Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in <i>E</i>. <i>coli</i>

<div><p>Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro) inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in <i>E</i>. <i>coli</i>, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an <i>in vitro</i> gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42–175 (MBP-Pro42-175) exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42–115 (MBP-Pro42-115) or 42–98 (MBP-Pro42-98) also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity.</p></div

Binding of MBP-MSTNpro proteins to MSTN in a pull-down assay.

<p><b>A)</b> Fifty microliters of amylose resin was mixed with either 480 nM of MBP-Pro42-175, MBP-Pro42-115, or MBP-Pro42-98 plus 48 nM of MSTN, separately to a final volume of 1 mL pull-down assay. The column was then washed with column buffer three times. Proteins bound to the amylose resin were then eluted with reducing SDS-PAGE loading buffer, and subjected to SDS-PAGE analysis. The commercial MSTN (lane 1) added in the pull-down experiment contained about 50-fold excess amount of BSA (*) as a carrier protein (R&D Systems). Arrows indicate MSTN bands. <b>B)</b> The same procedure describe in A was employed except that five different concentrations (1, 15, 30, 45, 60 nM) of either MBP-Pro42-175 or MBP-Pro42-98 with fixed concentration of MSTN (6 nM) were examined in the pull-down experiment. MBP (60 nM) binding to MSTN (6 nM) was also examined in the pull-down assay to serve as a negative control. Proteins bound to the amylose resin were eluted and subjected to immuno-blot analysis against monoclonal anti-MSTN antibody.</p

Inhibition of GDF-11 activity by MBP-pMSTNpro proteins.

<p>The assay procedure was the same as the MSTN inhibition assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174956#pone.0174956.g003" target="_blank">Fig 3</a>) except that GDF-11 was used as a competing ligand instead of MSTN. Error bars represent ± SEM (n = 6). The means of IC₅₀ not sharing the same superscript are different at P<0.05.</p

SDS-PAGE analysis of MBP-Pro42-98 after factor Xa cleavage and examination of MSTN-inhibitory capacity of Pro42-98.

<p><b>(A)</b> After incubation of MBP-Pro42-98 with factor Xa, the total reaction mixture was centrifuged at 10,000 g for 3 min. The supernatant (soluble fraction) was subjected to 15% SDS-PAGE analysis under reduced and unreduced conditions, then visualized with Coomassie blue. *, Pro42-98. <b>(B)</b> The supernatant was subjected to gel filtration, and fractions 16 (f16) and 22 (f22) were subjected to SDS-PAGE analysis under reduced condition. *, Pro42-98. <b>(C)</b> MSTN-inhibitory capacity of purified Pro42-98 (f16) was measured using the (CAGA) ₁₂-luciferase reporter gene assay. The means of IC₅₀ not sharing the same superscript are different at P<0.05.</p

Inhibition of MSTN activity by MBP-pMSTNpro proteins.

<p>HEK293 cells stably expressing (CAGA) ₁₂-luciferase gene construct were seeded on a 96-well culture plate at 40,000 cells/well, and grown for 24 hr in DMEM with 10% fetal calf serum, antibiotic and antimycotic. Medium was removed, and MSTN (1 nM) and various concentrations (60–0 nM) of commercial MSTNpro (Commercial Pro) or MBP-MSTNpro proteins in DMEM were added to each well, followed by incubation for 24 hr. Medium was removed, and 60 μL of luminescence substrate were added and incubated for 3 minutes, followed by luminescence measurement. The % inhibition of MSTN activity was calculated by the following formula: % inhibition = (luminescence at 1 nM MSTN—luminescence at each ligand concentration)*100/(luminescence at 1 nM MSTN—luminescence at 0 nM MSTN). The error bars represent ± SEM (n = 6). The means of IC₅₀ not sharing the same superscript are different at P<0.05.</p

SDS-PAGE analysis of commercial MSTNpro after BMP-1 digestion and examination of MSTN-inhibitory capacity of BMP-1 digested commercial MSTNpro.

<p><b>(A)</b> After incubation of commercial MSTN-pro with BMP-1, the total reaction mixture was centrifuged at 10,000 g for 3 min. The supernatant (soluble fraction) was subjected to 15% SDS-PAGE analysis under reduced and unreduced conditions, then visualized with Coomassie blue. <b>(B)</b> MSTN-inhibitory capacity of BMP-1 digested commercial MSTN-pro was measured using the (CAGA) ₁₂-luciferase reporter gene assay.</p

Yield of MBP-fused pig MSTNpro proteins recovered from amylose affinity purification.

<p>Yield of MBP-fused pig MSTNpro proteins recovered from amylose affinity purification.</p

SDS-PAGE analysis of MBP-Pro42-175 after factor Xa cleavage and examination of MSTN-inhibitory capacity of Pro42-175.

<p><b>(A)</b> After incubation of MBP-Pro42-175 with factor Xa, the supernatant was subjected to amylose affinity purification, followed by removal of factor Xa using a factor Xa affinity column. Protein samples collected during the cleavage and purification steps were subjected to 15% SDS-PAGE analysis, and visualized with Coomassie blue staining. Lane 1, MBP-Pro42-175; lane 2, MBP-Pro42-175 digested with factor Xa; lane 3, Pro42-175 purified by amylose affinity; lane 4, Pro42-175 with factor Xa being removed; #, MBP; +, factor Xa (disulfide linked heterodimer under non-reduced condition); *, Pro42-175. <b>(B)</b> MSTN-inhibitory capacity of purified Pro42-175 was measured using the (CAGA) ₁₂-luciferase reporter gene assay. The means of IC₅₀ not sharing the same superscript are different at P<0.05.</p

Pyrosequencing data in bacterial community composition in pig slurry by storage period.

<p>Pyrosequencing data in bacterial community composition in pig slurry by storage period.</p

Bacterial taxonomic composition of phylum level in pig slurry by storage period.

<p>Sequences were classified using the EzTaxon-e database with an 80% confidence threshold. “wk” is an abbreviation to weeks.</p