P-00133497
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`Production of the Basic Fibroblast Growth Factor
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`(bFGF) in a High Cell Density Process by means of
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`Recombinant Escherichia coli
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`From the Department of Mechanical Engineering and Electrical Engineering
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`of the Technical University Carolo-Wilhelmina
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`in Braunschweig
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`Approved
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`Doctor of Engineering
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`(Dr.-Ing.)
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`Dissertation
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`by
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`Dipl.-Ing. Anke Seeger
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`of Rathenow
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`BEQ 1020
`Page 1
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`Production of the Basic Fibroblast Growth Factor
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`(bFGF) in a High Cell Density Process by means of
`
`Recombinant Escherichia coli
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`
`
`From the Department of Mechanical Engineering and Electrical Engineering
`
`of the Technical University Carolo-Wilhelmina
`
`in Braunschweig
`
`
`
`Approved
`
`Doctor of Engineering
`
`(Dr.-Ing.)
`
`Dissertation
`
`
`
`by
`
`Dipl.-Ing. Anke Seeger
`
`of Rathenow
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`
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`BEQ 1020
`Page 2
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`1st Referee
`2nd Referee
`Submitted on:
`Oral Examination:
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`Prof. Dr. D. C. Hempel
`Prof. Dr. W.-D. Deckwer
`05/31/1995
`08/30/1995
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`1995
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`BEQ 1020
`Page 3
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`The present work was produced during the period from April, 1992 to April, 1995 at the
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`Gesellschaft für Biotechnologische Forschung mbH [Society for Biotechnological Research
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`mbH], Braunschweig, under the direction of Prof. Dr. W.-D. Deckwer, whom I thank for
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`suggesting the thesis and supporting my work.
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`Furthermore, I would like to thank Prof. Dr. D. C. Hempel for conducting the review.
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` A
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` special thanks goes to Dr. U. Rinas, who helped me familiarize myself with this subject
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`matter, and provided supervision during this study. She provided a great deal of help through her
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`willingness to engage in discussion, in resolving problems that arose, or preventing them from
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`occurring.
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`Furthermore, I would like to thank all of my co-workers in the study group for developing
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`methods, for the pleasant work climate and for their assistance. I would like to thank Mrs. M.
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`Schreiner for her excellent technical assistance.
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`Pero vull també agrair molt especialment a en David per la seva ajuda i comprensió mostrada
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`durant la meva promoció.
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` I
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` would like to thank my family with all my heart for the support they have provided me during
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`my education, without whom I could never have completed this study.
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`Table of Contents
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`I
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`1.
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`Introduction ............................................................................................................1
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`1.1
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`1.2
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`Introduction and Overview of References ..................................................1
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`Presentation .................................................................................................6
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`2.
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`Theoretical Background ........................................................................................7
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`2.1
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`2.2
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`2.3
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`Basic Fibroblast Growth Factor (bFGF) .....................................................7
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`Cultivation of Escherichia coli in a High Cell Density Process ..................9
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`Intracellular Reactions to Stress Situations ...............................................11
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`2.3.1
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`Influence of Temperature on Growth ...........................................11
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`2.3.2
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`Influence of Temperature on Protein Synthesis ............................11
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`2.3.3 Degradation of RNA Components in Reaction to Stress ..............13
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`2.4
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`Folding of Recombinant Proteins .............................................................17
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`2.4.1 Molecular Mechanisms .................................................................17
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`2.4.2 Kinetics of Protein Folding ...........................................................18
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`2.5
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`Purification of Basic Fibroblast Growth Factors ......................................20
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`3.
