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B - eTheses Repository - University of Birmingham

B - eTheses Repository - University of Birmingham

B - eTheses Repository - University of

Physiological aspects underpinning recombinant protein production in Escherichia coli by Sara Nuri Alfasi A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Biosciences The University of Birmingham December 2010

  • Page 2 and 3: University of Birmingham Research A
  • Page 4 and 5: Dedicated with love and thanks to M
  • Page 6 and 7: Lists Table of contents List of fig
  • Page 8 and 9: Buffers and solutions 56 Buffers fo
  • Page 10 and 11: Western blotting analysis of the T7
  • Page 12 and 13: Figure 1.1 1.2 List of figures Phys
  • Page 14 and 15: 4.7 Yields of soluble and insoluble
  • Page 16 and 17: Table List of tables 2.1 E. coli st
  • Page 18 and 19: CHAPTER 1 Introduction 1
  • Page 20 and 21: ecombinant gene is expressed at hig
  • Page 22 and 23: Physiological responses of bacteria
  • Page 24 and 25: lives such as those required for th
  • Page 26 and 27: accumulation of unfolded protein in
  • Page 28 and 29: Figure 1.1: Physiological response
  • Page 30 and 31: eneficial to RPP in the dual stress
  • Page 32 and 33: Villaverde, 2001; Yoon et al., 2010
  • Page 34 and 35: Nascent polypeptide Unfolded/ misfo
  • Page 36 and 37: Effect of inclusion body formation
  • Page 38 and 39: Further investigation revealed that
  • Page 40 and 41: The divergence of solubility and fu
  • Page 42 and 43: probably by generally stimulating p
  • Page 44 and 45: tight control of gene expression an
  • Page 46 and 47: stress is characterised by down-reg
  • Page 48 and 49: and Valax, 1996). This has been use
  • Page 50 and 51: Plasmid maintenance The loss of the
  • Page 52 and 53:

    ather than 0.1 mM IPTG, increased t

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    accumulation of native recombinant

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    al., 1999; Jones, 2007). Linear pos

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    fluorescence could be used to estim

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    In contrast to an online GFP sensor

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    A B Forward Forward scatter scatter

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    parameter flow cytometry and FACS a

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    possesses two very important outer

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    produced. This has been demonstrate

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    CHAPTER 2 Materials and Methods 53

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    with glucose at 2% (w/v) final conc

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    Buffers and solutions for SDS-PAGE

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    Table 2.1: E. coli strains and plas

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    concentration (pO2) throughout the

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    chromosomal DNA were used as a temp

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    uffer, BSA and sterile distilled wa

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    pellet containing the insoluble pro

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    instructions and contained 10% meth

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    Fluorescence was measured from seri

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    sterilising oven overnight. After i

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    To prepare an ethanol killed contro

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    The production of soluble CheY as a

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    Total fluorescence (units) A OD 650

  • Page 98 and 99:

    Cfu x 10 8 / ml A B 1000 100 10 1 -

  • Page 100 and 101:

    from the pET vector (Figure 3.3 B).

  • Page 102 and 103:

    Dissolved oxygen (%) 120 100 80 60

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    Figure 3.6: Flow cytometry analysis

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    In conclusion, the standard protoco

  • Page 108 and 109:

    A 175 83 62 47.5 32.5 25 16.5 6.5 B

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    subsequently replica plated onto se

  • Page 112 and 113:

    induction protocol that minimises s

  • Page 114 and 115:

    The number of cfu was highest in th

  • Page 116 and 117:

    Plasmid retention (%) 120 100 80 60

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    of soluble CheYGFP is in the range

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    expression system. This prompted an

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    concentration each around 5 h post-

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    Dissolved oxygen (%) 120 100 80 60

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    A Before induction 4 h post-inducti

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    Comparison of levels of T7 RNA poly

  • Page 130 and 131:

    to scale-up CheYGFP production by u

  • Page 132 and 133:

    Dissolved oxygen (%) 120 100 80 60

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    protocol. An aeration of 6 L/ min (

  • Page 136 and 137:

    increased to almost 100%, correspon

  • Page 138 and 139:

    Cfu x 10 8 / ml 10000 1000 100 10 1

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    and perhaps the only compromise is

  • Page 142 and 143:

    The improved protocol has been deve

  • Page 144 and 145:

    OD 650 20 18 16 14 12 10 8 6 4 2 0

  • Page 146 and 147:

