High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography.
A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.
Alm
Tove
T
School of Biotechnology, Department of Proteomics, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden.
Steen
Johanna
J
Ottosson
Jenny
J
Hober
Sophia
S
D002411:Cation Exchange Resins / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012371:Robotics / Q000379:methods*; D013048:Specimen Handling / Q000379:methods
D002411:Cation Exchange Resins; D011506:Proteins