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Title: Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli

Abstract

Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated several more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides andmore » for diagnostic purposes in the GM2 gangliosidoses.« less

Authors:
;
Publication Date:
Research Org.:
Virginia Polytechnic Institute and State Univ., Blacksburg
OSTI Identifier:
5126992
Report Number(s):
CONF-8606151-
Journal ID: CODEN: FEPRA; TRN: 86-034820
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CLONE CELLS; AUTORADIOGRAPHY; ESCHERICHIA COLI; DNA-CLONING; GLYCOSYL HYDROLASES; IODINE 125; LIVER; MAN; ANIMALS; BACTERIA; BETA DECAY RADIOISOTOPES; BODY; CELL CULTURES; CLONING; DAYS LIVING RADIOISOTOPES; DIGESTIVE SYSTEM; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; GLANDS; HYDROLASES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPES; MAMMALS; MICROORGANISMS; NUCLEI; ODD-EVEN NUCLEI; ORGANS; PRIMATES; RADIOISOTOPES; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Wiktorowicz, J E, and Whitman, J M. Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli. United States: N. p., 1986. Web.
Wiktorowicz, J E, & Whitman, J M. Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli. United States.
Wiktorowicz, J E, and Whitman, J M. 1986. "Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli". United States.
@article{osti_5126992,
title = {Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli},
author = {Wiktorowicz, J E and Whitman, J M},
abstractNote = {Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated several more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides and for diagnostic purposes in the GM2 gangliosidoses.},
doi = {},
url = {https://www.osti.gov/biblio/5126992}, journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:6,
place = {United States},
year = {Thu May 01 00:00:00 EDT 1986},
month = {Thu May 01 00:00:00 EDT 1986}
}

Conference:
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