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`Materials and Methods .......................................................................................22
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`3.1
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`Production of bFGF ..................................................................................22
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`3.2
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`3.3
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`3.1.1 Microorganisms and Plasmids ......................................................22
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`3.1.2 Stock Culture ................................................................................23
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`3.1.3 Cultivation Media .........................................................................23
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`3.1.3.1 LB-Medium ....................................................................23
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`3.1.3.2 Media for High Cell Density Cultivation .......................24
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`3.1.4 Preparatory Cultures .....................................................................25
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`3.1.5 Experiment Set-up and Comparison of Reactor Types Used .......25
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`3.1.6 Course of the High Cell Density Cultivation for
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`Production of bFGF ......................................................................28
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`On-line Analysis during High Cell Density Cultivation ...........................31
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`Off-line Analysis during High Cell Density Cultivation ..........................33
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`3.3.1 Determination of Cell Growth ......................................................33
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`Table of Contents
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`3.3.2 Determination of Glucose, Ammonia and Phosphate Ions ...........33
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`3.3.3 Determination of Osmotic Strength ..............................................34
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`3.4
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`Determination of Metabolism By-products through
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`High-Performance Liquid Chromatography (HPLC) ...............................35
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`Quantitative bFGF determination .............................................................37
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`Qualitative Protein Analysis .....................................................................38
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`3.6.1 Goal, Sample Preparation, and Chemicals Used ..........................38
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`3.6.2 Gradient Gels ................................................................................39
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`3.6.3 2-D Gel Electrophoresis for Isolating bFGF .................................42
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`3.6.4 Transference of Proteins to 1-D or 2-D gels
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`to Membranes by Means of Blotting for Antibody-Specific
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`Dying 44
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`3.6.5 Antibody Dying ............................................................................44
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`3.5
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`3.6
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`3.7
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`Regeneration Strategy for bFGF ...............................................................45
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`3.7.1 Cell Harvest ..................................................................................45
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`3.7.2 Cell Degradation ...........................................................................45
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`3.7.3 Separation of the Insoluble Protein Fractions from the
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`Soluble Proteins ............................................................................46
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`3.7.4
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`Ion Exchange Chromatography ....................................................46
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`3.7.5 Dialysis .........................................................................................47
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`3.7.6 Heparin Affinity Chromatography ................................................48
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`3.7.7 Recording of Adsorption Isotherms for bFGF
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`on CM-sepharose and Heparin ......................................................48
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`3.8 Measurement of the Circular Dichroism (CD Spectra) .............................49
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`3.9
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`Sequence Analysis ....................................................................................50
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`3.10 Biological Activity Test for bFGF ............................................................51
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`4.
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`Results and Discussion ........................................................................................52
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`4.1
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`Examination of the Plasmids Used with Regard to
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`their Expression Behavior .........................................................................52
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`4.1.1 Shaking Flask Test ........................................................................52
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`4.1.2 Comparison of the Expression System in
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`High Cell Density Cultivations .....................................................54
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`BEQ 1020
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`Table of Contents
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`III
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`4.2
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`Production of bFGF by Means of Thermal Induction ..............................61
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`4.2.1
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`Influence of the Induction Point in Time ......................................61
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`4.2.2 Kinetic Considerations ...................................................................68
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`4.2.2.1 Depiction of the Expression Rate ..................................68
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`4.2.2.2 Depiction of the in vivo Folding and
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`4.3
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`Production of bFGF in 50 l Measures .......................................................80
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`Stability of bFGF ............................................................73
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`4.4 Metabolism Examinations of E. coli During
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`High Cell Density Cultivations .................................................................84
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`4.4.1 Enrichment of Metabolism By-Products in the Medium ..............84
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`4.4.2 Enrichment of Uracil in the Medium ............................................88
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`4.4.3
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`Influence of Osmotic strength of the Medium ..............................93
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`Results of Discussion of the Chromatographic Purification Steps ...........95
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`Product Characterization .........................................................................106
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`4.6.1 2-D Gel Electrophoresis ..............................................................106
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`4.6.2 Measurement of the Circular Dichroism (CD) of bFGF .............109
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`4.6.3 Sequence Analysis of the Recombinant Proteins bFGF ..............112
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`4.6.4 Biological Activity Test for bFGF ..............................................114
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`4.5
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`4.6
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`Summary ............................................................................................................117
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`Symbol Index .....................................................................................................120
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`6.1
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`6.2
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`Abbreviations ..........................................................................................120
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`Formula Symbols ....................................................................................121
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`Bibliography ......................................................................................................123
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`Appendix ............................................................................................................133
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`8.1
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`8.2
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`High Cell Density Cultivations ...............................................................133
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`Batch Tests for Bonding bFGF to CM-sepharose and Heparin ..............150
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`5.
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`6.
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`7.
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`8.
