Production of bioactive myostaitn Production of bioactive myostaitn propeptide of variouis animal species propeptide of variouis animal species
in E. coliin E. coli
Yong soo Kim, ProfessorDepartment of Human Nutrition,
Food and Animal SciencesUniversity of Hawaii, Manoa
Myostatin (MSTN)Myostatin (MSTN) 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
MSTN is a potent negative regulator of muscle growth
MSTN suppression can be a strategy to improve skeletal muscle growth in livestock and to treat muscle atrophic conditions in human
Genetic approach• Transgenesis• siRNA• Transient transfection
Protein administration• Antibodies• MSTN suppressors: propeptide, ActRIIB, follistatin, FLRG
(FSTL3)
• Production of Bioactive MSTN-suppressing proteins in E. coli
• Myostatin Propeptide (MSTNPro)• Follistatin
MSTNPro: $329/25 ugFollistatin: $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 MSTNBiosynthesis and processing of MSTN
Pre-propeptide, 375 aa (42 – 50 kDa)• Synthesized as a pre-propeptide
Propeptide complex formation with MSTNPropeptide complex formation with MSTN
ER secretion signal (23 aa)
Prodomain (243 aa)Myostatin (109 aa)
Proteolytic processing site (aa 266) N-glycosylation site (aa 71)
NH3 COOH
Production of pig MSTNPro in Production of pig MSTNPro in E. coliE. coli
Unprocessed MSTN (pig)
TOPO-TA clone
MSTNproW MSTNproM
pMAL-c5 vectorpMAL-c5 vector
PCR cloningPCR Mutagenesis (D99A)
RE digestion(Xmn1/BamHI)
RE digestion(Xmn1/BamHI)
ligationligation
pMALc5-MSTNproW pMALc5-MSTNproM
E. coli expression
SDS-PAGE Analysis of the expression of MBP-pMSTNpro proteins
K12TB1 (NEB), at 0.7-0.8 OD600, 4 hr at 37oC
Haq et al., 2013 Appl Microbiol. Biotechnol
Purification of MBP-pMSTNPro proteinsPurification of MBP-pMSTNPro proteins
kDa
Amylsoe affinity
Ion-exchange
Haq et al., 2013 Appl Microbiol. Biotechnol
SDS-PAGE and Western blot analysis of the SDS-PAGE and Western blot analysis of the pMSTNPro proteins after Factor Xa cleavagepMSTNPro proteins after Factor Xa cleavage
(B) Anti-MBP (C) Anti-prodomain
(A) Coomassie Blue
M IU IC AF2 IU IC AF2 Pro MBP FXa
Factor Xa − + − +
58 kDa
46 kDa
30 kDa25 kDa
80 kDa
IU IC AF2 IU IC AF2 Pro MBP FXa
Factor Xa − + − +
58kDa
46 kDa
30 kDa25 kDa
80kDa
IU IC AF2 IU IC AF2 Pro MBP FXa
Factor Xa − + − +
58kDa
46 kDa
30 kDa25 kDa
80kDa
W
W WM
M
M
*
*
Mutant confirmation and bioactivity Mutant confirmation and bioactivity examinationexamination
BMP-1 digestion
pGL3-(CAGA)12 luciferase assay
Yields of pMSTNPro proteins recovered from Yields of pMSTNPro proteins recovered from each purification stepeach purification step
Table 1. Yields of pMSTNPro proteins recovered from each purification step.
pMSTNProW pMSTNProM
Steps µg/ml culture Yield (%) µg/ml culture Yield (%)
Total protein 98.9 ± 4.9 100.0 95.5 ± 2.1 100.0
Soluble protein 84.6 ± 3.1 85.8 81.1 ± 2.2 84.9
Affinity chromatography 38.7 ± 7.9 39.1 36.9 ± 1.5 38.7
Ion-exchange chromatography 10.4 ± 0.4 10.5 9.3 ± 0.8 9.8
pMSTNPro after MBP removal1 4.2 ± 0.3 4.3 4.2 ± 0.3 4.4
Protein concentration was measured by Bradford method using BSA as a standard. The protein
recovery was calculated as the mean ± SEM from the purifications of a triplicate of 250 ml
culture of each type of pMSTNPro proteins.
