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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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Comparison of two methods used for testing protein expression from different cell densities. (a): Test protein expression level by gel filtration chromatography method different cell densities expression level (from high to low: A, 2 × 106 cells/mL; B, 4 × 106 cells/mL; C, 1 × 106 cells/mL). (b): Test protein expression level by SDS-PAGE method from different cell densities (lane A, 2 × 106 cells/mL; lane B, 4 × 106 cells/mL; lane C, 1 × 106 cells/mL. RA, RB, and RC indicated reduced stated of Ac-AChBP expression samples, respectively, from lanes A, B, and C), the result of test expression level method analysis was the same as gel filtration chromatography.
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fig3: Comparison of two methods used for testing protein expression from different cell densities. (a): Test protein expression level by gel filtration chromatography method different cell densities expression level (from high to low: A, 2 × 106 cells/mL; B, 4 × 106 cells/mL; C, 1 × 106 cells/mL). (b): Test protein expression level by SDS-PAGE method from different cell densities (lane A, 2 × 106 cells/mL; lane B, 4 × 106 cells/mL; lane C, 1 × 106 cells/mL. RA, RB, and RC indicated reduced stated of Ac-AChBP expression samples, respectively, from lanes A, B, and C), the result of test expression level method analysis was the same as gel filtration chromatography.

Mentions: For expression of Ac-AChBP, we found it was obvious that different conditions had different expression levels, which included the bacmid virus titers, volumes of inoculums, and the postinfection time. Figure 3(a) shows different cell densities (1 × 106, 2 × 106, 4 × 106 cells/mL) for different Ac-AChBP protein expression levels which were analyzed by gel filtration chromatography. The results indicated that 2 × 106 cells/mL was the highest protein expression level.


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)

Comparison of two methods used for testing protein expression from different cell densities. (a): Test protein expression level by gel filtration chromatography method different cell densities expression level (from high to low: A, 2 × 106 cells/mL; B, 4 × 106 cells/mL; C, 1 × 106 cells/mL). (b): Test protein expression level by SDS-PAGE method from different cell densities (lane A, 2 × 106 cells/mL; lane B, 4 × 106 cells/mL; lane C, 1 × 106 cells/mL. RA, RB, and RC indicated reduced stated of Ac-AChBP expression samples, respectively, from lanes A, B, and C), the result of test expression level method analysis was the same as gel filtration chromatography.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Comparison of two methods used for testing protein expression from different cell densities. (a): Test protein expression level by gel filtration chromatography method different cell densities expression level (from high to low: A, 2 × 106 cells/mL; B, 4 × 106 cells/mL; C, 1 × 106 cells/mL). (b): Test protein expression level by SDS-PAGE method from different cell densities (lane A, 2 × 106 cells/mL; lane B, 4 × 106 cells/mL; lane C, 1 × 106 cells/mL. RA, RB, and RC indicated reduced stated of Ac-AChBP expression samples, respectively, from lanes A, B, and C), the result of test expression level method analysis was the same as gel filtration chromatography.
Mentions: For expression of Ac-AChBP, we found it was obvious that different conditions had different expression levels, which included the bacmid virus titers, volumes of inoculums, and the postinfection time. Figure 3(a) shows different cell densities (1 × 106, 2 × 106, 4 × 106 cells/mL) for different Ac-AChBP protein expression levels which were analyzed by gel filtration chromatography. The results indicated that 2 × 106 cells/mL was the highest protein expression level.

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