IP Library › Granted Patent US 7,947,480

Granted Patent B2

US 7,947,480 · App. 12/875,342 · Granted May 24, 2011

Everninomicin biosynthetic genes

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Quick Facts

Patent No.: US 7,947,480
App. No.: 12/875,342
Granted: May 24, 2011
Kind: B2

External Links

USPTO Patent Center↗ Full Patent PDF↗

Abstract

This invention is directed to nucleic acids which encode the proteins that direct the synthesis of the orthosomycin everninomicin and to use of the nucleic acids and proteins to produce compounds exhibiting antibiotic activity based on the everninomycin structure. The DNA sequence for the gene clusters responsible for encoding everninomicin biosynthetic genes, which provide the machinery for producing everninomicin, are provided. Thus, this invention provides the nucleic acid sequences needed to synthesize novel everninomicin-related compounds based on everninomicin, arising from modifications of the DNA sequence designed to change glycosyl and modified orsellinic acid groups contained in everninomicin. A Micromonospora site-specific integrase gene is also provided, which can be incorporated in a vector for integration into any actinomycete, and, particularly into Monospora . Thus, the invention further provides methods for introducing heterologous genes into an actinomycete chromosome using this particular vector.

Claims (205)

1. An isolated polypeptide comprising the amino acid sequence:

(SEQ ID NO: 7)

Val Ala Ala Asp Leu Arg Ala Pro Leu Thr Pro Ala

1 5 10

Gly Arg Thr Val Val Asp Leu Leu Ala Gly Val Ile

15 20

Pro Arg Ile Ser Ala Glu Ala Ala Asp Arg Asp Arg

25 30 35

Thr Gly Thr Phe Pro Val Glu Ala Phe Glu Gln Phe

40 45

Ala Lys Leu Gly Leu Met Gly Ala Thr Val Pro Ala

50 55 60

Glu Leu Gly Gly Leu Gly Leu Thr Arg Leu Tyr Asp

65 70

Val Ala Thr Ala Leu Met Arg Leu Ala Glu Ala Asp

75 80

Ala Ser Thr Ala Leu Ala Trp His Val Gln Leu Ser

85 90 95

Arg Gly Leu Thr Leu Thr Tyr Glu Trp Gln His Gly

100 105

Thr Pro Pro Val Arg Ala Met Ala Glu Arg Leu Leu

110 115 120

Arg Ala Met Ala Glu Gly Glu Ala Ala Val Cys Gly

125 130

Ala Leu Lys Asp Ala Pro Gly Val Val Thr Glu Leu

135 140

His Ser Asp Gly Ala Gly Gly Trp Leu Leu Ser Gly

145 150 155

Arg Lys Val Leu Val Ser Met Ala Pro Ile Ala Thr

160 165

His Phe Phe Val His Ala Gln Arg Arg Asp Asp Asp

170 175 180

Gly Ser Val Phe Leu Ala Val Pro Val Val His Arg

185 190

Asp Ala Pro Gly Leu Thr Val Leu Asp Asn Trp Asp

195 200

Gly Leu Gly Met Arg Ala Ser Gly Thr Leu Glu Val

205 210 215

Val Phe Asp Arg Cys Pro Val Arg Ala Asp Glu Leu

220 225

Leu Glu Arg Gly Pro Val Gly Ala Arg Arg Asp Ala

230 235 240

Val Leu Ala Gly Gln Thr Val Ser Ser Ile Thr Met

245 250

Leu Gly Ile Tyr Ala Gly Ile Ala Gln Ala Ala Arg

255 260

Asp Ile Ala Val Gly Phe Cys Ala Gly Arg Gly Gly

265 270 275

Glu Pro Arg Ala Gly Ala Arg Ala Leu Val Ala Gly

280 285

Leu Asp Thr Arg Leu Tyr Ala Leu Arg Thr Thr Val

290 295 300

Gly Ala Ala Leu Thr Asn Ala Asp Ala Ala Ser Val

305 310

Asp Leu Ser Gly Asp Pro Asp Glu Arg Gly Arg Arg

315 320

Met Met Thr Pro Phe Gln Tyr Ala Lys Met Thr Val

325 330 335

Asn Glu Leu Ala Pro Ala Val Val Asp Asp Cys Leu

340 345

Ser Leu Val Gly Gly Leu Ala Tyr Thr Ala Gly His

350 355 360

Pro Leu Ser Arg Leu Tyr Arg Asp Val Arg Ala Gly

365 370

Gly Phe Met Gln Pro Tyr Ser Tyr Val Asp Ala Val

375 380

Asp Tyr Leu Ser Gly Gln Ala Leu Gly Leu Asp Arg

385 390 395

Asp Asn Asp Tyr Met Ser Val Arg Ala Leu Arg Ser

400 405

Arg Thr Ser Ala.