Potency of MSTNPro from different animal species
Production of truncated and Fc-fused pig MSTNPro
Ligation
# NEB5a
SequencingK12 TB1 / NEB
Transformation
Expression and purification of Proteins
pMALc5x plasmid
Plasmid extractionTransformation
Untruncated 23-266 (MBP-pMSTNProM)
42-218 (MBP-pMSTNPro42-218)
42-175 (MBP-pMSTNPro42-175)
42-115 (MBP-pMSTNPro42-115)42-98 (MBP-pMSTNPro42-98)
SDS-PAGE analysis of truncated MBP-pMSTNProM proteinsSDS-PAGE analysis of truncated MBP-pMSTNProM proteins
250150
100
75
50
37
kDa M 1 2 3 4 5 M : protein marker (bio-rad)1 : MBP-pMSTNProM2 : MBP-pMSTNProM 42-2183 : MBP-pMSTNProM 42-1754 : MBP-pMSTNProM 42-1155 : MBP-pMSTNProM 42-98
At 25oC for 15 hr, 0.4 -0.6 OD600
StepMBP-pMSTNProM42-218
MBP-pMSTNProM42-175
MBP-pMSTNProM42-115
MBP-pMSTNPro42-98
mg/L culture
Yield(%)
mg/L culture
Yield(%)
mg/L culture
Yield(%)
mg/L culture
Yield(%)
Soluble 110 ± 2.0 100 103 ± 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
BioactivityBioactivity of truncated pig MBP- of truncated pig MBP-MSTNProMSTNPro
IC50 for 1 nM MSTN inhibition, nM
1 2 42-218 42-175 42-115 42-98 2.2 a 2.8a 3.3a 2.7a 14.7b 24.1b
Ligand concentration (Log pM)
% I
nh
ibit
ion
0 1 2 3 4 50
20
40
60
80
100
1 Commercial Pro2 MBP-pigMSTNProM3 MBP-pigMSTNProM 42-2184 MBP-pigMSTNProM 42-1755 MBP-pigMSTNProM 42-1156 MBP-pigMSTNPro 42-98
Jiang et al., 2004
Jiang et al., 2004
production of Fc-fused pig MSTNPro
• Advantage of IgG Fc fusion• Increase …
• half-life of recombinant protein in circulation• therapeutic duration of treatment
Bioactivity of pig MBP-MSTNPro-Fc of pig MBP-MSTNPro-Fc
Production of Follistatin (FST) in E. coliProduction of Follistatin (FST) in E. coli
FST315FST303FST288
Production of chicken FST315 in E. coliProduction of chicken FST315 in E. coli(Lee er al., 2014. Appl. Microbiol. Biotech 98:10041-10051)(Lee er al., 2014. Appl. Microbiol. Biotech 98:10041-10051)
0 1 2 3 4 50
20
40
60
80
100
¨¨
¨¨
¨¨
¨
¨
¨
¨
Ligand concentration, nM
% I
nh
ibit
ion
Commercial rhFST315 (1)Commercial rhFST288 E. coli (2)
MBP-chFST315 (H-A) (3)MBP-chFST315 (H-A-G) (4)
chFST315 (5)¨
1 3 4 5 0.32a 8.85b 5.89b 16.46b
IC50 for 1 nM MSTN inhibition, nM
1 3 4 5 0.17a 2.47b 1.86b 6.67b
IC50 for 1 nM activin inhibition, nM
Ligand concentration (Log pM)
% I
nh
ibit
ion
0 1 2 3 4 50
20
40
60
80
100
0 1 2 3 4 50
20
40
60
80
100
¨¨
¨¨
¨¨
¨
¨
¨
¨
% In
hibi
tion
Ligand concentration (Log pM)
A) MSTN inhibition B) Activin inhibition
(5.8 mg/L)
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.
