Advanced

Physicochemical and biochemical characterization of human alpha 1-microglobulin expressed in baculovirus-infected insect cells

Wester, L; Johansson, M U LU and Åkerström, B LU (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)
Please use this url to cite or link to this publication:
author
organization
publishing date
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
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
2019-11-13 05:33:55
@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},
  keyword      = {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},
  volume       = {11},
  year         = {1997},
}