Highly branched poly(N-isopropylacrylamide) for use in protein purification.
Poly(N-isopropylacrylamide)s with imidazole endgroups were used to separate a histidine-tagged protein fragment directly from a crude cell lysate. The polymers display a lower critical solution temperature that can be tuned to occur at a range of subambient temperatures. UV-visible spectra indicated differences in the binding in aqueous media of Cu(II) and Ni(II) to the imidazole endgroups. These changes in the UV-visible spectra were reflected in the solution/aggregation behavior of the polymers as studied by dynamic light scattering. The addition of Cu(II) disaggregated the polymers, and the polymer coil swelled. On the other hand, when Ni(II) was added the polymers remained aggregated in aqueous media. The polymers were used to purify residues 230-534 of the histidine-tagged breast cancer susceptibility protein his6-BRCA1. Cu(II) was found to be better suited to the formation of useful polymer-metal ion-protein complexes that display cloud points, since Ni(II)/polymer mixtures generated very little purified protein. The polymers were synthesized using a previously reported variation of the reversible addition-fragmentation chain termination (RAFT) methodology, using the chain transfer agent 3H-imidazole-4-carbodithioic acid 4-vinyl benzyl ester with N-isopropylacrylamide (NIPAM).
Carter
Steven
S
The Polymer and Biomaterials Chemistry Laboratories, Department of Chemistry (Polymer Centre), Brook Hill, University of Sheffield, Sheffield, South Yorkshire, S3 7HF, UK.
Rimmer
Stephen
S
Rutkaite
Ramune
R
Swanson
Linda
L
Fairclough
J P A
JP
Sturdy
Alice
A
Webb
Michelle
M
D000180:Acrylic Resins / Q000138:chemical synthesis / Q000737:chemistry*; D019313:BRCA1 Protein / Q000737:chemistry* / Q000302:isolation & purification*; D001665:Binding Sites; D003300:Copper / Q000737:chemistry; D005260:Female; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015394:Molecular Structure; D009532:Nickel / Q000737:chemistry; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D009994:Osmolar Concentration; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D000180:Acrylic Resins; D019313:BRCA1 Protein; D009942:Organometallic Compounds; C052970:poly-N-isopropylacrylamide; D006639:Histidine; D003300:Copper; D009532:Nickel