Derecho Comparado

To examine the insert size distribution of the affinity-selected phagemid clones, bacterial colony PCR was performed (as described in section 2.2.2.5.1) on 20 randomly picked colonies from each of the 19 transformations. In total, 380 independent colonies of transformants were analysed (Figure 4.2). The insert sizes ranged from 0.3 – 2.5 Kb. Forty randomly selected recombinant phagemids (Figure 4.2, red arrows: 5 from the first round of panning and 19 from the fourth round of panning on AXYL, and 7 from first round of panning and 9 from fourth round of panning on RAC) contained inserts of varying sizes, and were further analysed by DNA sequencing.

Figure 4.2 Bacterial colony PCR of 380 clones selected from the metagenomic phage display library by affinity screening against complex carbohydrate substrates.

Lanes 1 – 380 correspond to PCR amplicons from randomly picked colonies obtained through transformations with 19 extracted DNA bands indicated in Figure 4.1. Abbreviations: RAC,

2kb 1kb 0.3kb + 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 L + 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 L + - 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 9 9 100 L L + 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 L +141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 1 71 172 173 174 175 176 177 178 179 180 L + 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 L + 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 2 51 252 253 254 255 256 257 258 259 260 L + 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 L +301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 33 1 332 333 334 335 336 337 338 339 340 L +341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 37 0 371 372 373 374 375 376 377 378 379 380 2kb 1kb 0.3kb 2kb 1kb 0.3kb 2kb 1kb 0.3kb 3kb 2kb 1kb 0.3kb 3kb 2kb 1kb 0.3kb 3kb 2kb 1kb 0.3kb 2kb 1kb 0.3kb 2kb 1kb 0.3kb 2kb 1kb 0.3kb

157 regenerated amorphous cellulose; AXYL, insoluble wheat arabinoxylan; A, acidic elution; B, basic elution; T, elution with host E. coli TG1; 1 – 4, first to fourth round of panning. Lanes: 1 – 60 RAC T4; 61 – 100 and 361 – 380 AXYL B4; 101 – 140 RAC A4; 141 – 200 and 301 – 320 RAC B4; 201 – 260 AXYL T4; 261 – 280 RAC A1; 281 – 300 RAC B1; 321 – 340 RAC T1; 341 – 360 AXYL B1; L, 1 Kb Plus DNA ladder (Life Technologies); +, PCR positive control (PCR amplicon from TG1 colony transformed with vector pDJ01); –, PCR negative control. Red arrows indicate sequenced inserts.

Sequencing was performed with an antisense primer (pspR03; see section 2.1.4) complementary to the pDJ01 vector sequence downstream of the insert. This primer anneals

within the vector gIII gene, encoding virion protein pIII that serves as a platform for display of

peptides in fusion with pIII. Thus, sequencing with pspR03 allows the joint between the insert

and the vector in the insert-gIII fusion to be determined. If the 5' end of the fusion ORF was not

reached, sequencing with a sense primer (pspF03; see section 2.1.4) was performed. The 40 sequenced recombinant phagemid inserts were analysed as described in section 2.2.7.3, and a detailed overview is represented in Appendix 3.

Of the 40 sequenced inserts, 24 contained ORFs in frame with gIII. Of these, 13 inserts

contained distinct ORF encoding putative proteins longer than 24 amino acid residues. Eleven inserts contained 10 short distinct ORFs encoding putative peptides and proteins (≤24 amino acid residues long) that were considered ‘background’ (Table 4.4). A large proportion of inserts (40%) contained distinct ORFs out of frame with gIII, that were not expected to be captured by affinity selection, and they also represent ‘background’.

Table 4.4 Distribution of 40 analysed inserts in regard to ORF frame status.

Abbreviations: RAC, regenerated amorphous cellulose; AXYL, insoluble wheat arabinoxylan; aa, amino acid residues. a In frame, ORFs encoding putative proteins >24 aa in frame with

vector-encoded pIII; b Background (≤24 aa), ORFs encoding putative proteins and peptides ≤24

aa in frame with vector-encoded pIII; c Background (Out of frame), ‘background’ inserts

containing ORFs encoding putative proteins and peptides that are not in frame with vector- encoded pIII; Total, total ORFs analysed per round.

