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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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Purification and identification of the secreted Ac-AChBP. (a) Gel filtration chromatography of the Ac-AChBP purification. (b) SDS-PAGE analysis of the Ac-AChBP purification. The nonreduced band was at the top (lane 1), which indicated Ac-AChBP formed pentamer. The reduced band was about 27 KD (lane 2).
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fig2: Purification and identification of the secreted Ac-AChBP. (a) Gel filtration chromatography of the Ac-AChBP purification. (b) SDS-PAGE analysis of the Ac-AChBP purification. The nonreduced band was at the top (lane 1), which indicated Ac-AChBP formed pentamer. The reduced band was about 27 KD (lane 2).

Mentions: The secreted soluble Ac-AChBP was purified by gel filtration chromatography. The eluted chromatography position of Ac-AChBP was in Figure 2(a) at column volume 12.5 mL. The position of 12.5 mL indicated molecular weight of ~13.5 KD according to the instruction of Superdex 200 column (GE Healthcare), proving Ac-AChBP formed pentamer. The small peak at 27 mL indicated imidazole, the following gel filtration chromatography indicated imidazole at the same position.


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)

Purification and identification of the secreted Ac-AChBP. (a) Gel filtration chromatography of the Ac-AChBP purification. (b) SDS-PAGE analysis of the Ac-AChBP purification. The nonreduced band was at the top (lane 1), which indicated Ac-AChBP formed pentamer. The reduced band was about 27 KD (lane 2).
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Purification and identification of the secreted Ac-AChBP. (a) Gel filtration chromatography of the Ac-AChBP purification. (b) SDS-PAGE analysis of the Ac-AChBP purification. The nonreduced band was at the top (lane 1), which indicated Ac-AChBP formed pentamer. The reduced band was about 27 KD (lane 2).
Mentions: The secreted soluble Ac-AChBP was purified by gel filtration chromatography. The eluted chromatography position of Ac-AChBP was in Figure 2(a) at column volume 12.5 mL. The position of 12.5 mL indicated molecular weight of ~13.5 KD according to the instruction of Superdex 200 column (GE Healthcare), proving Ac-AChBP formed pentamer. The small peak at 27 mL indicated imidazole, the following gel filtration chromatography indicated imidazole at the same position.

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