410

2. An isolated polypeptide that is encoded by a polynucleotide that hybridizes to the full length complement of a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 6 under high stringency conditions, said conditions being in the presence of 50% formamide and 0.2×SSC at 68° C.; wherein SCC is a 0.15 M NaCl, 0.015 M Na + citrate.

3. The polypeptide of claim 1 which is a chimeric polypeptide fused to a heterologous polypeptide.

4. The polypeptide of claim 3 wherein the heterologous polypeptide is a member selected from the group consisting of a poly-histidine tag, a FLAG tag, a glutathione-S-transferase tag and a myc epitope tag.

5. A method for making an isolated polypeptide comprising the amino acid sequence:

Val Ala Ala Asp Leu Arg Ala Pro Leu Thr Pro Ala Gly Arg Thr Val

1 5 10 15

Val Asp Leu Leu Ala Gly Val Ile Pro Arg Ile Ser Ala Glu Ala Ala

20 25 30

Asp Arg Asp Arg Thr Gly Thr Phe Pro Val Glu Ala Phe Glu Gln Phe

35 40 45

Ala Lys Leu Gly Leu Met Gly Ala Thr Val Pro Ala Glu Leu Gly Gly

50 55 60

Leu Gly Leu Thr Arg Leu Tyr Asp Val Ala Thr Ala Leu Met Arg Leu

65 70 75 80

Ala Glu Ala Asp Ala Ser Thr Ala Leu Ala Trp His Val Gln Leu Ser

85 90 95

Arg Gly Leu Thr Leu Thr Tyr Glu Trp Gln His Gly Thr Pro Pro Val

100 105 110

Arg Ala Met Ala Glu Arg Leu Leu Arg Ala Met Ala Glu Gly Glu Ala

115 120 125

Ala Val Cys Gly Ala Leu Lys Asp Ala Pro Gly Val Val Thr Glu Leu

130 135 140

His Ser Asp Gly Ala Gly Gly Trp Leu Leu Ser Gly Arg Lys Val Leu

145 150 155 160

Val Ser Met Ala Pro Ile Ala Thr His Phe Phe Val His Ala Gln Arg

165 170 175

Arg Asp Asp Asp Gly Ser Val Phe Leu Ala Val Pro Val Val His Arg

180 185 190

Asp Ala Pro Gly Leu Thr Val Leu Asp Asn Trp Asp Gly Leu Gly Met

195 200 205

Arg Ala Ser Gly Thr Leu Glu Val Val Phe Asp Arg Cys Pro Val Arg

210 215 220

Ala Asp Glu Leu Leu Glu Arg Gly Pro Val Gly Ala Arg Arg Asp Ala

225 230 235 240

Val Leu Ala Gly Gln Thr Val Ser Ser Ile Thr Met Leu Gly Ile Tyr

245 250 255

Ala Gly Ile Ala Gln Ala Ala Arg Asp Ile Ala Val Gly Phe Cys Ala

260 265 270

Gly Arg Gly Gly Glu Pro Arg Ala Gly Ala Arg Ala Leu Val Ala Gly

275 280 285

Leu Asp Thr Arg Leu Tyr Ala Leu Arg Thr Thr Val Gly Ala Ala Leu

290 295 300

Thr Asn Ala Asp Ala Ala Ser Val Asp Leu Ser Gly Asp Pro Asp Glu

305 310 315 320

Arg Gly Arg Arg Met Met Thr Pro Phe Gln Tyr Ala Lys Met Thr Val

325 330 335

Asn Glu Leu Ala Pro Ala Val Val Asp Asp Cys Leu Ser Leu Val Gly

340 345 350

Gly Leu Ala Tyr Thr Ala Gly His Pro Leu Ser Arg Leu Tyr Arg Asp

355 360 365

Val Arg Ala Gly Gly Phe Met Gln Pro Tyr Ser Tyr Val Asp Ala Val

370 375 380

Asp Tyr Leu Ser Gly Gln Ala Leu Gly Leu Asp Arg Asp Asn Asp Tyr

385 390 395 400

Met Ser Val Arg Ala Leu Arg Ser Arg Thr Ser Ala

405 410

(SEQ ID NO: 7), said method comprising transforming an isolated host cell with a vector comprising a polynucleotide encoding the polypeptide under conditions wherein the polypeptide is expressed by the host cell.