    Specific fluorescence (units) 300 2

  • Page 148 and 149:

    Serial dilutions of samples from al

  • Page 150 and 151:

    Plasmid retention (%) 120 100 80 60

  • Page 152 and 153:

    A B C 175 175 83 83 62 62 47.5 47.5

  • Page 154 and 155:

    The location of mutation in P2 BL21

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    Effect of combining both the improv

  • Page 158 and 159:

    used for induction, and indicates t

  • Page 160 and 161:

    Figure 4.10: Comparison 6.5 of yiel

  • Page 162 and 163:

    Figure 4.11: Western blotting analy

  • Page 164 and 165:

    Figure 4.12: CheYGFP production usi

  • Page 166 and 167:

    Fluorescence units 6000 5000 4000 3

  • Page 168 and 169:

    compared to using the improved prot

  • Page 170 and 171:

    Before induction 24 h post-inductio

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    were called C41 and C43, with C43 r

  • Page 174 and 175:

    Specific fluorescence (units) 300 2

  • Page 176 and 177:

    Total fluorescence (units) 5000 450

  • Page 178 and 179:

    175 83 62 47.5 32.5 25 16.5 Figure

  • Page 180 and 181:

    OD 650 14 12 10 8 6 4 2 0 0 10 20 3

  • Page 182 and 183:

    Specific fluorescence (units) 350 3

  • Page 184 and 185:

    In conclusion, using either the imp

  • Page 186 and 187:

    highlighted by differences in CheYG

  • Page 188 and 189:

    Gonorrhoea is characterized by the

  • Page 190 and 191:

    Freshly transformed cultures of BL2

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    Cfu x 10 8 / ml 140 120 100 80 60 4

  • Page 194 and 195:

    Cfu x 10 8 / ml 100 10 1 0.1 0.01 B

  • Page 196 and 197:

    apo-CCP when bacteria were either u

  • Page 198 and 199:

    the physiological impact on bacteri

  • Page 200 and 201:

    concentrations of 0.025 mM or more

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    introduce stress on the host that i

  • Page 204 and 205:

    175 83 62 47.5 32.5 A B B.I 5.5 24

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    using both the standard and improve

  • Page 208 and 209:

    OD 650 20 18 16 14 12 10 8 6 4 2 0

  • Page 210 and 211:

    Cfu x 10 8 / ml 250 200 150 100 50

  • Page 212 and 213:

    175 83 62 47.5 32.5 25 Figure 5.13:

  • Page 214 and 215:

    Cfu x 10 8 / ml OD 650 A B 9 8 7 6

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    Wild type BL21*; improved protocol

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    A B Stress-resistant candidates Con

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    generated by the wild type strain u

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    Cfu x 10 8 / ml 250 200 150 100 50

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    likely that there are additional di

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    Post-induction (h) B.I 6 9 24 33 49

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    Improving RPP by decreasing the rat

  • Page 230 and 231:

    Another important facet of the impr

  • Page 232 and 233:

    number of colony forming units of w

  • Page 234 and 235:

    The broad-range applicability and u

  • Page 236 and 237:

    T7 RNA polymerase activity, which w

  • Page 238 and 239:

    accumulate high yields of the prote

  • Page 240 and 241:

    esearch, which demonstrate the link

  • Page 242 and 243:

    higher specific fluorescence in P2

  • Page 244 and 245:

    Table 6.1. The production of CheYGF

  • Page 246 and 247:

    esulted in decreased levels of inso

  • Page 248 and 249:

    Role of DnaK in enhancing solubilit

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    facilitate the isolation of bacteri

  • Page 252 and 253:

    Target proteins Outer membrane Anti

  • Page 254 and 255:

    protein will be obtained in a relat

  • Page 256 and 257:

    the directed evolution of bacterial

  • Page 258 and 259:

    Albano, C.R., Randers-Eichhorn, L.,

  • Page 260 and 261:

    Caspers, P., Stieger, M. and Burn,

  • Page 262 and 263:

    Gadgil, M., Kapur, V. and Hu, W.S.

  • Page 264 and 265:

    Hedhammar, M., Stenvall, M., Lonneb

  • Page 266 and 267:

    Kurland, C.G. and Dong, H.J. (1996)

  • Page 268 and 269:

    Perederina, A., Svetlov, V., Vassyl

  • Page 270 and 271:

    Terpe, K. (2006) Overview of bacter

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