Limits...
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.

Show MeSH

Related in: MedlinePlus

Gel filtration chromatography and crystals of Ac-AChBP complex with α-conotoxin LtIA. (a) Comparison of Ac-AChBP (red line) and Ac-AChBP/LtIA (blue line) complex by gel filtration chromatography. (b) The crystals of co-crystallized receptor Ac-AChBP with its ligand α-conotoxin LtIA from Conus litteratus.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4127255&req=5

fig9: Gel filtration chromatography and crystals of Ac-AChBP complex with α-conotoxin LtIA. (a) Comparison of Ac-AChBP (red line) and Ac-AChBP/LtIA (blue line) complex by gel filtration chromatography. (b) The crystals of co-crystallized receptor Ac-AChBP with its ligand α-conotoxin LtIA from Conus litteratus.

Mentions: The gel filtration chromatography clearly showed the difference of binding and nonbinding of the ligand of α-conotoxin LtIA to the Ac-AChBP receptor (Figure 9(a)). Comparing Ac-AChBP (red line) to Ac-AChBP/LtIA (blue line) complex, both the peak values and the positions are changed by the chromatography. The Ac-AChBP/LtIA (blue line) complex showed molecular weight larger than Ac-AChBP which indicated its binding with α-conotoxin LtIA.


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 and crystals of Ac-AChBP complex with α-conotoxin LtIA. (a) Comparison of Ac-AChBP (red line) and Ac-AChBP/LtIA (blue line) complex by gel filtration chromatography. (b) The crystals of co-crystallized receptor Ac-AChBP with its ligand α-conotoxin LtIA from Conus litteratus.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Gel filtration chromatography and crystals of Ac-AChBP complex with α-conotoxin LtIA. (a) Comparison of Ac-AChBP (red line) and Ac-AChBP/LtIA (blue line) complex by gel filtration chromatography. (b) The crystals of co-crystallized receptor Ac-AChBP with its ligand α-conotoxin LtIA from Conus litteratus.
Mentions: The gel filtration chromatography clearly showed the difference of binding and nonbinding of the ligand of α-conotoxin LtIA to the Ac-AChBP receptor (Figure 9(a)). Comparing Ac-AChBP (red line) to Ac-AChBP/LtIA (blue line) complex, both the peak values and the positions are changed by the chromatography. The Ac-AChBP/LtIA (blue line) complex showed molecular weight larger than Ac-AChBP which indicated its binding with α-conotoxin LtIA.

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