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Use of bacterial DHFR-II fusion proteins to elicit specific antibodies

Authors
Journal
Gene
0378-1119
Publisher
Elsevier
Publication Date
Volume
41
Identifiers
DOI: 10.1016/0378-1119(86)90109-5
Keywords
  • Recombinant Dna
  • Plasmid Vectors
  • Translation
  • Major Sperm Protein
  • Caenorhabditis Elegans
  • Trimethoprim Resistance
Disciplines
  • Biology

Abstract

Abstract Plasmids containing the coding region of the type II dihydrofolate reductase (DHFR) specified by R388 have been used to alter the amino acid (aa) sequence at the C-terminus of this protein. These plasmids have a unique cloning site in the C-terminal portion of the 78-aa coding region. Insertions of DNA fragments into this site produced plasmids that code for proteins with 6- to 80-aa extensions. The vectors were constructed to terminate translation in all three phases beyond the position of insertion of foreign DNA. Random DNA fragments from the major sperm protein (MSP) gene of Caenorhabditis elegans produced by DNase I cleavage were inserted into these vectors. Cell extracts from colonies containing MSP sequences were examined by gel electrophoresis and immunoblotting. One of the hybrid DHFR-MSP proteins was isolated and antibody was prepared to it. This antibody preparation reacted with MSP in immunoblots of purified MSP and whole cell extracts of the worm. A rapid purification procedure for the DHFR is presented.

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