Cloning and expression of human neutrophil lipocalin cDNA derived from bone marrow and ovarian cancer cells

Abstract

Human neutrophil lipocalin (HNL) cDNA was amplified by PCR technology in combination with deoxyinosine containing oligonucleotides for cloning, sequencing and production of the recombinant protein in E. coli. The primers were targeted to the corresponding DNA backtranslate of the mature protein resulting in a PCR amplified 534 bp cDNA from different reverse transcripts of ovarian cancer cell line and bone marrow cell mRNAs. Sequence analysis revealed that two different cDNAs from ovarian cancer and bone marrow cells could be obtained. Cloning and expression of HNL cDNAs were performed in E. coli strain HMS 174 [DE3] using the pET system yielding in two recombinant proteins with a molecular weight of 21 kDa which is consistent with an 178 amino acid residues containing sequence of the mature HNL protein. N-terminal amino acid sequence analysis of the expression products showed an identical polypeptide sequence missing the E. coli processed starting methionine.