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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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General procedures for expressing Ac-AChBP in the bac-to-bac baculovirus expression system. The Bacmid verification is by agarose gel, but the protein expression test is by SDS-PAGE. P1 indicates passage one, and so on.
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fig1: General procedures for expressing Ac-AChBP in the bac-to-bac baculovirus expression system. The Bacmid verification is by agarose gel, but the protein expression test is by SDS-PAGE. P1 indicates passage one, and so on.

Mentions: As shown in Figure 1, the recombinant bacmid containing Ac-AChBP gene was obtained through transformation into E. coli DH10Bac, which was isolated from white colonies and tested by agarose gel electrophoresis (Figure 1, the picture of agarose gel). Then, the bacmid was transfected into insect cells (SF9) to obtain baculovirus passage 1 (P1).


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)

General procedures for expressing Ac-AChBP in the bac-to-bac baculovirus expression system. The Bacmid verification is by agarose gel, but the protein expression test is by SDS-PAGE. P1 indicates passage one, and so on.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: General procedures for expressing Ac-AChBP in the bac-to-bac baculovirus expression system. The Bacmid verification is by agarose gel, but the protein expression test is by SDS-PAGE. P1 indicates passage one, and so on.
Mentions: As shown in Figure 1, the recombinant bacmid containing Ac-AChBP gene was obtained through transformation into E. coli DH10Bac, which was isolated from white colonies and tested by agarose gel electrophoresis (Figure 1, the picture of agarose gel). Then, the bacmid was transfected into insect cells (SF9) to obtain baculovirus passage 1 (P1).

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