6. An isolated antibody or fragment thereof that binds specifically to the polypeptide of claim 1 which consists of the amino acid sequence:

(SEQ ID NO: 7)

Val Ala Ala Asp Leu Arg Ala Pro Leu Thr Pro Ala

1 5 10

Gly Arg Thr Val Val Asp Leu Leu Ala Gly Val Ile

15 20

Pro Arg Ile Ser Ala Glu Ala Ala Asp Arg Asp Arg

25 30 35

Thr Gly Thr Phe Pro Val Glu Ala Phe Glu Gln Phe

40 45

Ala Lys Leu Gly Leu Met Gly Ala Thr Val Pro Ala

50 55 60

Glu Leu Gly Gly Leu Gly Leu Thr Arg Leu Tyr Asp

65 70

Val Ala Thr Ala Leu Met Arg Leu Ala Glu Ala Asp

75 80

Ala Ser Thr Ala Leu Ala Trp His Val Gln Leu Ser

85 90 95

Arg Gly Leu Thr Leu Thr Tyr Glu Trp Gln His Gly

100 105

Thr Pro Pro Val Arg Ala Met Ala Glu Arg Leu Leu

110 115 120

Arg Ala Met Ala Glu Gly Glu Ala Ala Val Cys Gly

125 130

Ala Leu Lys Asp Ala Pro Gly Val Val Thr Glu Leu

135 140

His Ser Asp Gly Ala Gly Gly Trp Leu Leu Ser Gly

145 150 155

Arg Lys Val Leu Val Ser Met Ala Pro Ile Ala Thr

160 165

His Phe Phe Val His Ala Gln Arg Arg Asp Asp Asp

170 175 180

Gly Ser Val Phe Leu Ala Val Pro Val Val His Arg

185 190

Asp Ala Pro Gly Leu Thr Val Leu Asp Asn Trp Asp

195 200

Gly Leu Gly Met Arg Ala Ser Gly Thr Leu Glu Val

205 210 215

Val Phe Asp Arg Cys Pro Val Arg Ala Asp Glu Leu

220 225

Leu Glu Arg Gly Pro Val Gly Ala Arg Arg Asp Ala

230 235 240

Val Leu Ala Gly Gln Thr Val Ser Ser Ile Thr Met

245 250

Leu Gly Ile Tyr Ala Gly Ile Ala Gln Ala Ala Arg

255 260

Asp Ile Ala Val Gly Phe Cys Ala Gly Arg Gly Gly

265 270 275

Glu Pro Arg Ala Gly Ala Arg Ala Leu Val Ala Gly

280 285

Leu Asp Thr Arg Leu Tyr Ala Leu Arg Thr Thr Val

290 295 300

Gly Ala Ala Leu Thr Asn Ala Asp Ala Ala Ser Val

305 310

Asp Leu Ser Gly Asp Pro Asp Glu Arg Gly Arg Arg

315 320

Met Met Thr Pro Phe Gln Tyr Ala Lys Met Thr Val

325 330 335

Asn Glu Leu Ala Pro Ala Val Val Asp Asp Cys Leu

340 345

Ser Leu Val Gly Gly Leu Ala Tyr Thr Ala Gly His

350 355 360

Pro Leu Ser Arg Leu Tyr Arg Asp Val Arg Ala Gly

365 370

Gly Phe Met Gln Pro Tyr Ser Tyr Val Asp Ala Val

375 380

Asp Tyr Leu Ser Gly Gln Ala Leu Gly Leu Asp Arg

385 390 395

Asp Asn Asp Tyr Met Ser Val Arg Ala Leu Arg Ser

400 405

Arg Thr Ser Ala.

410

7. A method for making an antibody that specifically binds to the polypeptide of claim 1 comprising immunizing a host animal with said polypeptide.

8. The method of claim 7 wherein the host animal is a rabbit, mouse, rat, sheep or goat.

9. The method of claim 7 wherein the polypeptide is conjugated to an immunogenic carrier.

10. The method of claim 7 further comprising generating a hybridoma from cells of the host animal.

Assignments (1)

CHANGE OF NAME Recorded Aug 30, 2012

From: SCHERING CORPORATION

To: MERCK SHARP & DOHME CORP.

Reel/Frame 028884/0151 →