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Influence of caspase inhibitor (CI) on the expression yield of representative model proteins.

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posted on 07.05.2014, 03:43 by Marlitt Stech, Robert B. Quast, Rita Sachse, Corina Schulze, Doreen A. Wüstenhagen, Stefan Kubick

The membrane proteins Mel-Hb-EGF-eYFP (51 kDa), bacteriorhodopsin (27 kDa) and endothelin-B receptor (49 kDa), the glycoproteins Mel-EPO (21 kDa, protein in its non-glycosylated form) and Mel-vtPA (41 kDa, protein in its non-glycosylated form) as well as the protein eYFP (29 kDa) were synthesized in batch (B) and CECF (C) reactions in the presence (+) and absence (−) of CI using insect lysate. Reactions were carried out for 48 h in the absence of the reducing agent DTT and in presence of 14C-leucine. A) Diagram showing total protein yields which were determined by incorporation of 14C-leucine and liquid scintillation counting. Standard deviations were calculated from triplicate analysis (n = 3). B) Fluorescence imaging of eYFP and Mel-Hb-EGF-eYFP using a phosphorimager system. NTC  =  No template control; translation reaction without addition of a DNA template. C) Qualitative analysis of cell-free synthesized proteins by SDS-PAGE and autoradiography. Addition of CI significantly increased the protein yield of all target proteins analyzed in this study.