Identification of a collagen-binding protein from Necator americanus by using a cDNA-expression phage display library.
A phage display library was made starting from a cDNA library from the hematophagous human parasite Necator americanus. The cDNA library was transferred by polymerase chain reaction (PCR) cloning into phage display vectors (phagemids), using specially designed primers such that proteins would be expressed as fusions with the C-terminal part of the phage coat protein pVI. The vectors used are multicloning site variants of the original pDONG vectors described by Jespers et al. (1995). Electroporation of the ligation mixtures into electrocompetent Escherichia coli TGI cells yielded 3 x 10(8) pG6A, 1.9 x 10(8) pG6B, and 1 x 10(8) pG6C transfectants for N. americanus. The final libraries consisted of a mix of equal numbers of insert-containing phages from the A, B, and C libraries. Selection of phages for binding to human collagen was performed. Four rounds of panning on human collagens I and III resulted in a significant enrichment of collagen-binding phages from the N. americanus libraries. PCR analysis revealed various insert lengths; however, sequence determination indicated that all phages contained the same protein, albeit with different poly-A tail lengths. The encoded protein itself is a 135-amino acid protein (15 kDa), with no apparent homology to any other known protein. Next the protein was recloned into E. coli using the pET-15b-vector. Upon isopropyl-1-thio-beta-D-galactopyranoside induction, the recombinant protein, rNecH1, could be recovered by urea treatment from inclusion bodies. The rNecH1 protein binds to different collagens: human I > rat I > human III = calf skin I in a specific, dose-dependent, and saturable manner.
Viaene
A
A
Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium.
Crab
A
A
Meiring
M
M
Pritchard
D
D
Deckmyn
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D018509:DNA, Helminth / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D017221:Necator americanus / Q000737:chemistry* / Q000235:genetics; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D051381:Rats; D038981:Receptors, Collagen
D018076:DNA, Complementary; D018509:DNA, Helminth; D016023:Integrins; D019151:Peptide Library; D038981:Receptors, Collagen; D003094:Collagen