Tuning permeabilization of microbial cells by three-phase partitioning.
Three-phase partitioning of cells was carried out by mixing t-butanol and ammonium sulfate with aqueous suspension of cells. Permeabilized cells formed the interface between aqueous and alcohol layers. A preincubation step in which cells were exposed to just t-butanol was found to tune the selectivity of permeabilized cells of Thermus thermophilus,Saccharomyces cerevisiae, and Escherichia coli. Smaller proteins (green fluorescent protein and lipase with molecular weights of 29 and 34 kDa, respectively) were released with preincubation of 15 min, and penicillin G acylase ( approximately 85 kDa) was released with preincubation of 30 min. The high-molecular-weight proteins (alcohol dehydrogenase from S. cerevisiae and T. thermophilus with molecular weights of 150 and 170 kDa, respectively) were retained even after preincubation of 60 min. The specific activities and electrophoretic analysis of some of the proteins obtained reflected their high purity.
Raghava
Smita
S
Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
Gupta
Munishwar N
MN
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000645:Ammonium Sulfate / Q000737:chemistry* / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis; D008049:Lipase / Q000378:metabolism; D008970:Molecular Weight; D010399:Penicillin Amidase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D016963:Thermus thermophilus / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D014867:Water / Q000737:chemistry; D020002:tert-Butyl Alcohol / Q000737:chemistry* / Q000494:pharmacology
D014867:Water; D049452:Green Fluorescent Proteins; D000426:Alcohol Dehydrogenase; D008049:Lipase; D010399:Penicillin Amidase; D020002:tert-Butyl Alcohol; D000645:Ammonium Sulfate