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