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ID 40762
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Title Alternative
Expression and purification of the HIV-1 env gene products in Escherichia coli
Author
Zhang, Bo
Abstract
To purify the HIV-1 envelope protein with antigenic reactivity, the Pvu II-Bgl II fragment of the HIV-1 env gene, from the Pvu II site to the second Bgl II site, encoding the carboxyl terminal 180 amino acids of the viral surface protein (SU, gp120) was molecularly cloned in Escherichia coli strain HB101 using protein A expression-shuttle vector pRIT5. The pRIT5 contains the protein A gene, encoding the secretion signal and IgG binding domain of protein A with the upstream promoter and the downstream multicloning sites, as well as the two replication sites for Escherichia coli and Staphylococcus aureus. A fused protein with the molecular weight of about 55 kilodaltons was produced, which showed the same reactivity as the native protein A against rabbit serum IgG on Western blotting analysis. Most of the fused protein in the periplasmic space was degraded, while the complete fused protein inside the cells was recovered as an insoluble protein. The fused protein was solubilized with sodium dodecylsulfate (SDS), partially purified by IgG sepharose affinity chromatography, and completely purified by SDS-polyacrylamide gel electrophoresis. The quantity of the expressed fused protein was estimated about 1% of the total proteins. The purified fused protein contained 516 amino acids with Mr54, 976, consisting of 305 amino acids of the IgG binding domain of protein A, 5 amino acids derived from polylinker, a carboxyl terminal 180 amino acids of the HIV-1 envelope surface protein gp120, and 26 amino acids derived from the pUC19 sequence.
Keywords
HIV-1
エンベロープ蛋白質
発現ベクター
Protein A
IgG-Sepharoseカラム
Published Date
1989-06
Publication Title
岡山医学会雑誌
Publication Title Alternative
Journal of Okayama Medical Association
Volume
volume101
Issue
issue5-6
Publisher
岡山医学会
Publisher Alternative
Okayama Medical Association
Start Page
659
End Page
672
ISSN
0030-1558
NCID
AN00032489
Content Type
Journal Article
Official Url
https://www.jstage.jst.go.jp/article/joma1947/101/5-6/101_5-6_659/_article/-char/ja/
Related Url
http://www.okayama-u.ac.jp/user/oma/
language
日本語
Copyright Holders
岡山医学会
File Version
publisher
Refereed
True
Eprints Journal Name
joma