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Chapter 5 The protocol used for gel filtration has been described in section 2.6. The concentrated sample from the ion exchange column was loaded into the gel filtration column and the filtration process was started with a flow rate of 1ml/min. Based on the results obtained from the UV detector (fig 5.2a), the samples between peaks A and D (columns 5-18) were analysed on a SDS-PAGE gel. The results showed that the fractions containing JNL still had a large concentration of smaller proteins present (fig 5.2b). 5.1.3 Second Ion exchange purification: DEAE sepharose As better separation had been obtained with the ion exchange column, this method was repeated with a shallower buffer gradient. This was done as an attempt to improve the separation of JNL from the smaller proteins. As can be seen in figure 5.3a, this approach was largely successful. Samples from tubes 15 to 18 (lanes 2 to 5 in figure 5.3a) were then collected and pooled together. To ensure JNL had not eluted in fraction tubes prior to tube 15, another SDS-PAGE gel was run and included samples from tubes 10 to 14 (fig 5.3b). This SDS-PAGE gel also had the pooled product from tubes 15 to 18 (purified JNL) as well as running samples from the unbound collection to ensure no JNL was present (5.3b). 191
Chapter 5 (a) A B C D E (b) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 72 kDa 55 kDa 17 kDa 11 kDa Fig 5.2: UV graph and SDS-PAGE page analysis of JNL from gel filtration column. (a) UV graph showing regions of protein elution. (b) SDS-PAGE gel. Lane 1: Prestained marker (Fermentas); Lane 2: Tube 5; Lane 3: Tube 6; Lane 4: Tube 7; Lane 5: Tube 8; Lane 6: Tube 9; Lane 7: Tube 10; Lane 8: Tube 11; Lane 9: Tube 12; Lane 10: Tube 13; Lane 11: Tube 14; Lane 12: Tube 15; Lane 13: Tube 16; Lane 14: Tube 17; Lane 15: Tube 18. 192
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FUNCTIONAL AND STRUCTURAL CHARACTER
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Abstract Proteins are involved in a
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Dedicated to Mum, Dad, and Sharique
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Index of contents Chapter 1: Introd
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2.4.5: Bacteriophage P1 Transductio
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4.1.5: Chimeras DHF & GEI 162 4.2:
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List of illustrations Chapter 1: In
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Fig 4.1: Schematic representation o
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List of tables Chapter 1: Introduct
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SDW STPKs TAE TEMED TBS Sterile dis
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Chapter 1 1.1 Protein Folding The g
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Chapter 1 Fig 1.1: The energy lands
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Chapter 1 To try and escape from th
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Chapter 1 Fig 1.2: Pathways regulat
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Chapter 1 As protein folding in Myc
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Chapter 1 1.4 The molecular chapero
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Chapter 1 1.4.1 Classes of molecula
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Chapter 1 Hsp40 DnaJ CbpA DjlA Ydj1
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Chapter 1 obtained. These revertant
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Chapter 1 1.4.3.1 The ribosome asso
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Chapter 1 (A) (B) Fig 1.7: Structur
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Chapter 1 complete the reaction cyc
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Chapter 1 1.4.3.4 The small Hsps On
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Chapter 1 1.4.3.5 Hsp90 family of c
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Chapter 1 Fig 1.11: Structure of Ht
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Chapter 1 1.4.3.6 Hsp100 family of
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Chapter 1 Fig 1.13: The different r
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Chapter 1 encoded by more than one
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Chapter 1 Fig 1.15: The illustratio
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Chapter 1 (Rommelaere et al., 1993,
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Chapter 1 Fig 1.16: Octameric struc
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Chapter 1 main difference between t
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Chapter 1 1.5.3.3 Prefoldin It has
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Chapter 1 result the substrate prot
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Chapter 1 Fig 1.18: The structures
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Chapter 1 6- If the released peptid
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Chapter 1 1.5.4.3 GroEL substrates
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Chapter 1 Recent work has suggested
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Chapter 1 (A) cpn10 cpn60.1 cpn60.2
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Chapter 1 1998). Cpn60.1 however ha
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Chapter 1 1.6 Aims of the project T
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Chapter 2 Materials and Methods: 2.
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Chapter 2 pET-cpn10- cpn60.1 Deriva
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Chapter 2 2.3 Primers: Below is a l
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Chapter 2 Mut EL.3 Rev groEL 1401-1
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Chapter 2 29R-EL-60.2 groEL 432-412
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Chapter 2 Biofilm base media: 13.6g
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Chapter 2 Preparation of bacterioph
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Chapter 2 then resuspended in 200µ
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Chapter 2 - 10X BSA (depending on t
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Chapter 2 - Colony/culture 1μl - S
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Chapter 2 The PCR cycling parameter
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Chapter 2 tubes were placed on ice
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Chapter 2 2.6 Protein analysis 2.6.
