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

Test expression level method analysis different time expression level postinfection. Expression Ac-AChBP level from high to low, 35 h (lane 1); 55 h (lane 2); 75 h (lane 3); 95 h (lane 4). R1, R2, R3, and R4 indicated reduced stated of Ac-AChBP from lanes 1, 2, 3, and 4.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Test expression level method analysis different time expression level postinfection. Expression Ac-AChBP level from high to low, 35 h (lane 1); 55 h (lane 2); 75 h (lane 3); 95 h (lane 4). R1, R2, R3, and R4 indicated reduced stated of Ac-AChBP from lanes 1, 2, 3, and 4.

Mentions: The protein expression levels were evaluated by the SDS-PAGE gels (Figure 5); it also showed that when the postinfection time was more than 55 h, the cells were dying (lane 3 and lane 4), and the quantity of recombinant protein expression also increased very slowly. So considering pure and high yield of Ac-AChBP protein sample, the best harvest time was 55 h of postinfection, and it can get about 5 mg/L of Ac-AChBP protein under the optimized conditions.


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)

Test expression level method analysis different time expression level postinfection. Expression Ac-AChBP level from high to low, 35 h (lane 1); 55 h (lane 2); 75 h (lane 3); 95 h (lane 4). R1, R2, R3, and R4 indicated reduced stated of Ac-AChBP from lanes 1, 2, 3, and 4.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Test expression level method analysis different time expression level postinfection. Expression Ac-AChBP level from high to low, 35 h (lane 1); 55 h (lane 2); 75 h (lane 3); 95 h (lane 4). R1, R2, R3, and R4 indicated reduced stated of Ac-AChBP from lanes 1, 2, 3, and 4.
Mentions: The protein expression levels were evaluated by the SDS-PAGE gels (Figure 5); it also showed that when the postinfection time was more than 55 h, the cells were dying (lane 3 and lane 4), and the quantity of recombinant protein expression also increased very slowly. So considering pure and high yield of Ac-AChBP protein sample, the best harvest time was 55 h of postinfection, and it can get about 5 mg/L of Ac-AChBP protein under the optimized conditions.

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