Cell-free synthesis and affinity isolation of proteins on a nanomole scale.
The performance of conventional cell-free gene expression systems based on the Escherichia coli S30 extract can be significantly improved by using expression vectors that encode viral structural elements known to enhance translation in vivo and to protect mRNA from ribonuclease action. The expression vectors reported here are designed to produce a functionally active protein carrying the Strep-tag oligopeptide at its C-terminus. They can be used in translation, transcription-translation or replication-translation reactions. Depending on its type, the reaction yields up to 40 micrograms per mL, or about 1 nmol of a standard protein. The presence of Strep-tag allows the synthesized protein to be easily isolated on a streptavidin-agarose column under mild conditions and the entire procedure to be completed within one working day. The results show that standard low-cost, cell-free systems can serve for rapid preparation of purified proteins in amounts that can satisfy a number of needs of a research laboratory.
Alimov
A P
AP
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Khmelnitsky AYu
Simonenko
P N
PN
Spirin
A S
AS
Chetverin
A B
AB
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D004274:DNA, Recombinant; D012333:RNA, Messenger; D011994:Recombinant Proteins