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Chapter 2 2.6.2- Native gel Reagent
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Chapter 2 - Diluted primary antibod
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Chapter 2 supernatant was decanted
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Chapter 2 2.6.6- Analytical ultrace
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Chapter 3 Background As discussed a
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Chapter 3 the -35 region of the trp
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Chapter 3 20 mins 40 mins 60 mins 8
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Chapter 3 (a) 60 mins 90 mins 120 m
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Chapter 3 For this experiment, MGM1
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Chapter 3 When IPTG was not include
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Chapter 3 The results from these co
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Chapter 3 3.2.1 The effect of chang
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Chapter 3 3.2.2 Using pRARE plasmid
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Chapter 3 3.3 Complementation and e
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Chapter 3 of MGM100. The use of Tab
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Chapter 3 3.3.1 Using pET plasmid i
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Chapter 3 1 2 3 4 5 6 7 8 95 kDa 72
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Chapter 3 it only digests the paren
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Chapter 3 72 kDa 1 2 3 4 5 55 kDa 1
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Chapter 3 30°C/26°C 37°C MGM100
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Chapter 3 Fig 3.12: Comparison of t
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Chapter 3 AI90/pACYC-Ptrc-GroESL-Sa
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Chapter 3 1 2 3 4 5 6 7 1000 bp 600
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Chapter 3 10 -1 10 -2 10 -3 10 -4 1
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Chapter 3 this observation. The res
Page 159 and 160: Chapter 3 expression. However, from
Page 161 and 162: Chapter 4 Construction and analysis
Page 163 and 164: Chapter 4 Background In the last ch
Page 165 and 166: Chapter 4 Experimental design To ma
Page 167 and 168: Chapter 4 Cpn60.2 7 8 9 Cpn60.1 1 2
Page 169 and 170: Chapter 4 The full list of all the
Page 171 and 172: Chapter 4 4.1 Imprecise domain swap
Page 173 and 174: Chapter 4 4.1.1 Chimera AEC This is
Page 175 and 176: Chapter 4 (a) Dilutions 10 -1 10 -2
Page 177 and 178: Chapter 4 of GroEL (fig 4.7). This
Page 179 and 180: Chapter 4 (a) Dilutions 10 -1 10 -2
Page 181 and 182: Chapter 4 4.1.5 Chimeras DHF & GEI
Page 183 and 184: Chapter 4 4.2 Precise domain swaps
Page 185 and 186: Chapter 4 intermediate domains were
Page 187 and 188: Chapter 4 1 2 800 kDa 480 kDa 146 k
Page 189 and 190: Chapter 4 Dilutions 10 -2 10 -3 10
Page 191 and 192: Chapter 4 is important to note that
Page 193 and 194: Chapter 4 Summary the complementati
Page 195 and 196: Chapter 4 The other aspect of this
Page 197 and 198: Chapter 4 caused due to lack of exp
Page 199 and 200: Chapter 4 structures were not expec
Page 201 and 202: Chapter 4 Name Construct Blue -Cpn6
Page 203 and 204: Chapter 5 Background As discussed i
Page 205 and 206: Chapter 5 macromolecules at equilib
Page 207 and 208: Chapter 5 5.1 Purification of JNL 5
Page 209: Chapter 5 (a) 1 2 3 4 5 6 7 8 9 10
Page 213 and 214: Chapter 5 5.2 AUC analysis of JNL U
Page 215 and 216: Chapter 5 (a) 1 JNL in Buffer 5 0.9
Page 217 and 218: Chapter 5 (a) (b) 100 mM P i 75 mM
Page 219 and 220: Chapter 5 In this experiment GroEL
Page 221 and 222: Chapter 5 1 2 3 Corrected values Av
Page 223 and 224: Chapter 5 expected, evidence of lar
Page 225 and 226: Chapter 5 In summary, based on the
Page 227 and 228: Chapter 6 In this chapter I will be
Page 229 and 230: Chapter 6 of Mscpn60.1 can indirect
Page 231 and 232: Chapter 6 can produce products with
Page 233 and 234: Chapter 6 As the group in Pittsburg
Page 235 and 236: Chapter 6 Fig 6.3: Masses of 4 day
Page 237 and 238: Chapter 6 than 7 days, so there is
Page 239 and 240: Chapter 6 are required to facilitat
Page 241 and 242: Chapter 6 Fig 6.4: Sequence alignme
Page 243 and 244: Chapter 6 To ensure that the lack o
Page 245 and 246: Chapter 6 6.2.3 Complementation ana
Page 247 and 248: Chapter 6 Discussion The double rin
Page 249 and 250: Chapter 6 Section 6.3: Testing the
Page 251 and 252: Chapter 6 Experimental design For t
Page 253 and 254: Chapter 6 EcoRV groEL (1.6kb) HindI
Page 255 and 256: Chapter 6 6.3.2 Complementation res
Page 257 and 258: Chapter 6 1 2 3 4 72 kDa 55 kDa Fig
Page 259 and 260: Chapter 6 10 -1 10 -2 10 -3 10 -4 1
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Chapter 6 Discussion The results th
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Summary The main aim of this study
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together, these results strongly su
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Basha, E., K. L. Friedrich & E. Vie
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Cehovin, A., A. R. Coates, Y. Hu, Y
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Diaz-Acosta, A., M. L. Sandoval, L.
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Fux, C. A., J. W. Costerton, P. S.
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Gupta, P., S. Mishra & T. K. Chaudh
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Hoffmann, A., F. Merz, A. Rutkowska
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Ito, K., Y. Akiyama, T. Yura & K. S
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Krzewska, J., G. Konopa & K. Libere
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Lin, Z., D. Madan & H. S. Rye, (200
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Mogk, A. & B. Bukau, (2004) Molecul
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Patzelt, H., G. Kramer, T. Rauch, H
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Rommelaere, H., M. Van Troys, Y. Ga
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Strickland, E., B. H. Qu, L. Millen
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Tyagi, N. K., W. A. Fenton & A. L.
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Yamamori, T. & T. Yura, (1982) Gene
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