Establishment Of A Visible Gel Filtration Chromatography And Preliminary Studies On Leucine Zipper As A Tool To Change Protein Conformation

Gel filtration chromatography is one of a commonly used methods for protein separation and purification, and for determination of native protein molecular weight. It used spherical gel which has mesh as the stationary phase, so the protein which had larger molecular weight can not enter the sieve, it went through the shorter path and flows out earlier. On the contrary, smaller protein entered the sieve and flow out later. The proteins can be isolated depending on the size of the molecular weight. This strategy was widely used not only in researches, but also in the Biochemistry courses demonstrations.Four fluorescent proteins or tandem fusion proteins, RFP (red,26.9kDa), RC(orange,54.9kDa), YRY (pink,84.3kDa) and PKII-EGFP (green,84.3kDa), with different sizes and different colors were expressed and purified.The proteins with good solubility and strong stability are distinct clearly by color characteristics and legible to the naked eye. The colorful proteins were also separated well in superdex200column, and successfully used as chromatography demonstration. Proteins were separated in gel filtration chromatography, based not only on the native molecular weight, but also the shape of proteins. The tandem fusion proteins, non-globular proteins, are not perfect for gel filtration chromatography. The best choice might be to change chain-like protein into globular ones by end to end connection. Leucine zippers were tested as a tool for protein conformational change. Cis or trans leucine zipper sequence (from GCN4) were fused to the N-or C-ends of CFP-YFP which connected by a protease3C recognition sites. CFP-3C-YFP without leucine zipper sequence was used as a control.The purified proteins were detected by FRET after fully digestion with3C protease. The trans leucine zippers were found working well for the association of CFP and YFP based FRET analysis after protease treatment, while no FRET signal was detected with the cis leucine zippers. This showed that the trans leucine zippers can be used as a tool to change protein conformation.To further optimized this tool and explore the minimum desired length of leucine zipper for changing protein conformation, the length of the basic region or the zipper repeats was shortened. FRET was not detected after protease treatment for the zipper repeats shortened proteins.In summary, we established a visible gel filtration chromatography and found that the trans leucine zipper could be used as a tool for changing protein conformations.