Prokaryotic expression of bovine lactoferrin deletion mutants that bind to the Ca2+-dependent lactoferrin receptor on isolated rat hepatocytes.
We generated a series of recombinant variants of bovine lactoferrin (Lf) as fusion proteins using two prokaryotic expression vectors and examined the ability of the expressed proteins to compete with native Lf for binding to the Ca2+-dependent Lf receptor on isolated rat hepatocytes. A near-full-length bovine Lf cDNA (pN16b) was expressed in pGEMEX-2 as a gene 10 fusion protein (r-bLf10/-70). Deletions of pN16b were cloned into the HindIII/NotI and BamHI/NotI restriction sites of expression vector pET 32 and expressed as thioredoxin fusion proteins, r-bLfT/-271 and r-bLfT/-310, respectively. r-bLf10/-70, r-bLfT/-271, and r-bLfT/-310 lacked, respectively, the NH2-terminal 70, 271, and 310 amino acids of Lf. Expression of recombinant proteins in Escherichia coli BL21-DE3 strain was monitored by denaturing gel electrophoresis or by immunoblot with anti-Lf antibodies. The yield of each of the soluble recombinant proteins was approximately 10 mg/L of BL21-DE3 suspension. r-bLf10/-70 and r-bLfT/-271 competed strongly with 125I-Lf for binding to hepatocytes but r-bLfT/-310 did not. Our findings are consistent with the conclusion that Lf binds to its Ca2+-dependent receptor on hepatocytes via noncarbohydrate determinants contained within its C-lobe.
Sitaram
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MP
Department of Chemistry and Biochemistry, California State University at Long Beach, 1250 Bellflower Boulevard, Long Beach, California, 90840, USA.
Moloney
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B
McAbee
D D
DD
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007781:Lactoferrin / Q000737:chemistry*; D008099:Liver / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013879:Thioredoxins / Q000235:genetics
D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C038340:lactoferrin receptors; D013879:Thioredoxins; D007781:Lactoferrin; D002118:Calcium