High-throughput liberation of water-soluble yeast content by irreversible electropermeation (HT-irEP).
The article describes a high-throughput method for the liberation of water-soluble cell contents by exploiting the phenomenon of irreversible membrane electropermeation (HT-irEP). The method is exemplified in recombinant proteins and plasmid liberation from yeast Saccharomyces cerevisiae on the detectable level. Obtained extracts are pure enough to be readily applied for further analytical analysis such as enzyme assay, PCR, and so on. From the same HT-irEP extract, one can measure activity of the target protein and perform amplification of the corresponding gene from the DNA vector by PCR for recombinant protein with intracellular expression. Therefore, the method is suitable for the high-throughput screening (HTS) of yeast libraries where extracellular expression of recombinant protein is problematic. The method can be easily automated and integrated into existing HTS systems.
Zakhartsev
Maxim
M
Biochemical Engineering, School of Engineering and Science, International University Bremen, Bremen, Germany. maxim.zakhartsev@awi.de
Momeu
Carmen
C
Ganeva
Valentina
V
D002463:Cell Membrane Permeability; D004271:DNA, Fungal; D018274:Electroporation / Q000295:instrumentation / Q000379:methods; D015870:Gene Expression; D005822:Genetic Vectors; D005949:Glucose Oxidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013053:Spectrophotometry; D014170:Transformation, Genetic; D014867:Water / Q000737:chemistry
D004271:DNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014867:Water; D049452:Green Fluorescent Proteins; D005949:Glucose Oxidase