ORF status RAC 1st RAC 4th AXYL 1st AXYL 4th

Count % Count % Count % Count % In frame a 3 42.9 1 11.1 2 40 7 36.8 Background b (≤24 aa) 3 42.9 2 22.2 2 40 4 21.1 Background c (Out of frame) 1 14.3 6 66.7 1 20 8 42.1 Total 7 100 9 100 5 100 19 100

Selection for ‘background’ inserts containing ‘out of frame’ ORFs during panning procedure has been reported previously [437-439]. It has been hypothesised that some of these ‘out of frame’ constructs may have a selective advantage, and programmed ribosomal frameshifting (PRF) events have been implicated [440, 441]. PRF is a translational recoding mechanism involving ribosomal slippage that leads to a switch of reading frame in –1 and +1 directions, or translation past stop codons when a ribosome encounters a specific signal in the mRNA sequence [442]. Strong stimulators of –1 PRF are a combination of a heptameric ‘slippery sequence’ of the type X XXY YYZ (where X represents any nucleotide, Y represents A or U and Z represents A, U, or C), a 1 – 15 nucleotide long spacer sequence, and a sequence that can form a stable secondary hairpin structure (pseudoknot).

To investigate whether the putative proteins encoded by ‘out of frame’ ORFs could have been brought in frame with pIII due to PRF events, resulting in their display and capture by affinity selection, inserts were inspected for putative sites that can stimulate –1 PRF using the KnotInFrame algorithm [407]. Around 31% of all inserts that contained ORFs out of frame with gIII had predicted –1 frameshift sites. However, none of the –1 PRF events stimulated from these putative sites could have led to a frameshift into the pIII frame and, for this reason, could have not contributed to display of putative proteins encoded by the ‘out of frame’ ORFs.

Putative proteins longer than 24 amino acid residues in frame with pIII were further analysed for the presence of membrane-targeting signals as described in section 2.2.7.3 (Appendix 3). None of the analysed putative proteins captured in the first and the last round of panning on RAC were predicted to contain membrane-targeting signals. In contrast, among the 19 putative proteins captured in the last round of panning on AXYL, two contained putative type I signal sequences, and one contained a predicted N-terminal transmembrane helix. In addition, four putative proteins (two captured in first, and two captured in the last round of panning on AXYL), that did not contain a signal sequence, or transmembrane helices, had

SecretomeP 2.0 [259] scores higher than 0.5, indicating their possible secretion via non-classical

pathways. Based on this analysis, five recombinant phagemids were selected for the affinity binding assays to AXYL.

To explain the high proportion of putative proteins without predicted membrane- targeting signals, or shorter than 24 amino acid residues, putative proteins encoded by ORFs in

frame with gIII were scanned for motifs conferring possible competitive advantage and/or

substrate-unrelated binding as described in section 2.2.7.3. Around 30% of all putative proteins in frame with pIII from the first round and 46% from the fourth round of panning on both substrates were predicted to confer possible propagation advantage and/or putative motifs for binding to unrelated targets such as plastic or IgG class of antibodies, which are present as low-level contaminants in the BSA used for substrate blocking during panning (Appendix 3). When putative proteins containing these motifs from the first and last round of panning are

159 compared, the overall proportion of putative proteins encoded by background ORFs increased from 10% in the first, to 23% in the last round, while the proportion of putative proteins longer than 24 amino acid residues was the same.

Functional annotation of the 13 identified ORFs encoding putative proteins longer than 24 amino acid residues in frame with pIII, based on best BLASTP (E-value <1e-05) against the sequences in the NCBI nr protein database, resulted in a large number of assignments to conserved hypothetical (5) and hypothetical (3) proteins. One ORF (AXYL236) encoded a putative protein involved in protein transport across the membrane, while four ORFs (AXYL225, RAC261, RAC263 and AXYL342 were predicted to encode putative enzymes

(Appendix 3). No putative CBMs were identified through a HMM-based search via dbCAN

web server and BLASTP searches against CAZymes in the dbCAN and CAT databases.