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Efficient expression of acetylcholine-binding protein from Aplysia californica in Bac-to-Bac system.

Lin B, Meng H, Bing H, Zhangsun D, Luo S - Biomed Res Int (2014)

Bottom Line: The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins.The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully.Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drug of Haikou, Hainan University, Haikou, Hainan 570228, China.

ABSTRACT
The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 10(6) cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

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Gel filtration chromatography analysis correlation of Ac-AChBP intracellular and extracellular expression. (a): about 500 mAU peak value expression, the blue line was intracellular expression and the red line was extracellular expression. (b): about 1500 mAU peak value expression, the blue line was intracellular expression and the red line was extracellular expression.
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fig8: Gel filtration chromatography analysis correlation of Ac-AChBP intracellular and extracellular expression. (a): about 500 mAU peak value expression, the blue line was intracellular expression and the red line was extracellular expression. (b): about 1500 mAU peak value expression, the blue line was intracellular expression and the red line was extracellular expression.

Mentions: The Ac-AChBP intracellular and extracellular expression was analyzed by SDS-PAGE gel (Figure 7). Although intracellular expressed Ac-AChBP showed as the pentamer, the band was faint. So the intracellular expression was not pure and the expression level was also lower than extracellular. The Ac-AChBP expression in intracellular and extracellular was also analyzed by gel filtration chromatography (Figure 8). It shows that the intracellular and extracellular peak value was correlative; when the intracellular peak value was 500 mAU, the extracellular peak was about 500 mAU too (equivalent to 1.7 mg/L expression level); when the intracellular peak value was 1500 mAU, the extracellular peak was also about 1500 mAU (equivalent to 5 mg/L expression level). It could be concluded that the intracellular expression manipulated extracellular expression or extracellular expression depended on baculovirus amplification. After 55 h of postinfection, the cells were unhealthy and dying, so the extracellular expression was slowed down.


Efficient expression of acetylcholine-binding protein from Aplysia californica in Bac-to-Bac system.

Lin B, Meng H, Bing H, Zhangsun D, Luo S - Biomed Res Int (2014)

Gel filtration chromatography analysis correlation of Ac-AChBP intracellular and extracellular expression. (a): about 500 mAU peak value expression, the blue line was intracellular expression and the red line was extracellular expression. (b): about 1500 mAU peak value expression, the blue line was intracellular expression and the red line was extracellular expression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127255&req=5

fig8: Gel filtration chromatography analysis correlation of Ac-AChBP intracellular and extracellular expression. (a): about 500 mAU peak value expression, the blue line was intracellular expression and the red line was extracellular expression. (b): about 1500 mAU peak value expression, the blue line was intracellular expression and the red line was extracellular expression.
Mentions: The Ac-AChBP intracellular and extracellular expression was analyzed by SDS-PAGE gel (Figure 7). Although intracellular expressed Ac-AChBP showed as the pentamer, the band was faint. So the intracellular expression was not pure and the expression level was also lower than extracellular. The Ac-AChBP expression in intracellular and extracellular was also analyzed by gel filtration chromatography (Figure 8). It shows that the intracellular and extracellular peak value was correlative; when the intracellular peak value was 500 mAU, the extracellular peak was about 500 mAU too (equivalent to 1.7 mg/L expression level); when the intracellular peak value was 1500 mAU, the extracellular peak was also about 1500 mAU (equivalent to 5 mg/L expression level). It could be concluded that the intracellular expression manipulated extracellular expression or extracellular expression depended on baculovirus amplification. After 55 h of postinfection, the cells were unhealthy and dying, so the extracellular expression was slowed down.

Bottom Line: The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins.The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully.Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drug of Haikou, Hainan University, Haikou, Hainan 570228, China.

ABSTRACT
The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 10(6) cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

Show MeSH
Related in: MedlinePlus