Journal: Scientific reports

Article Title: Cell-free expression of functional receptor tyrosine kinases.

doi: 10.1038/srep12896

Figure Lengend Snippet: Figure 1. (a) Schematic of cell-free co-translation of ERBB2-NLP and EGFR-NLP. (b) ERBB2 was cell-free produced in the presence and absence of DMPC or with DMPC and co-expressed Δ 49A1. FluoroTect™ GreenLys (Promega) was added for visualizing newly synthesized ERBB2 protein. After 4 hours of expression, cell-free reactions were centrifuged at 14,000 rpm for 10 minutes. Small aliquots of sample before centrifuging (total, T), and the supernatant (soluble, S) and pellet (P) after centrifuging, were collected. All samples were loaded along with a cell-free reaction mixture only (-). Gel images were taken using Molecular Dynamics Typhoon 9410 Molecular Imager from GE Healthcare. Full-length versions of gel images are presented in Supplementary Figure 5. (c) The ERBB2-NLP complex shows greatly enhanced solubility as compared to protein only or protein DMPC vesicles.

Article Snippet: ELISA was carried out using DuoSet IC ERBB2 ELISA kit from R&D Systems.

Techniques: Produced, Synthesized, Expressing, Solubility

Journal: Scientific reports

Article Title: Cell-free expression of functional receptor tyrosine kinases.

doi: 10.1038/srep12896

Figure Lengend Snippet: Figure 2. (a) NLP associated ERBB2 is tyrosine phosphorylated. Cell-free expressions were set up with and without (-) ERBB2 plasmid. Samples were collected at 2 hr, 5 hr, 8 hr and overnight (18 hr), resolved by SDS- PAGE and western blotted with anti-phospho-tyrosine ERBB2 antibody pY1248 and anti-ERBB2 antibody Ab-3 after stripping. (b) The NLP associated ERBB2 is phosphorylated independent of protein expression. Cell-free expressed ERBB2 (in replicate) was treated with calf-intestinal alkaline phosphatase (CIP) to remove the phosphate group and then Ni purified. The purified ERBB2-NLPs were incubated with ATP, Mn2+, Mg2+ and buffer to allow for re-phosphorylation. Samples were resolved by SDS-PAGE and western blotted with anti-phospho-tyrosine antibody 4G10 and anti-ERBB2 antibody Ab-3. (c) Binding of trastuzumab to cell-free ERBB2-NLPs and cellular ERBB2 extract were measured by ELISA as described. Cell-free ERBB2-NLPs or ERBB2 extracted from HEK293 cells over-expressing ERBB2 receptor were exposed to varying concentrations of trastuzumab (0–500 nM). The Kds for the trastuzumab binding to cell-free ERBB2-NLP and cellular ERBB2 extract were 4.4 nM and 2.7 nM, respectively. Each data point represents the average value of triplicate measurements. (d) NLP associated EGFR is phosphorylated, and the presence of EGF in the cell-free reaction increases the level of phosphorylation. EGFR-NLPs showed low level of phosphorylation during cell-free expression. Adding EGF, the natural ligand of EGFR, increases the phosphorylation. Cell-free expressions were set up with and without (-) EGFR plasmid, with and without EGF. After an 8 hr reaction, cell-free mixtures were resolved by SDS-PAGE and western blotted with anti-phospho-tyrosine EGFR antibody pY1110 and anti-EGFR. The white vertical line represents a splicing event of lanes from the same Western blot. Full-length versions of all western blots are presented in Supplementary Figure 6.

Article Snippet: ELISA was carried out using DuoSet IC ERBB2 ELISA kit from R&D Systems.

Techniques: Plasmid Preparation, SDS Page, Western Blot, Stripping Membranes, Expressing, Purification, Incubation, Phospho-proteomics, Binding Assay, Enzyme-linked Immunosorbent Assay