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`BEQ 1020
`Page 7
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`1. Introduction and Goal
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`
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`1.1
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`Introduction and Overview of References
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`
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`As a result of the strong dissemination of the genetic engineering methods for targeted
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`modification of nucleic acid sequences of proteins, so-called “genetic engineering” has become a
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`primary tool in bioengineering in recent years. The production of commercially interesting
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`proteins is of primary interest thereby, wherein, by means of the production of these substances,
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`new perspectives have also been quite frequently explored in the medical field thereby. A few
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`examples of commercially important proteins produced in genetically modified microorganisms
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`are listed below (Georgiou, 1988).
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`Hormones:
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`Enzymes:
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`Growth hormones, endorphin, insulin, Factor VIII
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`Protease, cellulase, pullulanase, prochymosin, elastase, urokinase,
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`streptokinase, superoxide dismutase
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`Phys. active substances:
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`Interferons, interleukins, tumor necrosis factor
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`Inoculants:
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`Hepatitis B surface antigen
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` A
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` market for 1995 in the USA of 2,400 million dollars was predicted thereby, just for the group
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`of growth hormones (Robinson et al., 1992). It is therefore not surprising that the production of
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`these proteins in recombinant microorganisms was preferred over the comparably difficult
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`isolation thereof from the tissues in which they originate, which produces a lower yield
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`(Gospodarowicz et al., 1985; Gospodarowicz et al., 1978).
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`In the framework of planning a process for the production of recombinant proteins, in general,
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`the selection of suitable host organisms, an induction system, and a process strategy had to be
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`taken into account. The selection of a suitable host strain from the numerous known
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`microorganisms was strongly limited by the quantity of available biological data regarding a
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`strain. For this reason, the well researched Gram-negative bacteria Escherichia coli remains the
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`BEQ 1020
`Page 8
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`favorite when selecting an easily cultivated host organism. Likewise widely disseminated and
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`used specifically for the production of enzymes is the strain Bacillus sp. (Georgiou, 1988).
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`For commercial production of a recombinant protein with a high yield in recombinant E. coli
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`strains, it is furthermore necessary to combine an effective induction system with a high cell
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`density process, and to ensure a high degree of stability of the recombinant protein through the
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`selection of a suitable cultivation parameter. The various induction systems have thus been the
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`subject of numerous studies in recent years. A good summary of induction systems frequently
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`used in E. coli is provided in the publication by Georgiou (1988).
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`As a prerequisite for a cultivation of E. coli to a high cell density of up to 148 g/l, a fed-batch
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`method has been developed at the GBF, which is based on a carbon limitation of the cells during
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`the fed-batch phase (Korz et al., 1994; Korz, 1992). The constant growth rate reduced in this
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`manner prevents an oxygen limitation and an enrichment of by-products that have a detrimental
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`effect on metabolism. Numerous pharmaceutically important proteins have already been
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`produced in cultivations with relatively high cell densities. Some of these are listed in Table 1.
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`Such a process for a high cell density cultivation with the synthesis of foreign proteins
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`furthermore offers the possibility of examining the host organism in stress situations, such as
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`higher temperatures, or carbon deficits, with respect to its metabolism. As a result, conclusions
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`can be drawn regarding the fundamental microbial processes in bacteria, which in part can be
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`generalized for other organisms. As a result, to date the questions regarding the purpose and
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`effects of the production of additional proteins induced in E. coli at higher temperatures, the so-
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`called heat shock proteins, have not been fully resolved; a phenomenon that extends from the
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`bacteria to human organisms. Due to the very high cell densities during the cultivation,
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`measurable parameters, which indicate a change in metabolism, e.g. increased heat generation,
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`Page 9
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`release of proteins or metabolism by-products, are increased, and thus made more quickly
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`available (possibly on-line).
