Nucleic Acids Research, 1992, Vol. 20, No. 1 33-39
© 1992
MOLECULAR BIOLOGY |
Expression of active iron regulatory factor from a full length human cDNA by in vitro transcription/translation
Swiss Institute for Experimental Cancer Research, Genetics Unit CH-1066 Epalinges, Switzerland
*To whom correspondence should be addressed
Received November 11, 1991. Accepted December 6, 1991.
Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98, 400 dalton. By in vitro transcription of a assembled DRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized DRF that bound specifically to a human ferritin IRE. in vitro translated DRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by ß-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental DRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene.
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