Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner.
To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained alpha-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI approximately/= 5.8) and ZZ-Cutinase-Zbasic (pI approximately/= 7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.
Gräslund
Torbjörn
T
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Ehn
Maria
M
Lundin
Gunnel
G
Hedhammar
My
M
Uhlén
Mathias
M
Nygren
Per-Ake
PA
Hober
Sophia
S
D001483:Base Sequence; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004256:DNA Polymerase I / Q000302:isolation & purification; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel
D002411:Cation Exchange Resins; D017931:DNA Primers; D004256:DNA Polymerase I