenter Control Software’ (MFCS) untuk suapan secaraautomatik. Dengan mempelbagaikan strategi suapan kadar pertumbuhan spesifik (µ)dan strategi induksi, hasil biomass sebanyak 19.43 g.L-1 dan 11.16 mg.g DCW-1antigen rekombinan BmSXP telah diperolehi hasil daripada strategi suapan secaraeksponen pada µ sebanyak 0.20 h-1, dan dengan aruhan tunggal 1 mM IPTG padaakhir fasa log pertengahan pertumbuhan bakteria. Selain itu juga, dapat dilihatbahawa pada kadar suapan ini, pekali fermentasi hasil produktiviti (YP/X), hasilbiomass (YX/S) dan hasil produk (YP/S) adalah tinggi. Strategi ini telah berjayamengawal pengumpulan produk rencatan asid asetik di bawah tahap rencatanpertumbuhan yang dilaporkan sebanyak 2 g.L-1 dan kestabilan plasmid didapatiberada dalam keadaan baik. Antigen rekombinan BmSXP kemudian ing)denganmenggunakankromatografi afiniti tidak bergerak. Demi meningkatkan keberkesanan prosespenulenan, pelbagai isipadu penimbal basuhan, kepekatan imidazol dan garamdilakukan. Didapati kepekatan garam pada 300 mM NaCl dan 30 mM imidazolmemberikan hasil terbaik, dan bersama dengan 10 isipadu penimbal basuhan telahmemberikan hasil antigen rekombinan BmSXP yang tertinggi dengan ketulenan yangbaik. Tindak balas imuno dari antigen rekombinan BmSXP yang dihasilkanmenunjukkan ia adalah 100% sensitif dan spesifik apabila diuji dengan ELISA danPemblotan Western menggunakan sampel serum daripada 32 pesakit LF (16Wuchereria bancrofti, 16 Brugia malayi) dan 32 serum kawalan yang lain (16penyakit nematoda yang lain, 16 individu sihat). Keseluruhan pengeluaran proteinxxiii
sasaran dapat ditingkatkan hampir 20-kali ganda berbanding dengan kaedahpengkulturan konvensional di dalam kelalang. Kesimpulannya, kajian ini telahmemberikan kaedah yang lebih baik, lebih efisien dan menjimatkan kos untuk prosespengeluaran dan penulenan protein rekombinan BmSXP.xxiv
FERMENTATION OF RECOMBINANT E. coliTOP10F’/pPROEX HTa/BmSXP TO ACHIEVE HIGH YIELDOF BIOMASS AND RECOMBINANT ANTIGEN FORDIAGNOSTIC APPLICATIONABSTRACTpanLF Rapid is a rapid IgG4 antibody detection test which is based on thedetection of anti-filarial IgG4 antibodies that react with recombinant B. malayiantigens, BmR1 and BmSXP. This diagnostic kit is very useful for the detection oflymphatic filariasis (LF), especially in assisting the WHO on its certification andsurveillance activities of post-mass drug administration that is in relation to its‘Global Programme to Eliminate Lymphatic Filariasis’ effort. The production of thistest kit has received a significant demand in the market, hence there is a need to upscale the production of the recombinant antigens and increase the purificationefficiency in order to increase the production rate and also reduce the cost ofproduction. In this study the yield of BmSXP recombinant antigen was maximized byachieving high biomass yield using fed-batch culture in a bioreactor, and therecovery rate of the protein of interest was optimized in the downstream X acteriasmall-scalefermentation using shake flask culture where it yielded 4.2 g.L-1 and0.576 mg.g DCW-1 of BmSXP. The up-scaling process was then performed using fedbatch fermentation where cells were grown in modified Terrific broth medium andxxv
glucose was fed exponentially at a controlled rate using Multifermenter ControlSoftware (MFCS) for automated feeding. Varying an assortment of feedingstrategies, specific growth rate (µ) and induction strategies, biomass concentration of19.43 g.L-1 and 11.16 mg.g DCW-1 of BmSXP were obtained based on exponentialfeeding strategy at µ of 0.20 h-1, and with 1 mM single pulse IPTG induction at thelate-log phase of the bacterial growth curve. It was also observed that at this feedingrate, the fermentation yield coefficients of overall specific productivity (YP/X),biomass yield (YX/S) and product yield (YP/S) were high. This strategy hassuccessfully controlled the accumulation of acetic acid by-inhibitory product belowthe reported growth inhibitory level of 2 g.L-1 and plasmid stability was found to begood. The BmSXP recombinant antigen was then purified under non-denaturingconditions using immobilized metal affinity chromatography. In order to increase theefficiency of the purification process, various volumes of wash buffer, imidazole andsalt concentrations were performed. Salt at 300 mM and imidazole at 30 mM werefound to be the best concentrations, and along with 10 column volumes of washingbuffers gave the best yield of BmSXP recombinant antigen while achieving sufficientpurity. Immunoreactivity of the recovered BmSXP recombinant antigen was found tobe 100% sensitive and specific when tested with ELISA and Western blot usingserum samples from 32 LF patients (16 Wuchereria bancrofti, 16 Brugia malayi) and32 other control sera (16 other nematode disease, 16 healthy individuals). The overallproduction of the target protein was improved to almost 20-fold compared to theconventional flask cultivation method. In conclusion, this study provided animproved, more efficient and cost-saving method for the production and downstreamprocessing of BmSXP recombinant protein.xxvi
