Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63 Ag8 cells |
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Authors: | M R Michel M Elgizoli H Koblet Ch Kempf |
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Institution: | (1) Institute for Hygiene and Medical Microbiology, University of Bern, Friedbühlstraße 51, CH-3010 Bern, (Switzerland) |
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Abstract: | Summary Using the suspension cell line P3X63 Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0°C. Subsequent resealing of the pores was completed after a 5-min incubation at 37°C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37°C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K+, Mg2+, and Ca2+) and resealing (in the presence of K+, Mg2+, and Ca2+), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure. |
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Keywords: | Electroporation diffusion loading electropore resealing protein synthesis |
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