Lactoferrin cDNA. Expression and in vitro mutagenesis.
The full length copy DNA (cDNA) for human lactoferrin has been synthesised by the polymerase chain reaction (PCR) using sequence specific primers. The template was first strand cDNA, synthesised from human bone marrow RNA using oligo(dT) to prime DNA synthesis by MMLV reverse transcriptase. The full-length human lactoferrin cDNA has been expressed in baby hamster kidney (BHK) cells using the expression vector pNUT. The protein expressed from the cloned cDNA is secreted into the culture medium and yields of up to 40 mg per litre have been obtained. A mutant protein corresponding to the N-lobe of human lactoferrin (LfN) has also been expressed in BHK cells. The cDNA coding for this protein was produced by the introduction of stop codons into the region of the cDNA corresponding to the helix linking the N- and C-lobes of the native protein. LfN is also expressed as a secreted protein and has been obtained in high yield. LfN binds iron and has UV/Vis and ESR spectra which are virtually identical to the native protein. However, the pH at which iron is released from LfN is quite different to the pH of iron release from the native and the full-length recombinant protein. A number of mutations have been introduced into LfN by site-directed mutagenesis and the mutant proteins expressed in BHK cells. These mutations involve the iron binding ligands and have been designed to introduce some of the changes found in the C-lobe of melanotransferrin into LfN. An attempt has been made to express a protein corresponding to the C-lobe of human lactoferrin (LfC) by attaching the sequence for the signal peptide of lactoferrin to the cDNA sequences coding for the C-lobe.
Tweedie
J W
JW
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Bain
H B
HB
Day
C L
CL
Nicholson
H H
HH
Mead
P E
PE
Sheth
B
B
Stowell
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D007501:Iron / Q000378:metabolism; D007668:Kidney; D007781:Lactoferrin / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D007501:Iron; D007781:Lactoferrin