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`Product
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`Max. Product
`Concentration
`Interleukin-2
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`1.02 g/l
`Interleukin-2
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`40 mg/gBTM
`Human Epidermal Growth
`Factor
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`60 mg/l
`Interferon-Alpha1
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`2x7 107U/gBTM
`Interferon-Alpha1
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`55x107U/gBTM
`bFGF
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`23.2 mg/l
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`Malaria antigen
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`12 mg/g
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`Strain
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`Plasmid
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`Promoter
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`Induction system
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`M5248
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`pNKM21
`HW21-2
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`pFC54
`HB 101
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`pTRLBT1
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`BMH7118
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`PBV-867
`TG1
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`pBB210
`MH294
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`pTB669
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`AR58
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`pR32tet32
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`PL
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`30° C to 42° C
`PL
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`30° C to 37° C
`Ptrp
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`Removal of Tryptophan from
`the medium
`PL
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`30° C to 37° C
`constitutive
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`Ptrp
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`Addition of 3--indoleacrylic
`acid
`PL
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`30° C to 42° C
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`Process
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`Max. BTM
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`DO-stat
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`29 g/l
`Fed-batch
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`42 g/l
`Fed-batch
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`21 g/l
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`Fed-batch
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`50-80 g/l
`Fed-batch
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`60 g/l
`Batch
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`no
`information
`Fed-batch
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`50 g/l
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`Reference
`
`Seo et al.
`(1992)
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`Macdonnald et al.
`(1990)
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`Shimizu et al.
`(1990)
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`Yang et al.
`(1992)
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`Riesenberg et al.
`(1990)
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`Iwane et al.
`(1987)
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`Zabriskie et al.
`(1987)
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`Table 1.1: Pharmaceutically important proteins, produced in recombinant E. coli strains.
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`With the production of pharmaceutically important proteins, one main problem thereby is the
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`purification of proteins in their native forms. Many of the recombinant proteins are preferably
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`deposited in their insoluble form, the so-called inclusion bodies, in the cells (Schein, 1989;
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`Shimizu et al., 1991). These are normally biologically inactive, and require extensive cleansing,
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`denaturing and renaturation steps (Bernardez-Clark et al., 1991).
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`The frequently occurring proteolytic degradation in the cells may also be another obstacle in the
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`production of recombinant proteins. As such, Talmadge et el. (1982) has shown that the half-life
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`period of the pharmaceutically important protein preproinsulin, a preliminary stage of insulin, is
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`less than two minutes in cytoplasm of E. coli. Some recombinant proteins are recognized as
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`abnormal and degraded by the cells – in comparison with the native proteins – wherein this is
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`clearly not observed with all recombinant proteins, and appears to be dependent on the sequence
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`of its first amino acids (Georgiou, 1988).
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`
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`The regeneration of recombinant produced proteins is also problematic, because important
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`environmental parameters, such as the pH value and the temperature, can only be varied within
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`narrow ranges, because this would otherwise lead to an aggregation of the protein molecules.
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`Furthermore, the use of organic solvents may lead to a denaturing of the proteins.
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` A
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` major problem is quality control for the recombinant proteins. In order to ensure that the use
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`thereof does not present a risk in human medicine, all of the possible contaminants must be
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`eliminated. Contaminants may have a directly damaging effect on test subjects thereby, or they
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`may have a detrimental effect on the effect of the therapeutic agent that is being administered.
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`Frequently occurring contaminants having negative effects are, e.g. oncogene DNA, endotoxins,
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`infectious agents or materials that give rise to an immune reaction (Anicetti et al., 1989). The
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`protein itself can also be a starting point for contaminants as a result of undesired
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`heterogeneities, e.g. deamidation or amino acid substitution (Manning et al., 1989; Liu, 1992). A
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`few typical contaminants, frequently found in pharmaceutical proteins, are listed in the following
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`Table, according to the corresponding detection methods.
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`Contaminant
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`Endotoxins
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`Cell or media proteins
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`Infectious agents
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`Product variants:
`deamidation products
`oxidation products
`amino acid substitutions
`aggregated forms
`proteolytic products
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`Detection method
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`LAL1, rabbit pyrogen
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`SDS-Page2, Immune test
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`Reverse transcriptase test
`Cell cultures; cytopathic effects, electron microscope
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`SDS-Page2, isoelectric focusing
`HPLC
`Edman degradation analysis
`SDS-Page2
`SDS-Page2, HPLC
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`Table 1.2: Frequently occurring contaminants in pharmaceutical proteins and the corresponding
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`detection methods (Anicetti et al., 1989), LAL1 limulus amebocyte lysate, SDS-Page2 Sodium
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`dodecyl sulfate polyacrylamide gel electrophoresis
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`
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`These types of contaminants in the protein solution are problematic because they are difficult to
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`detect, and can only be detected by means of respective very specific analytical methods. Of
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`course, this does not only apply to proteins that are produced through genetic engineering
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`approaches, but rather for pharmaceutical products that are isolated directly from the initial
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`tissues as well. As a consequence, this has resulted in the past in infections of personnel and
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`patients through undetected viral contaminants, e.g. AIDS or Ebola. For this reason, it is very
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`important that the information regarding the purity of a protein must always be regarded in
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`conjunction with the analytical methods that are used, the sensitivity thereof regarding specific
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`heterogeneities, and the reliability thereof.
