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.

scholarly journals Receptor recognition sites reside in both lobes of human serum transferrin

1997 ◽  
Vol 326 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Anne B. MASON ◽  
Beatrice M. TAM ◽  
Robert C. WOODWORTH ◽  
Ronald W. A. OLIVER ◽  
Brian N. GREEN ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Serum ◽  
Transferrin Receptor ◽  
Expression System ◽  
Cell System ◽  
Serum Transferrin ◽  
Human Serum Transferrin ◽  
Human Transferrin ◽  
Domain Specific ◽  
Mammalian Expression

The binding of iron by transferrin leads to a significant conformational change in each lobe of the protein. Numerous studies have shown that the transferrin receptor discriminates between iron-saturated and iron-free transferrin and that it modulates the release of iron. Given these observations, it seems likely that there is contact between each lobe of transferrin and the receptor. This is the case with chicken transferrin, in which it has been demonstrated unambiguously that both lobes are required for binding and iron donation to occur [Brown-Mason and Woodworth (1984) J. Biol. Chem. 259, 1866–1873]. Further support to this contention is added by the ability of both N- and C-domain-specific monoclonal antibodies to block the binding of a solution containing both lobes [Mason, Brown and Church (1987) J. Biol. Chem. 262, 9011–9015]. In the present study a similar conclusion is reached for the binding of human serum transferrin to the transferrin receptor. With the use of recombinant N- and C-lobes of human transferrin produced in a mammalian expression system, we show that both lobes are required to achieve full binding. (Production of recombinant C-lobe in the baby hamster kidney cell system is reported here for the first time.) Each lobe is able to donate iron to transferrin receptors on HeLa S3 cells in the presence of the contralateral lobe. The results are not identical with the chicken system, because the C-lobe alone shows a limited ability to bind to receptors and to donate iron. Further complications arise from the relatively weak re-association between the two lobes of human transferrin compared with the re-association of the ovotransferrin lobes. However, domain-specific monoclonal antibodies to either lobe block the binding of N- and C-lobe mixtures in the human system, thus substantiating the need for both.

scholarly journals Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system

2016 ◽  
Vol 11 ◽  
pp. 86-89 ◽  
Author(s):  
Tsuyoshi Ikehara ◽  
Shihoko Nakashima ◽  
Junichi Nakashima ◽  
Tsubasa Kinoshita ◽  
Takeshi Yasumoto
Keyword(s):  
Low Temperature ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Efficient Production ◽  
Baculovirus Expression

scholarly journals Purification and characterization of the seven cyanogen bromide fragments of human serum transferrin

1974 ◽  
Vol 139 (1) ◽  
pp. 163-168 ◽  
Author(s):  
Michael R. Sutton ◽  
K. Brew
Keyword(s):  
Human Serum ◽  
Cyanogen Bromide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
High Yield ◽  
Serum Transferrin ◽  
Human Serum Transferrin ◽  
Equilibrium Ultracentrifugation

1. Procedures are described for the isolation of seven distinct cyanogen bromide fragments in high yield from human serum transferrin. 2. Cyanogen bromide-cleaved transferrin is separated into three fragments (CN-A, CN-B and CN-C) by gel filtration with Sephadex G-100. 3. Four peptides are obtained from CN-A (the largest fragment) after reduction and carboxamidomethylation, by gel filtration in acidic solvents. Two peptides are similarly obtained from fragment CN-B, whereas fragment CN-C is a single cystine-free peptide. 4. The molecular weights of the seven peptides, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, by sedimentation-equilibrium ultracentrifugation and by sequence studies, range from 3100 to 27000. Together they account for a molecular weight of 76200 for transferrin. 5. The two largest fragments contain the carbohydrate attachment sites of the protein, and the smallest fragment is derived from the N-terminus. 6. The amino acid compositions and N-terminal groups of the fragments are reported and the results compared with those of previous investigations.

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
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