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Production of bioactive myostaitn propeptide of variouis animal species in E. coli Yong soo Production of bioactive myostaitn propeptide of variouis animal species in E. coli Yong soo Kim, Professor Department of Human Nutrition, Food and Animal Sciences University of Hawaii, Manoa

Myostatin (MSTN) n n 1997, Se-jin Lee’s Lab at the Johns Hopkins University A Myostatin (MSTN) n n 1997, Se-jin Lee’s Lab at the Johns Hopkins University A member of the TGF-β superfamily growth and differentiation proteins, also called GDF-8 Natural, non-functional mutation

Ø MSTN is a potent negative regulator of muscle growth Ø MSTN suppression can Ø MSTN is a potent negative regulator of muscle growth Ø MSTN suppression can be a strategy to improve skeletal muscle growth in livestock and to treat muscle atrophic conditions in human n Genetic approach • Transgenesis • si. RNA • Transient transfection n Protein administration • Antibodies • MSTN suppressors: propeptide, Act. RIIB, follistatin, FLRG (FSTL 3)

 • Production of Bioactive MSTN-suppressing proteins in E. coli • Myostatin Propeptide (MSTNPro) • Production of Bioactive MSTN-suppressing proteins in E. coli • Myostatin Propeptide (MSTNPro) • Follistatin MSTNPro: $329/25 ug Follistatin: $329/25 ug Mouse study: 2, 400 ug ($31, 584) 6 animals x 4 injections x 100 ug/10 g mouse (10 mg/kg body wt) Pig study: 204 mg ($2. 68 Mil) 6 animals x 2 injections x 17 mg/1. 7 kg new-born pig (10 mg/kg body wt)

Biosynthesis and processing of MSTN n Pre-propeptide, 375 aa (42 – 50 k. Da) Biosynthesis and processing of MSTN n Pre-propeptide, 375 aa (42 – 50 k. Da) • Synthesized as a pre-propeptide ER secretion signal (23 aa) Prodomain (243 aa) Myostatin (109 aa) COOH NH 3 N-glycosylation site (aa 71) n Proteolytic processing site (aa 266) Propeptide complex formation with MSTN

Production of pig MSTNPro in E. coli Unprocessed MSTN (pig) TOPO-TA clone PCR cloning Production of pig MSTNPro in E. coli Unprocessed MSTN (pig) TOPO-TA clone PCR cloning PCR Mutagenesis (D 99 A) MSTNpro. M MSTNpro. W p. MAL-c 5 vector RE digestion (Xmn 1/Bam. HI) ligation p. MALc 5 -MSTNpro. W p. MALc 5 -MSTNpro. M E. coli expression

SDS-PAGE Analysis of the expression of MBP-p. MSTNpro proteins K 12 TB 1 (NEB), SDS-PAGE Analysis of the expression of MBP-p. MSTNpro proteins K 12 TB 1 (NEB), at 0. 7 -0. 8 OD 600, 4 hr at 37 o. C Haq et al. , 2013 Appl Microbiol. Biotechnol

Purification of MBP-p. MSTNPro proteins Amylsoe affinity k. Da Ion-exchange Haq et al. , Purification of MBP-p. MSTNPro proteins Amylsoe affinity k. Da Ion-exchange Haq et al. , 2013 Appl Microbiol. Biotechnol

SDS-PAGE and Western blot analysis of the p. MSTNPro proteins after Factor Xa cleavage SDS-PAGE and Western blot analysis of the p. MSTNPro proteins after Factor Xa cleavage (A) Coomassie Blue W M Factor Xa IU IC − M AF 2 + IU IC − AF 2 Pro MBP FXa + 80 k. Da 58 k. Da * 46 k. Da 30 k. Da 25 k. Da (C) Anti-prodomain (B) Anti-MBP W IU Factor Xa IC − + M AF 2 IU IC − + M W AF 2 IU Pro MBP FXa Factor Xa 80 k. Da 58 k. Da 46 k. Da 30 k. Da 25 k. Da IC − + AF 2 IU − IC AF 2 Pro MBP FXa + *

Mutant confirmation and bioactivity examination BMP-1 digestion p. GL 3 -(CAGA)12 luciferase assay Mutant confirmation and bioactivity examination BMP-1 digestion p. GL 3 -(CAGA)12 luciferase assay

Yields of p. MSTNPro proteins recovered from each purification step Yields of p. MSTNPro proteins recovered from each purification step

Potency of MSTNPro from different animal species Potency of MSTNPro from different animal species

Production of truncated and Fc-fused pig MSTNPro p. MALc 5 x plasmid Untruncated 23 Production of truncated and Fc-fused pig MSTNPro p. MALc 5 x plasmid Untruncated 23 -266 (MBP-p. MSTNPro. M) 42 -218 (MBP-p. MSTNPro 42 -218) 42 -175 (MBP-p. MSTNPro 42 -175) 42 -115 (MBP-p. MSTNPro 42 -115) 42 -98 (MBP-p. MSTNPro 42 -98) Ligation Transformation # NEB 5 a Transformation Expression and purification of Proteins K 12 TB 1 / NEB Plasmid extraction Sequencing

