Soluble expression of porcine myostatin propeptide in an escherichia coli expression system using maltose binding protein as a fusion protein

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2011-12

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[Honolulu] : [University of Hawaii at Manoa], [December 2011]

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Abstract

Myostatin (MSTN) is a potent negative regulator of skeletal muscle mass. The activity of MSTN is inhibited by MSTN propeptide, the N-terminal fragment of unprocessed MSTN cleaved off during post-translational MSTN processing, indicating the potential of MSTN propeptide to enhance skeletal muscle growth in livestock. The objective of this project was to produce bioactive wild-type porcine MSTN propeptide (pMSTNProW) and its mutated form at the cleavage site (pMSTNProM) in E. coli. The pMSTNProW and pMSTNProM genes were separately cloned into pMAL-c5X vector downstream of the maltose-binding protein (MBP) gene, and were transformed and expressed in E. coli Top10F' strain. The MBP-pMSTNPro proteins were purified by the amylose-resin affinity chromatography, followed by the anion exchange chromatography. For each mL of cell culture, about 38.7 ug of soluble MBP-pMSTNProW protein and 37.0 ug of soluble MBP-pMSTNProM protein were purified by amylose-resin affinity chromatography. By using anion exchange chromatography, about 10.4 ug of MBP-pMSTNProW protein and 9.3 ug of MBP-pMSTNProM protein per mL cell culture were recovered and further purified. MBP was removed from the pMSTNPro proteins by Factor Xa digestion. Approximately 4.2 ug of pMSTNProW and pMSTNProM proteins were purified per mL of cell culture. Western blot analysis further confirmed the soluble expression of pMSTNPro proteins. Bioactivity tests verified the MSTN-suppressing capacity of six different forms of pMSTNPro proteins in vitro. All the pMSTNPro proteins demonstrated a similar tendency on inhibiting MSTN activity, indicating they were bioactive and their inhibitory effects on MSTN activity were dosage-dependent. Further studies are needed to examine the potency and binding characteristics of different forms of the pMSTNPro proteins and the interaction between metalloprotease and MBP-pMSTNPro proteins. The results of this study demonstrate that a large production of pMSTNPro is possible using an E. coli system to study the potential of pMSTNPro to improve muscle growth in pigs.

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M.S. University of Hawaii at Manoa 2011.
Includes bibliographical references.

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Myostatin

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Theses for the degree of Master of Science (University of Hawaii at Manoa). Animal Sciences.

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