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Expression of cloned human lactoferrin in baby-hamster kidney cells

1991; Portland Press; Volume: 276; Issue: 2 Linguagem: Inglês

10.1042/bj2760349

1470-8728

Kathryn M. Stowell, Thomas A. Rado, Walter D. Funk, John W. Tweedie,

Pediatric Hepatobiliary Diseases and Treatments

Human lactoferrin was expressed from a cloned cDNA introduced into mammalian cells in tissue culture. Total RNA was extracted from human bone marrow, and lactoferrin cDNA was synthesized by primer-specific polymerase chain reaction after oligo(dT)-primed first-strand synthesis. The cDNA was sequenced to confirm its identity with previously published human lactoferrin sequences and cloned into the eukaryotic expression vector pNUT. Recombinant vector DNA containing the human lactoferrin sequence was introduced into baby-hamster kidney (BHK) cells in culture, and stable transfectants were produced by dominant marker selection. Human lactoferrin was expressed from the metallothionein promoter of pNUT by Zn2+ induction. The protein was secreted into the tissue-culture medium and was subsequently purified to homogeneity in a single step. Initial characterization suggests that the protein expressed by BHK cells is identical with native human lactoferrin.