scholarly journals Efficient Expression of Acetylcholine-Binding Protein fromAplysia californicain Bac-to-Bac System

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Bo Lin ◽  
Hailing Meng ◽  
Hui Bing ◽  
Dongting Zhangsun ◽  
Sulan Luo
Keyword(s):  
Cell Density ◽  
Lymnaea Stagnalis ◽  
Expression System ◽  
Volume Ratio ◽  
Baculovirus Expression System ◽  
High Yield ◽  
Factors Affecting ◽  
Functional Studies ◽  
Efficient Expression ◽  
Inoculum Volume

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 haresAplysia 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 × 106 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 fromLymnaea stagnalissuccessfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

scholarly journals Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system

2017 ◽  
Vol 37 (1) ◽  
Author(s):  
Bo Lin ◽  
Kun Liu ◽  
Wenting Wang ◽  
Wei Li ◽  
Xu Dong ◽  
...  
Keyword(s):  
Cancer Cell Line ◽  
Volume Ratio ◽  
High Yield ◽  
Key Factors ◽  
Factors Affecting ◽  
Human Liver Cancer Cell ◽  
Time Post Infection ◽  
Human Liver Cancer ◽  
Inoculum Volume ◽  
High Yields

α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72–96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP.

scholarly journals A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

10.3791/52911 ◽  
2015 ◽  
Author(s):  
Farokh Dotiwala ◽  
Isabelle Fellay ◽  
Luis Filgueira ◽  
Denis Martinvalet ◽  
Judy Lieberman ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
High Yield ◽  
Efficient Expression ◽  
Cost Efficient

High yield of biologically active recombinant human amelogenin using the baculovirus expression system

2006 ◽  
Vol 45 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Angela L. Taylor ◽  
Amir Haze-Filderman ◽  
Anat Blumenfeld ◽  
Boaz Shay ◽  
Leah Dafni ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Biologically Active ◽  
High Yield ◽  
Baculovirus Expression

scholarly journals High-yield production of functionally active human serum transferrin using a baculovirus expression system, and its structural characterization

1996 ◽  
Vol 319 (1) ◽  
pp. 191-195 ◽  
Author(s):  
Stuart A. ALI ◽  
Heidi C JOAO ◽  
Robert CSONGA ◽  
Franz HAMMERSCHMID ◽  
Alexander STEINKASSERER
Keyword(s):  
Human Serum ◽  
Expression System ◽  
Baculovirus Expression System ◽  
High Yield ◽  
Efficient Production ◽  
Serum Transferrin ◽  
Human Serum Transferrin ◽  
Mammalian Expression ◽  
Baculovirus Expression ◽  
Wide Range

Recently, there has been much interest in expressing recombinant human serum transferrin (HST) and mutants thereof for structural and functional studies. We have developed a baculovirus expression system for the rapid and efficient production of large quantities of HST (> 20 mg/l). Like native HST, the recombinant protein can bind two ferric ions in the presence of bicarbonate, and is actively taken up by receptor-mediated endocytosis. Secondary structure calculations from CD measurements indicate a content of 42% α-helix and 28% β-sheet. This is the first reported use of a non-mammalian expression system to produce functional HST, and will provide a practical tool to allow expression of a wide range of HST variants for mutagenesis studies.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

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