Ph.D Thesis Efficient production of single-chain Fv antibody using recombinant Escherichia coli by DO-stat fed-batch culture NGUYEN HIEU NGHIA Kyoto Institute of Technology 2021 Abstract Recombinant protein production using Escherichia coli is one of the fundamental techniques in biotechnology In this study, we challenged the efficient production of soluble single-chain Fv (scFv) antibodies using E coli A scFv against C-reactive protein (anti-CRP scFv) that appears in blood plasma as a marker of systemic inflammation was employed as a model protein Dissolved oxygen (DO)stat fed-batch culture, which allows a high cell density culture of microorganisms under constant DO conditions, was developed and applied to the scFv production using recombinant E coli The DO-stat fed-batch culture was successfully performed under various DO conditions for more than 50 h, resulting in increased scFv production from 0.5–0.8 g/L by flask and batch cultures to 2.8–3.0 g/L by the fedbatch culture under the conditions of 5–40% of DO saturation In addition, the formation of inclusion bodies was effectively depressed during the DO-stat fed-batch operation; consequently, the solubility of scFv was significantly improved from 36– 43% by the flask and batch cultures to 96–98% by the DO-stat fed-batch culture under a wide range of DO conditions To achieve further increase in scFv production by a prolonged DO-stat fed-batch culture, the additional supply of yeast extract was investigated because serious degradation of expressed scFv was observed in the later phase of DO-stat fed-batch culture Therefore, the degradation of scFv was then successfully prevented by increasing the yeast extract content in the feeding medium, resulting in successful scFv production of 4.0–5.2 g/L with more than 97% solubility at 100 h DO-stat fed-batch culture Thus, it was found that the fed-batch culture operated by the DO-stat feeding strategy using yeast extract-enriched feeding medium was effective for the enhanced production of anti-CRP scFv with high solubility Acknowledgements This study was carried out at Chemical and Biochemical Engineering Laboratory, Department of Functional Chemistry, Kyoto Institute of Technology under the guidance and supervision of Professor Jun-ichi HORIUCHI This study was financially supported by the Japanese Government (MEXT) scholarship, JSPS KAKENHI (grant number JP 19K05162), and JST A-STEP (grant number JPMJTM19FQ), to which I would like to express my gratitude I would like to express my special gratitude to Prof Jun-ichi HORIUCHI for supporting me with the opportunity to complete this study, for discussions, dedicated teaching, and critical review of my writing, especially for extensively valuable knowledge in Biochemical Engineering, Bioprocess, and Fermentation Technology I would like to express my sincere appreciation to Assoc Prof Yoichi KUMADA, for his kindness, discussions, precise advice, and valuable background in Immunology, especially Antibody Engineering I wish to express my gratitude to Prof Kaeko KAMEI for offering me the opportunity to study at KIT I would like to thank Assoc Prof Dang Thi Phuong THAO for valuable background in Molecular and Microorganism Biology I need to express my gratitude to Lab A members – University of Science – VNUHCM for their kind support, especially Dr Nguyen Tri NHAN and Dr Nguyen Thi My TRINH I would like to thank Ms SUZUKI, Mr SAKAMOTO, Ms TAKEDA, Mr IJIMA, Ms KAWAMI, Mr NAKAO, Mr IBUKI, Mr MUKAI, Ms MICHELLE, members of the Jar-fermentor group, and all members of the Laboratory for their kind support and cooperation Finally, I want to express my deep gratitude to my entire family for everything and Ms Ngoc always for her encouragement during my doctoral course Kyoto, 2021 Nguyen Hieu NGHIA iii Table of Contents Nomenclature vii Greek letters viii Abbreviation ix Chapter 1: Introduction and literature review 1.1 Recombinant protein production using E coli 1.2 Host-vector system for recombinant protein expression using E coli 1.2.1 Host cells 1.2.2 Vector systems 1.2.3 Regulation of recombinant protein expression in E coli 1.3 E coli metabolism and growth 10 1.3.1 Culture medium 11 1.3.2 Glucose metabolism 11 1.3.3 Ammonia assimilation 14 1.3.4 Dissolved oxygen (DO) 17 1.4 E coli cultivation techniques for recombinant protein production 18 1.4.1 Batch culture 18 1.4.2 Fed-batch culture 18 1.5 Feeding strategies for fed-batch cultivation 22 1.5.1 Exponential feeding strategy 22 1.5.2 pH-stat feeding strategy 23 1.5.3 DO-stat feeding strategy 24 1.5.4 Application of PID controller in fed-batch cultivation 26 1.6 Single-chain Fv antibody (scFv) production using E coli 28 1.6.1 Characteristic structure of full-length antibody and scFv 28 iv 1.6.2 ScFv production using recombinant E coli 30 1.7 Research objectives of this thesis and strategies 32 Chapter 2: Materials and Methods 33 2.1 The bacterial strain, scFv, and expression vector 34 2.2 Culture medium 35 2.3 Seed culture 36 2.4 Flask and batch cultures 37 2.5 Analytical methods of cell concentration, acetate, and NH3 37 2.6 Determination of anti-CRP scFv concentration 38 2.7 Determination of intracellular protease activity 40 2.8 SDS-PAGE, western blotting, and ELISA 40 Chapter 3: Characteristic of anti-CRP scFv production using recombinant Escherichia coli by flask and batch cultures 42 3.1 Introduction 43 3.2 Results and discussion 44 3.2.1 Anti-CRP scFv production by flask culture 44 3.2.2 Anti-CRP scFv production by batch culture 47 3.3 Conclusions 51 Chapter 4: Application of DO-stat fed-batch culture for anti-CRP scFv production using recombinant E coli 52 4.1 Introduction 53 4.2 DO-stat fed-batch culture and its mathematical models 55 4.2.1 DO-stat fed-batch culture 55 4.2.2 Mathematical models for DO-stat fed-batch culture 59 4.3 Results and discussion 62 4.3.1 Anti-CRP scFv production by fed-batch culture with DO-stat 62 v 4.3.2 Effects of DO conditions on anti-CRP scFv production 66 4.3.3 Kinetic analysis of anti-CRP scFv production by DO-stat fed-batch culture under various DO conditions 70 4.4 Conclusions 75 Chapter 5: Stabilization of single-chain Fv antibody production using recombinant E coli by DO-stat fed-batch culture employing yeast extract-enriched feeding medium 76 5.1 Introduction 77 5.2 Determination of amount of NH3 and yeast extract supplied during DO-stat fed-batch culture 78 5.3 Results and discussion 79 5.3.1 Proteolytic degradation of anti-CRP scFv in DO-stat fed-batch culture 79 5.3.2 Effects of the increase of YE supply on anti-CRP scFv production 83 5.3.3 Stabilization of anti-CRP scFv production using yeast extract-enriched feeding medium 86 5.4 Conclusions 97 Chapter 6: Conclusions and future works 98 6.1 Conclusions 99 6.2 Future works 100 References 101 Publication list 127 vi ... culture 44 3.2.2 Anti-CRP scFv production by batch culture 47 3.3 Conclusions 51 Chapter 4: Application of DO-stat fed-batch culture for anti-CRP scFv production using recombinant. .. analysis of anti-CRP scFv production by DO-stat fed-batch culture under various DO conditions 70 4.4 Conclusions 75 Chapter 5: Stabilization of single-chain Fv antibody production. ..Abstract Recombinant protein production using Escherichia coli is one of the fundamental techniques in biotechnology In this study, we challenged the efficient production of soluble single-chain Fv