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 hema tophagous human parasite Necator americanus. The cDNA library was transferr ed by polymerase chain reaction (PCR) cloning into phage display Vectors (p hage-mids), using specially designed primers such that proteins would be ex pressed as fusions with the C-terminal part of the phage coat protein pVI. The vectors used are multicloning site variants of the original pDONG vecto rs described by Jespers el al. (1995). Electroporation of the ligation mixt ures into electrocompetent Escherichia coli TG1 cells yielded 3 x 10(8) pG6 A, 1.9 x 10(8) pG6B, and 1 X 10(8) pC6C transfectants for N. americanus. Th e 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 h uman collagen was performed. Four rounds of panning on human collagens I an d 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 sam e protein, albeit with different poly-A tail lengths. The encoded protein i tself is a 135-amino acid protein (15 kDa), with no apparent homology to an y 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, dosedependent, and satura ble manner.