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`1.2 Presentation
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`The goal of this work was the development of an economically reasonable process for the
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`production of the basic fibroblast growth factor (bFGF) as a protein of pharmaceutical interest
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`using a recombinant Escherichia coli strain. The high cell density process developed at the GBF
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`could be used for this (Korz, 1992). As an induction system, a temperature inducible system was
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`to be compared with a chemically inducible system. Both systems were to be compared with
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`regard to their influence on the cell growth, the product yield and the solubility – and thus to the
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`biological activity – of the synthesized pharmaceutical protein.
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`In the case of a deposit of the recombinant protein in insoluble inclusion bodies, it would be
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`desirable to characterize method and biological effect variables, and to optimize the process in
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`favor of the soluble bFGF fractions.
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`Furthermore, the high cell density cultivation is to be used for examining the metabolism of the
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`host organism with regard to its behavior in stress situations, e.g. glucose deficit, or high
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`temperatures.
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`Another primary focus is the purification of the soluble fraction of the target protein. For this, a
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`gentle product purification is sought for obtaining a high recovery rate in as few purification
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`steps as possible.
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`Lastly, the purified protein is to be examined with regard to potentially arising contaminants,
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`structural changes or micro-heterogeneities.
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`2. Theoretical Background
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`2.1 The Basic Fibroblast Groth Factor (bFGF)
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`
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`The basic fibroblast growth factor (bFGF) belongs to a class of growth hormones that can be
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`subdivided into six different factors, each of which has a different medical application potential.
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`The most common terminology for these most important growth factors are: epidermal growth
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`factor (EGF), fibroblast growth factor (FGF), transforming growth factor  (TGF-),
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`transforming growth factor  (TGF-), insulin-like growth factor (IGF), and platelet-derived
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`growth factor (PDGF) (Robinson et al., 1992).
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`The basic fibroblast growth factor (bFGF) is a single strain, non-glycosylated polypeptide, which
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`was originally isolated from cattle brain and the bovine pituitary gland (Gospodarowicz, 1974).
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`It was also found in numerous tissues and organs, such as human brain and placenta, cattle retina
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`and kidney, the hypothalamus, thymus, amygdala, and various types of tumors (Gospodarowicz,
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`1987). Due to the isolation of bFGF in numerous tissues and organs, it is described in the
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`relevant literature under various synonyms, such as endothelial growth factor, tumor angionesis
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`factor or hepatoma growth factor. The characterizing property of bFGF is its isoelectric point,
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`which lies at 9.6 (Gospodarowicz, 1987).
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`The amino acid sequences of bovine and human bFGF are known and exhibit a 98.7% homology
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`(Abraham et al., 1986). The molecular weight of the 155 amino acid forms of the bFGF is 18
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`kDa. The existence of numerous bFGF forms could be demonstrated in the various initial
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`tissues, which differ in terms of their numbers of amino acids and molecular weights. Thus, in
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`addition to the original 146 and 155 amino acid forms isolated in cattle brain and pituitary gland
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`having a molecular weight of 16.5 and 18 kDa, a 157 amino acid form of the bFGF was found in
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`the placenta, and a 163 amino acid form of bFGF was found in hepatoma cells (Brigstock et al.,
`
`1990). Sequencing has shown that these are not N-terminus extensions, which cannot be
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`explained through post-translational modifications. It is assumed that the longer bFGF forms are
`
`encoded with the same cDNA, but are initiated by the CUG codon that is an alternative to the
`
`AUG codon (Prats et al., 1989; Brigstock et al., 1990). So far, however, it has not been possible
`
`to find any indication of the function of the N-terminus extensions; the various bFGF forms do
`
`not differ in terms of their biological activity (Brigstock et al., 1990).