SDS-PAGE analysis of truncated MBP-p. MSTNPro. M proteins k. Da 250 150 M 1 SDS-PAGE analysis of truncated MBP-p. MSTNPro. M proteins k. Da 250 150 M 1 2 3 4 5 M : protein marker (bio-rad) 1 : MBP-p. MSTNPro. M 2 : MBP-p. MSTNPro. M 42 -218 3 : MBP-p. MSTNPro. M 42 -175 4 : MBP-p. MSTNPro. M 42 -115 5 : MBP-p. MSTNPro. M 42 -98 100 75 50 37 At 25 o. C for 15 hr, 0. 4 -0. 6 OD 600 MBP-p. MSTN Pro. M 42 -218 MBP-p. MSTN Pro. M 42 -175 mg/L culture Yield (%) Soluble 110 ± 2. 0 103 ± 5. 6 100 129 ± 1. 9 100 118 ± 3. 0 100 Amylose 7. 98 ± 0. 390 7. 3 9. 83 ± 0. 476 9. 5 32. 2 ± 1. 98 25 38 ± 1. 85 32 Step MBP-p. MSTN Pro. M 42 -115 MBP-p. MSTN Pro 42 -98

Bioactivity of truncated pig MBPMSTNPro IC 50 for 1 n. M MSTN inhibition, n. Bioactivity of truncated pig MBPMSTNPro IC 50 for 1 n. M MSTN inhibition, n. M 1 2. 2 a 2 2. 8 a 42 -218 3. 3 a 42 -175 2. 7 a 42 -115 14. 7 b 42 -98 24. 1 b Jiang et al. , 2004

production of Fc-fused pig MSTNPro • Advantage of Ig. G Fc fusion • Increase production of Fc-fused pig MSTNPro • Advantage of Ig. G Fc fusion • Increase … • half-life of recombinant protein in circulation • therapeutic duration of treatment

Bioactivity of pig MBP-MSTNPro-Fc Bioactivity of pig MBP-MSTNPro-Fc

Production of Follistatin (FST) in E. coli FST 315 FST 303 FST 288 Production of Follistatin (FST) in E. coli FST 315 FST 303 FST 288

Production of chicken FST 315 in E. coli (Lee er al. , 2014. Appl. Production of chicken FST 315 in E. coli (Lee er al. , 2014. Appl. Microbiol. Biotech 98: 10041 -10051) (5. 8 mg/L) A) MSTN inhibition B) Activin inhibition IC 50 for 1 n. M MSTN inhibition, n. M 1 0. 32 a 3 8. 85 b 4 5. 89 b 5 16. 46 b IC 50 for 1 n. M activin inhibition, n. M 1 0. 17 a 3 2. 47 b 4 1. 86 b 5 6. 67 b

Conclusion • Myostatin propetide (MSTNPro) can be produced in a cost-effective way in an Conclusion • Myostatin propetide (MSTNPro) can be produced in a cost-effective way in an E. coli system • The easy availability of MSTPro will allow us to investigate the potentials of MSTNPro as an agent to improve skeletal muscle growth of meat-producing animals.

Acknowledgement Dr. Dr. Shihuan Kuang, Purdue University Bruria Funkenstein, Israel Oceanographic and Limnological Research Acknowledgement Dr. Dr. Shihuan Kuang, Purdue University Bruria Funkenstein, Israel Oceanographic and Limnological Research Hyung. Joo Jin’s lab, Gangneung-Wonju National University, Korea Yun. Jaie Choi’s lab, Seoul National University, Korea Dr. Sang. Beum Lee Dr. Yunkyung Lee Naveen Bobbili Mandy Haq Rocky Choi Thanks 谢谢

Effects of In-ovo Injection of Monoclonal Anti-myostatin Antibody (m. Ab-c 134) on Post-hatch Chicken Effects of In-ovo Injection of Monoclonal Anti-myostatin Antibody (m. Ab-c 134) on Post-hatch Chicken Growth and Muscle Mass Y S Kim, N K Bobbili, K S Paek and H J Jin Poultry Sci. 85: 1062 -1071 (2006)

n Production of monoclonal anti-MSTN antibody (m. Abc 134 ) • Antigen: Semi-purified recombinant n Production of monoclonal anti-MSTN antibody (m. Abc 134 ) • Antigen: Semi-purified recombinant c-terminal fragment MSTN from E. coli • Production: UH Monoclonal Antibody Core Facility n n 6 positives from 120 hybridoma clones Ascites fluid at the UH Manoa Monoclonal Antibody Core Facility Affinity purification with protein A Binding to some members of TGF- superfamily rm. GDF 8, rh. TGF- 3, rh. BMP 2, and p. TGF- 1 (100 ng) Anti-MSTN antibodies: m. Ab-c 134 (B, 1 µg/ml), R&D Systems (C, 0. 3 µg/ml), Santa Cruz (D, 1 µg/ml) and polyclonal anti-MSTN Ab (E, 0. 1 µg/ml)

n Analysis of binding characteristics of m. Ab-c 134 in competitive ELISA • • n Analysis of binding characteristics of m. Ab-c 134 in competitive ELISA • • • n Coating antigen: solubilized recombinant mature MSTN (20 ug/ml) Competing antibody: m. Ab-c 134 (30 ug/ml) Competing ligand: GDF 8 in PBS containing 0. 1% BSA 800, 160, 32, 6. 4, 1. 28, 0. 256, 0. 0512 n. M Inhibition of MSTN activity by m. Ab-c 134 in p. GL 3(CAGA)12 -Lux reporter assay

Injection and hatching Injection: 40 µg/50 µl PBS at 3 d after incubation Areas Injection and hatching Injection: 40 µg/50 µl PBS at 3 d after incubation Areas of injection: albumen and yolk

Post hatch body weights of broilers n n In multi-stack wire cage in a Post hatch body weights of broilers n n In multi-stack wire cage in a temp-controlled room (26. 6 o. C) First 3 wk, 22% prot, 1% lysine, 0. 45 % methionine, then switched to 16% prot, 0. 5% lysine, and 0. 25% methionine Sacrifice at 5 wk Dressing %, wt of breast muscle, leg (thigh plus leg bone-in), other organs

On muscle and organ weights On muscle and organ weights

Feed consumption and feed efficiency Feed consumption and feed efficiency