Thanks
Acknowledgement
Dr. Shihuan Kuang, Purdue UniversityDr. Bruria Funkenstein, Israel Oceanographic and Limnological ResearchDr. HyungJoo Jin’s lab, Gangneung-Wonju National University, KoreaDr. YunJaie Choi’s lab, Seoul National University, Korea
Dr. SangBeum LeeDr. Yunkyung LeeNaveen BobbiliMandy Haq Rocky Choi
谢谢
Effects of In-ovo Injection of Monoclonal Effects of In-ovo Injection of Monoclonal Anti-myostatin Antibody (mAb-c134) on Anti-myostatin Antibody (mAb-c134) on Post-hatch Chicken Growth and Muscle Post-hatch Chicken Growth and Muscle
MassMass
Y S Kim, N K Bobbili, K S Paek and H J Jin Y S Kim, N K Bobbili, K S Paek and H J Jin Poultry Sci. Poultry Sci. 85:1062-107185:1062-1071 (2006)(2006)
Production of monoclonal anti-MSTN antibody (mAb-c134 )• Antigen: Semi-purified recombinant c-terminal fragment MSTN from E. coli• Production: UH Monoclonal Antibody Core Facility
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
rmGDF8, rhTGF-3, rhBMP2, and pTGF-1 (100 ng)
Anti-MSTN antibodies: mAb-c134 (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)
Analysis of binding characteristics of mAb-c134 in competitive ELISA
• Coating antigen: solubilized recombinant mature MSTN (20 ug/ml) • Competing antibody: mAb-c134 (30 ug/ml)• Competing ligand: GDF8 in PBS containing 0.1% BSA 800, 160, 32, 6.4, 1.28, 0.256, 0.0512 nM
10 -12.5 10 -10.0 10 -7.5 10 -5.0-25
0
25
50
75
100
GDF8, M
% i
nh
ibit
ion
Inhibition of MSTN activity by mAb-c134 in pGL3-(CAGA)12-Lux reporter assay
5 50 5000
10000
20000
30000
40000
50000
mAb-c134 concentration, ng/ml
RU
L
Injection and hatchingInjection and hatching
Injection: 40 µg/50 µl PBS at 3 d after incubation
Areas of injection: albumen and yolk
Hatchability and number of birds sacrificed at 5 wk after hatch Con Albumen Yolk Number of eggs incubated 40 60 60 Number of eggs hatched 36 45 40 % hatchability 90% 75% 67% Number of birds raised until sacrifice 36 42 36 Number of male 19 21 14 Number of female 17 21 22
Post hatch body weights of broilersPost hatch body weights of broilers
Treat1 Sex Statistical significance Age, d Con Albumen Yolk Male Female Treat Sex Treat x Sex 1 46.3a 44.1b 45.1a,b 45.5 44.9 ** NS ** 7 147.7a 150.0a,b 161.1b 153.1 152.8 + NS NS 14 457.0a 452.0a 488.5b 477.7 454.0 ** * NS 21 891.5a 887.9a 943.2b 942.3 872.8 ** ** NS 25 1209.1a,b 1169.6a 1252.2b 1257.0 1163.6 ** ** NS 28 1394.9a,b 1363.0a 1445.6b 1456.0 1346.3 ** ** NS 35 1778.7a 1760.0a 1854.0b 1844.6 1712.8 ** ** NS Data are least square mean (SEM). **, P<0.01; *, P<0.05; +, P<0.1; 1Mean difference was analyzed by Tukey HSD test: means not sharing the same superscript are differ at P<0.05.
In multi-stack wire cage in a temp-controlled room (26.6oC) 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 weightsOn muscle and organ weights
Treat1 Sex Significance Con Albumen Yolk Male Female Treat Sex Treat x Sex
35 d body wt, g 1778.7a 1759.7a 1853.9b 1844.6 1712.8 ** ** NS Carcass wt, g 1313.0a 1309.0a 1374.7b 1358.1 1268.0 ** ** NS Dressing % 73.5 74.4 74.1 73.6 74.1 NS NS NS Leg wt2, g 357.1a 361.6a,b 376.9b 374.2 340.0 * ** NS Breast muscle wt, g 286.1 285.8 301.8 280.6 291.5 + NS NS Liver wt, g 43.4a,b 41.7a 45.4b 43.4 43.4 * NS NS Heart wt, g 8.21a 8.20a 8.75b 8.69 7.74 ** ** NS Data are least square mean **, P<0.01; *, P<0.05; +, P<0.1 1Mean difference was analyzed by Tukey HSD test: means not sharing same superscript are differ at P<0.05. 2Includes thigh and leg with bone-in.
Feed consumption and feed efficiency
Con Albumen Yolk Average weight gain between 10-35 day 1651.58 1625.98 1798.22 Average feed intake between 10-25 day 2810.39 3473.81 2600.20 Gain/feed ratio 0.59 0.47 0.69