`
`
`The protein contains 4 cysteines, which, however, are not capable of forming disulfide bridges,
`
`due to their spatial arrangement. Examinations of three-dimensional structures of bFGF have
`
`furthermore shown that its topology has a strong homology to that of the interleukin 1, and
`
`consists mainly of -sheet structures (Erikson et al., 1991; Zhang et al., 1991).
`
`
`
`
`
`
`
`Fig. 2.1: The structure of the basic fibroblast growth factor. This is the bFGF form according to
`
`Erikson et al. (1991) consisting of 146 amino acids, recorded using the computer program
`
`BRAGI/GBF. The dark grey markings indicated -sheet structures, the black markings indicate
`
`the accordingly labeled cysteine side chains.
`
`
`
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`bFGF is a mitogen for endothelial cells as well as a large number of cells originating in the
`
`mesodermal and neural-ectodermal regions (Gospodarowicz, 1987; Gospodarowicz et al., 1987).
`
`In addition, it induces the differentiation of nerve cells and prevents the de-differentiation of
`
`vascular and corneal endothelial cells. bFGF is an angiogenetic factor, i.e. it stimulates the
`
`formation of blood vessels (Gospodarowicz et al., 1979; Togari et al., 1987). The molecular
`
`mechanism is based on the tyrosine-specific protein kinase activity of the bFGF receptor, which
`
`can catalyze the phosphorylation of a cellular substrate protein. So far, three bFGF receptors
`
`have been identified (Bradshaw et al., 1987). Baird et al. (1988) was able to show that the
`
`peptide fragments 24-68 and 106-115 bond with the bFGF receptors, of which 103-104 per cell
`
`have been found.
`
` A
`
` broad application spectrum, in particular in the field of medicine, is obtained from the various
`
`functions of bFGF. By stimulating fibroblasts, it causes an accelerated healing process for
`
`wounds, which is of particular interest with regard to chronic or poorly healing wounds. In the
`
`treatment of thrombosis, bFGF can induce a blood vessel regeneration, and thus offers new
`
`possibilities for the treatment thereof (Buntrock et al., 1982; Buntrock et al., 1984). Further
`
`clinical fields of application arise in tissue transplants and through the possibility of nerve cell
`
`regeneration. It is to be expected that the demand for growth factors will increase sharply in the
`
`future, due to the many various application possibilities (Prats et al., 1989).
`
`
`
`2.2 Cultivation of Escherichia coli in a High Cell Density Process
`
`
`
`The two-phase method developed at the GBF for cultivating the Gram-negative bacteria
`
`Escherichia coli up to a cell density of 148 g/l is based on a fed-batch strategy, in which a
`
`controlled feeding of the carbon source follows the batch phase. In order to prevent a substrate
`
`enrichment in the medium, and to ensure a growth having a constant growth rate, the feeding
`
`occurs with a defined, exponentially increasing speed (Korz, 1992; Korz et al., 1994). In
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`
`contrast to this simple method for maintaining a constant growth rate by means of a pre-
`
`calculated substrate mass flow, a complex regulating strategy has been proposed by Riesenberg
`
`et al. (1991). With this, the current growth rate of the culture can be continuously calculated
`
`from the data provided by the discharge gas analysis, and controlled via the speed of the stirrer.
`
`A second control loop guarantees the pO2 control through variation of the glucose feed.
`
` A
`
` prerequisite for the development of this process was the observations of the Pasteur and
`
`Crabtree effects in the bacterial metabolism of E. coli (Weide et al., 1979). Oxygen played an
`
`important role thereby as an end acceptor for electrons of the respiratory chain in the metabolism
`
`of the facultative anaerobic microorganism. The fact that, with an oxygen deficit caused by
`
`shortening the electron transport chain, this leads to a reduction of the ATP production and thus
`
`to a reduced biomass formation, emphasizes the necessity of a sufficient oxygen supply for the
`
`cells in a high cell density cultivation. By inhibiting the pyruvate dehydrogenase complex,
`
`pyruvate is reduced via acetyl-CoA, under anaerobic conditions, to acetate, a metabolism by-
`
`product that has an inhibiting effect on both growth as well as product formation (Pasteur effect).
