Purification of recombinant Lumican and the healing of scratched HTCE cells.

<p><i>Purification of GST-Lum and GST-LumC₅₀ recombinant proteins</i> Purification of recombinant GST-Lum and GST-LumC₅₀ was monitored by Coomassie Brilliant Blue (CBB) staining and western blot anlaysis. (A) CBB staining revealed two major bands eluted with glutathione. The upper band with a Mr. ∼70 kDa was GST-Lum and the lower band with a Mr. ∼25 kDa was GST. (B) Immunostaining with an anti-LumN oilgopeptide antibody (CDDLKLKSVPMVPPGIK) only labeled the 70 kDa band (GST-Lum fusion protein) while the lower band did not react to the antibody and is likely related to GST. (C) CBB stained two bands at 30 and 25 kDa from <i>E.coli</i> transfected with GST-LumC₅₀ plasmid. (D) Immunostaining with an anti-LumC peptide antibody (NPLTQSSLPPDMYEC) labeled the 30 kDa GST-LumC₅₀ fusion protein. <i>Effect of recombinant GST-Lum and GST-LumC₅₀ on healing of scratched HTCE cells</i> Confluent HTCE cells were wounded in CM (complete medium), BM (basic medium), BM + GST (glutathione S-transferase recombinant protein), BM+recombinant GST-Lum (0.15 µM) and GSTLumC_50. The wound gap was determined by time-lapse microscopy. (E) Representative time-lapse images of the healing of scratched HTCE cells; (F) The healing followed biphasic kinetics in cells treated with BM+GST-Lum and CM, whereas those of BM and BM+GST followed monophasic kinetics. R² values were as follows: BM 0.957; CM 0.994; GST 0.985; LumC₅₀ 0.985; Lum 0.995. The rate constants are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082730#pone-0082730-t001" target="_blank">Table 1</a>.