MOLECULAR CLONING AND EXPRESSION IN ESCHERICHIA COLI OF THE HUMAN ALPHA-GALACTOSIDASE A GENE.
Item
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Title
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MOLECULAR CLONING AND EXPRESSION IN ESCHERICHIA COLI OF THE HUMAN ALPHA-GALACTOSIDASE A GENE.
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Identifier
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AAI8713763
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identifier
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8713763
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Creator
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HANTZOPOULOS, PETROS A.
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Contributor
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David H. Calhoun
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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We have used recombinant DNA methods to isolate and characterize a human {dollar}\alpha{dollar}-galactosidase A cDNA clone from a {dollar}\lambda{dollar}gt11 human liver cDNA expression library. For the isolation of this clone, we used monospecific antibodies made against purified {dollar}\alpha{dollar}-galactosidase A, as well as oligonucleotide mixtures corresponding to amino terminal and internal amino acid sequences. The cDNA insert contains a 1234 bp sequence with an open reading frame encoding 398 amino acids of the propeptide, as well as five amino acids of the leader peptide. For the expression of the {dollar}\alpha{dollar}-galactosidase A propeptide in Escherichia coli, the 5{dollar}\sp\prime{dollar}-terminus of the cDNA insert was engineered so that an ATG initiator codon precedes the first codon of the mature form of the enzyme. The engineered cDNA insert was cloned into the Cla I site of the prokaryotic expression vector ptrpL1, and expression was monitored in maxicells. Clones that contained different sequences in the region separating the ribosome binding site and the ATG initiation codon were constructed and tested for efficiency of expression. Clones with {dollar}\alpha{dollar}-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kilodaltons (kDa), the size expected for the intact proenzyme. The 45 kDa protein is specifically precipitated by antibody to {dollar}\alpha{dollar}-galactosidase A, and its expression is repressed by tryptophan and induced by 3-{dollar}\beta{dollar}-indoleacrylic acid as expected for this expression vector. The human enzyme is produced in E. coli in a catalytically active form at levels sufficient to support the growth of cells in minimal media using {dollar}\alpha{dollar}-galactosides as sole sources of carbon and energy. In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-{dollar}\alpha{dollar}-D-galactopyranoside (X-{dollar}\alpha{dollar}-Gal). The entire {dollar}\alpha{dollar}-galactosidase A cDNA, including the 5{dollar}\sp\prime{dollar}-end untranslated sequence, and sequences encoding the complete prepropeptide was constructed when a genomic clone containing this segment was isolated. Primer extension experiments with poly(A){dollar}\sp +{dollar} RNA identified the probable CAP site for the {dollar}\alpha{dollar}-galactosidase A transcript. The clones constructed in this study will be useful to evaluate the production of human recombinant {dollar}\alpha{dollar}-galactosidase A for enzyme replacement therapy of Fabry disease using prokaryotic and eukaryotic systems. (Abstract shortened with permission of author.).
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
