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Expression of human Cu, Zn-superoxide dismutase in an insect cell-free system and its structural analysis by MALDI-TOF MS

Paper ID Volume ID Publish Year Pages File Format Full-Text
24410 43513 2009 6 PDF Available
Title
Expression of human Cu, Zn-superoxide dismutase in an insect cell-free system and its structural analysis by MALDI-TOF MS
Abstract

Human Cu, Zn-superoxide dismutase (hSOD1) is a homodimer that coordinates one copper and one zinc ion per monomer. These metal ions contribute to its enzymatic activity and structural stability. In addition, hSOD1 maintains an intra-subunit disulfide bond formed in the reducing environment of the cytosol and is active under a variety of stringent denaturing conditions. We report the expression of hSOD1 in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) insect cells, and its structural analysis including the status of the sole intra-subunit disulfide bond by mass spectrometry. By using this system hSOD1 was obtained in a soluble active form after addition of Cu2+ and Zn2+ and was purified with a yield of approximately 33 μg from 1 ml of reaction volume. Both enzymatic and structural analyses of the recombinant hSOD1 indicate that it was completely identical to the protein isolated from human erythrocytes.

Keywords
CAT, chloramphenicol acetyltransferase; FALS, familial amyotrophic lateral sclerosis; hSOD1, human Cu, Zn-superoxide dismutase; PDI, protein disulfide isomerase; PMF, peptide mass fingerprinting; Sf21, Spodoptera frugiperda 21Cell-free protein synthesis s
First Page Preview
Expression of human Cu, Zn-superoxide dismutase in an insect cell-free system and its structural analysis by MALDI-TOF MS
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 144, Issue 4, December 2009, Pages 287–292
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering