Identification of a Collagen-Binding Protein fromNecator americanusby Using acDNA-Expression Phage Display Library

Abstract

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.

Non-Invasive Molecular Imaging of Fibrosis Using a Collagen-Targeted Peptidomimetic of the Platelet Collagen Receptor Glycoprotein VI

“…Peptides with collagen-binding properties have previously been reported: cyclic peptides mimicking the collagen-binding site of vWF bound to rat tail collagen, and inhibited vWF binding to calfskin and human collagen, but only at high concentrations [24]; phage display also made it possible to identify the collagen-binding protein rNecH1 from Necator americanus [25], but no further use of these peptides or proteins has been reported. Recently, CNA35, the collagen binding part of a bacterial protein, has been successfully used as a fluorescent probe for collagen [26].…”

Human Hookworm Infection in the 21st Century

“…Measured IL-10 levels in humans infected with hookworm are extremely high (Bethony J., unpublished observation) suggesting that hookworm TIMP could have a role in host immunomodulation. In this case, hookworm TIMP would join a list of hookworm-derived molecules that have a role in either modulating or down-regulating the host inflammatory response, including neutrophil inhibitory factor (NIF), which serves as an integrin antagonist of host CD11b/CD18 (Moyle et al , 1994; Muchowski et al ., 1994); hookworm proteases, which cleave eotaxin (Culley et al ., 2000); a calreticulin that interacts with host C1q (Kasper et al ., 2001); a retinol binding protein (Basavaraju et al ., 2003); a collagen-binding protein (Viaene et al ., 2001); a C-type lectin (Loukas et al ., 2002); an acetylcholinesterase (Brown and Pritchard, 1993); a glutathione S-transferase (Brophy et al ., 1995); a Cu/Zn superoxide dismutase (Taiwo et al ., 1999); and molecules that induce host T cell apoptosis (Chow et al ., 2000). In addition adult, hookworms produce and release at least four different ASPs, which are similar in structure to the two L3 ASPs (Zhan et al ., 2003).…”

Oligopeptide M13 Phage Display in Pathogen Research

“…The main coat protein VIII was used for peptides, oligopeptides and antibody fragments [32,33,34,35], but besides other drawbacks the size of the fusion partner is limited [36]. The coat protein pVI was successfully used in some cases as fusion partner for oligopeptides, mainly from cDNA libraries [37,38,39,40,41]. Alternative fusion partners are pVII and pIX, but these fusion partners on the pIII opposing site were mainly used for antibody fragments [36,42,43,44,45,46].…”