`
`A defined growth rate ensures a controllable oxygen consumption of the cells thereby, and thus
`
`enables growth without oxygen limitation.
`
`
`
`The bacterial Crabtree effect relates to the interaction between glucose concentrations in media,
`
`and the glucose metabolism under aerobic conditions. Thus, a high concentration of glucose in
`
`the medium results in a repression of the formation of certain enzymes of the TCA cycle, and in
`
`particular, the NADH dehydrogenase and the succinate dehydrogenase are inhibited, which in
`
`turn leads to a greater enrichment of inhibiting metabolism by-products such as acetate
`
`(Hollywood et al., 1976).
`
`
`
`
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`2.3 Intracellular Reactions to Stress Situations
`
`
`
`2.3.1
`
`Influence of Temperature on Growth
`
`
`
`Increasing the cultivation temperature causes stress in the metabolism of the mesophilic
`
`organism E. coli. In the temperature range of 20°-37° C, the so-called normal or Arrhenius
`
`range, the growth rate represents a simple function of the temperature. With temperature
`
`fluctuations within this range, the growth rate that is characteristic of the current temperature is
`
`adjusted to without delay.
`
`
`
`With temperatures higher than 40° C, in contrast, the growth rate speed decreases until it reaches
`
`a full stop in growth in relation to the medium. It has been concluded from these observations
`
`that the effects of temperature fluctuations in the normal range can be compensated for through
`
`an adjustment of the enzyme activity, while at higher temperatures, changes in individual cell
`
`components, such as in the protein, fatty acids and phospholipid composition could be observed
`
`(Neidhardt et al., 1987).
`
`
`
`2.3.2
`
`Influence of Temperature on Protein Synthesis
`
`
`
`While proteins, which are involved in the transcription or the translation, occur at temperatures
`
`above 40° C in E. coli in a reduced quantity in the cells, a group of at least 17 polypeptides has
`
`been identified, the expression of which can be induced more strongly by a factor of up to 100
`
`when the temperature is increased (Reeve et al., 1984; Lemaux et al., 1978; Yamamori et al.,
`
`1982). A selection of the most important heat shock proteins identified so far is listed in the
`
`
`
`following Table.
`
`
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`Function
`
`Gene MG
`(kDa)
`
`25.300
`
`grpE
`
`Initiation of the DNA replication
`
`mopA 62.833 Stabilizing, complex forming in the protein folding, is necessary for 1-phage
`head assembly
`
`69.121 has 5’-nucleotidase and weak ATP-ase activity, is capable of auto-
`phosphorylation
`
`70.263 Sub-unit of the RNA polymerase, responsible for normal promoter detection,
`regulates the synthesis of other heat shock proteins
`
`mopB 10.670 Proteolysis, forms complex with GroEL, inhibits its ATP-ase activity
`
`60.500 participates in the formation of adenylated nucleotides.
`
`dnaK
`
`rpoD
`
`lysU
`
`Protein
`name
`
`GrpE
`
`GroEL
`
`DnaK
`
`Sigma
`
`GroES
`
`Lysyl-
`tRNA
`synthetase
`Form 2
`
`
`
`Table 2.1: List of selected heat shock protein of E. coli (Martin et al., 1991; DeBernardez-Clark
`
`et al., 1991; Niedhardt et al., 1987; Varshavsky, 1983)
`
`
`
`These so-called heat shock proteins (HSP) result in not only an improved heat tolerance of the
`
`cell, they are also identical in part to proteins induced through oxidative stress and the addition
`
`of ethanol. Their role in the survival mechanism of E. coli is a theme discussed at length in
`
`technical literature. VanBogelen et al. (1987) was able to demonstrate, through induction of the
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`regulons for SOS, heat shock and oxidative stress, that these stress regulons were induced
`
`independently of one another, wherein some stress factors, e.g. CdCl2, induce numerous
`
`regulons. A decisive function of these proteins induced through heat is, intracellularly, their
`
`influence on the correct folding of proteins.
`
`
`
`In numerous processes for the production of proteins, large quantit