CHAPTER IINTRODUCTION1.1Introduction to filariasis1.1.1Lymphatic filariasisLymphatic filariasis (LF) or elephantiasis as commonly known, is a parasitic diseasecaused by thread-like filarial nematodes or round worms that live in the humanlymphatic system. LF is mainly caused by three species of filarial nematodes namelyWuchereria bancrofti (W. bancrofti), Brugia malayi (B. malayi) and Brugia timori.This disease is widespread throughout countries located within the equator band,namely the tropical and sub-tropical regions of the world, such as Asia, Africa,Central and South America. An estimated 1.3 billion people around the world(approximately 19% of world population) are at risk of LF infection. Southern andSoutheast Asian regions have by far the greatest number of people (891 million) atrisk for LF (accounting for 68% globally), out of which 454 million people at risk arein India alone. Tropical Africa represents the second largest number of people at risk,estimated at 382 million in 2007 (30% globally). Currently over 120 million peoplein at least 83 countries are already infected, with more than 51 million in chronicstage whereby they have been incapacitated or disfigured with swollen breasts(lymphoedema) and genitals (hydrocele) or swollen limbs with thickened, hard,rough and fissured skin, a condition known as Elephantiasis (Michael & Bundy,1997; Lindsay & Thomas, 2000; Muturi et al., 2008; WHO, 2008; GAELF, 2010). Inaddition to the overt abnormalities, internal damage to the kidneys and lymphaticsystem is a common and hidden problem (Srivastavaa et al., 2010). The economic1
impact of LF is significant as it is one of the world’s most disabling and disfiguringdiseases. This disease strikes poverty ridden and underdeveloped countries, hence itis also known as the disease of poverty.In 1998, the World Health Organization (WHO) has identified lymphatic filariasis tobe one of the six infectious diseases that has the potential to be eliminated as a publichealth problem (WHO, 1998; Ottesen et al., 2008). In response to this, GlobalProgramme to Eliminate Lymphatic Filariasis (GPELF) was initiated in year 2000with two major objectives to achieve. Firstly is to interrupt transmission of theparasite and the other objective is to provide care for those who suffer thedevastating clinical manifestations of the disease (morbidity control). The ultimateambitious goal of this program is to relegate LF from the world as non-public healthpriorities by year 2020 (Addis & Brady, 2007).1.1.2Wuchereria bancroftiBancroftian filariasis is caused by W. bancrofti infection and it is responsible for90% (115 million) of all LF infections. W. bancro
inoculum age on growth characteristics of recombinant E. coli TOP10F'/pPROEX HTa/BmSXP 119 Table 3.7 Growth performance of recombinant E. coli in three different feeding strategies of fed-batch fermentation 124 Table 3.8 Growth performance of recombinant E. coli in exponential feeding strategy at varying specific growth rate (µ) during
examined for their potential to produce recombinant proteins in high titres. A comprehensive overview of all E. coli strains used in recombinant protein production processes and their characteristics is give n in (Waegeman & Soetaert, 2011). Although E. coli B and E. coli K12 strains are equally used as host for recombinant protein production (47%
2.4.2. Feeding strategy during fermentation 32 2.4.3. Induction strategy and effect of oxygen during fermentation 33 2.4.4. Scale up of fermentation process 35 2.5. Purification strategies for recombinant proteins 37 2.5.1. Recombinant protein as inclusion bodies 37 2.5.2. Inclusion body formation, isolation and solubilization 38 2.5.3.
most favourable E. coli strain for recombinant protein production (Ko et al., 2010; Ryu et al., 2010; Striedner et al., 2010). Many different strategies have been applied to increase recombinant protein formation and decrease acetate formation in E. coli K12 strains including optimisation of the bioprocess
B. Hindgut fermentation digestive system (example: horse) *Fermentation compartment Figure 2. Digestive tract for A) foregut fermentation and B) hindgut fermentation. Note the location of the fermentation compartment is the rumen in cattle, and it occurs before the small and large intestines (i.e., foregut). Note the location of the .
LAB 11: Fermentation I. Objectives: Upon completion of this topic you should be able to describe: o the role of glucose and ATP in the powering of cellular reactions o the different types of fermentation in metabolism o the products of fermentation in yeast o how different sugars, temperature, and pH affect the rate of fermentation II.
The manufacturing process of the drug substance consists of two main steps: 1) fermentation and harvest of the recombinant yeast cell slurry, and 2) purification of the VLPs and adsorption of the purified VLPs onto aluminium-containing adjuvant to form the MBAP. The fermentation process consists of a seed fermentation and a production fermentation.
Quanti-Tray Demonstration Add Colilert to sample and shake to dissolve Pour mixture into a Quanti-Tray. 13 25 Quanti-Tray Demonstration cont. Seal and then incubate at 35 C for 24 hours Count positive wells and refer to MPN table 26 E.coli- Blue Fluorescence- Quanti-Tray under a 365nm UV Light. 14 27File Size: 1MBPage Count: 23Explore furtherFecal coliform and E. coli Analysis in wastewater and .ohiowea.orgAddressing Total Coliform Positive or E.coli Positive .www.epa.govMethod 1604: Total Coliforms and Escherichia coli in Water .www.epa.gov5.11 Fecal Bacteria Monitoring & Assessment US EPAarchive.epa.govMost probable number (MPN) method for counting coliform .www.onlinebiologynotes.comRecommended to you based on what's popular Feedback
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