Bacterial bioreactors for high yield production of recombinant protein.
We developed a new bacterial expression system that utilizes a combination of attributes (low temperature, induction of an mRNA-specific endoribonuclease causing host cell growth arrest, and culture condensation) to facilitate stable, high level protein expression, almost 30% of total cellular protein, without background protein synthesis. With the use of an optimized vector, exponentially growing cultures could be condensed 40-fold without affecting protein yields, which lowered sample labeling costs to a few percent of the cost of a typical labeling experiment. Because the host cells were completely growth-arrested, toxic amino acids such as selenomethionine and fluorophenylalanine were efficiently incorporated into recombinant proteins in the absence of cytotoxicity. Therefore, this expression system using Escherichia coli as a bioreactor is especially well suited to structural genomics, large-scale protein expressions, and the production of cytotoxic proteins.
Suzuki
Motoo
M
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Roy
Rohini
R
Zheng
Haiyan
H
Woychik
Nancy
N
Inouye
Masayori
M
D019149:Bioreactors / Q000382:microbiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D009587:Nitrogen Isotopes; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012645:Selenomethionine / Q000378:metabolism; D010135:p-Fluorophenylalanine / Q000378:metabolism
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C422955:MazF protein, E coli; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D010135:p-Fluorophenylalanine; D012645:Selenomethionine; D004722:Endoribonucleases