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Heparin binding of the WMS mutant of fibrillin-1 protein fragment PF17-1. (

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posted on 2012-11-02, 02:40 authored by Stuart A. Cain, Amanda McGovern, Andrew K. Baldwin, Clair Baldock, Cay M. Kielty

A) Sequence trace from mammalian expression vector pCEP-pu/AC7 containing fibrillin-1 PF17-1 WMS mutant. The sequence trace generated by Finch TV V1.4 with the two codons either side of the 24 bp deletion (boxed) is shown. Below is a section of the resulting Blast search showing the alignment with human fibrillin-1, indicating the same two codons. Also shown is the corresponding amino acid sequence aligned with the last base pair of each codon. (B) Chromatographic trace of size exclusion chromatography of PF17-1 WMS fragment using Superdex S200 10/300 GL column (GE Healthcare), showing peaks and retention volume of monomer and dimer of PF17-1 WMS. (C) SDS-PAGE of all PF17-1 WMS monomer and dimer fragments run under non-reducing (NR) and reducing conditions (Red). (D) Heparin binding of PF17-1, PF17-1 WMS monomer and dimer protein fragments. Binding was analyzed using Surface Plasmon Resonance, and the response difference (Resp. Diff.) normalized to PF17-1 response level at 800 nM for each experiment was plotted against concentration. Resp. Diff. is the heparin-immobilized flow cell minus the control flow cell. The value shown is average normalized Resp. Diff. and SEM of three separate experiments. Representative sensorgrams are shown in Figure S3C.