Selection of aptamers for a protein target in cell lysate and their application to protein purification.
Functional genomics requires structural and functional studies of a large number of proteins. While the production of proteins through over-expression in cultured cells is a relatively routine procedure, the subsequent protein purification from the cell lysate often represents a significant challenge. The most direct way of protein purification from a cell lysate is affinity purification using an affinity probe to the target protein. It is extremely difficult to develop antibodies, classical affinity probes, for a protein in the cell lysate; their development requires a pure protein. Thus, isolating the protein from the cell lysate requires antibodies, while developing antibodies requires a pure protein. Here we resolve this loop problem. We introduce AptaPIC, Aptamer-facilitated Protein Isolation from Cells, a technology that integrates (i) the development of aptamers for a protein in cell lysate and (ii) the utilization of the developed aptamers for protein isolation from the cell lysate. Using MutS protein as a target, we demonstrate that this technology is applicable to the target protein being at an expression level as low as 0.8% of the total protein in the lysate. AptaPIC has the potential to considerably speed up the purification of proteins and, thus, accelerate their structural and functional studies.
Javaherian
Sahar
S
Department of Chemistry, York University, Toronto, Ontario, Canada.
Musheev
Michael U
MU
Kanoatov
Mirzo
M
Berezovski
Maxim V
MV
Krylov
Sergey N
SN
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D052156:SELEX Aptamer Technique
D052157:Aptamers, Nucleotide; D015345:Oligonucleotide Probes; D011506:Proteins; D051719:MutS DNA Mismatch-Binding Protein