Physicochemical and biochemical characterization of human alpha 1-microglobulin expressed in baculovirus-infected insect cells
(1997) In Protein Expression and Purification 11(1). p.95-103- Abstract
DNA encoding the signal peptide and the alpha 1-microglobulin part of the human alpha 1-microglobulin-bikunin gene was expressed in baculovirus-infected insect cells. Recombinant alpha 1-microglobulin was secreted and could be purified from the medium with a yield of 20-30 mg/ L. Biochemical and physicochemical characterization showed that the recombinant protein was very similar to alpha 1-microglobulin isolated from human urine and plasma, except that the recombinant protein had smaller N-linked oligosaccharides, lacked the O-linked oligosaccharide, and was devoid of sialic acid. Recombinant alpha 1-microglobulin migrated upon SDS-PAGE as two bands, 27 and 29 kDa, representing alpha 1-microglobulin with one and two N-linked... (More)
DNA encoding the signal peptide and the alpha 1-microglobulin part of the human alpha 1-microglobulin-bikunin gene was expressed in baculovirus-infected insect cells. Recombinant alpha 1-microglobulin was secreted and could be purified from the medium with a yield of 20-30 mg/ L. Biochemical and physicochemical characterization showed that the recombinant protein was very similar to alpha 1-microglobulin isolated from human urine and plasma, except that the recombinant protein had smaller N-linked oligosaccharides, lacked the O-linked oligosaccharide, and was devoid of sialic acid. Recombinant alpha 1-microglobulin migrated upon SDS-PAGE as two bands, 27 and 29 kDa, representing alpha 1-microglobulin with one and two N-linked carbohydrates, respectively. An overall structural similarity was indicated as antibodies raised against human urinary alpha 1-microglobulin were found to recognize recombinant, plasma, and urinary alpha 1-microglobulin in a similar manner. CD studies suggested an almost identical secondary structure for recombinant and urinary alpha 1-microglobulin but a slightly different structure for plasma alpha 1-microglobulin. The absorbance spectrum as well as visual examination demonstrated that recombinant, urinary, and plasma alpha 1-microglobulin carried a yellow-brown chromophore, but that plasma alpha 1-microglobulin was slightly less intensely colored. Although it is still a puzzle why the immunosuppressive plasma protein alpha 1-microglobulin and the protease inhibitor bikunin, which have no known function in common, are cotranslated from the same mRNA, it can be concluded that bikunin is not necessary for an adequate translation, folding, and secretion of alpha 1-microglobulin. Furthermore, since recombinant alpha 1-microglobulin was produced in large amounts and found to be very similar to plasma and urinary alpha 1-microglobulin, it may prove to be useful in structural and functional studies of the protein.
(Less)
- author
- Wester, L ; Johansson, M U LU and Åkerström, B LU
- organization
- publishing date
- 1997-10
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Alpha-Globulins/biosynthesis, Animals, Baculoviridae, Carbohydrates/chemistry, Cell Line, Circular Dichroism, Cloning, Molecular, Electrophoresis, Agar Gel, Humans, Molecular Weight, Moths, Protease Inhibitors/chemistry, Recombinant Proteins/chemistry, Spectrophotometry, Atomic
- in
- Protein Expression and Purification
- volume
- 11
- issue
- 1
- pages
- 95 - 103
- publisher
- Academic Press
- external identifiers
-
- scopus:0031260197
- pmid:9325144
- ISSN
- 1046-5928
- DOI
- 10.1006/prep.1997.0760
- language
- English
- LU publication?
- yes
- id
- 7fd9980b-e27f-459c-9689-2f5e8c5cce27
- date added to LUP
- 2019-05-22 10:15:44
- date last changed
- 2024-01-01 06:39:45
@article{7fd9980b-e27f-459c-9689-2f5e8c5cce27,
abstract = {{<p>DNA encoding the signal peptide and the alpha 1-microglobulin part of the human alpha 1-microglobulin-bikunin gene was expressed in baculovirus-infected insect cells. Recombinant alpha 1-microglobulin was secreted and could be purified from the medium with a yield of 20-30 mg/ L. Biochemical and physicochemical characterization showed that the recombinant protein was very similar to alpha 1-microglobulin isolated from human urine and plasma, except that the recombinant protein had smaller N-linked oligosaccharides, lacked the O-linked oligosaccharide, and was devoid of sialic acid. Recombinant alpha 1-microglobulin migrated upon SDS-PAGE as two bands, 27 and 29 kDa, representing alpha 1-microglobulin with one and two N-linked carbohydrates, respectively. An overall structural similarity was indicated as antibodies raised against human urinary alpha 1-microglobulin were found to recognize recombinant, plasma, and urinary alpha 1-microglobulin in a similar manner. CD studies suggested an almost identical secondary structure for recombinant and urinary alpha 1-microglobulin but a slightly different structure for plasma alpha 1-microglobulin. The absorbance spectrum as well as visual examination demonstrated that recombinant, urinary, and plasma alpha 1-microglobulin carried a yellow-brown chromophore, but that plasma alpha 1-microglobulin was slightly less intensely colored. Although it is still a puzzle why the immunosuppressive plasma protein alpha 1-microglobulin and the protease inhibitor bikunin, which have no known function in common, are cotranslated from the same mRNA, it can be concluded that bikunin is not necessary for an adequate translation, folding, and secretion of alpha 1-microglobulin. Furthermore, since recombinant alpha 1-microglobulin was produced in large amounts and found to be very similar to plasma and urinary alpha 1-microglobulin, it may prove to be useful in structural and functional studies of the protein.</p>}},
author = {{Wester, L and Johansson, M U and Åkerström, B}},
issn = {{1046-5928}},
keywords = {{Alpha-Globulins/biosynthesis; Animals; Baculoviridae; Carbohydrates/chemistry; Cell Line; Circular Dichroism; Cloning, Molecular; Electrophoresis, Agar Gel; Humans; Molecular Weight; Moths; Protease Inhibitors/chemistry; Recombinant Proteins/chemistry; Spectrophotometry, Atomic}},
language = {{eng}},
number = {{1}},
pages = {{95--103}},
publisher = {{Academic Press}},
series = {{Protein Expression and Purification}},
title = {{Physicochemical and biochemical characterization of human alpha 1-microglobulin expressed in baculovirus-infected insect cells}},
url = {{http://dx.doi.org/10.1006/prep.1997.0760}},
doi = {{10.1006/prep.1997.0760}},
volume = {{11}},
year = {{1997}},
}