[Effect of different tags on pulldown assays implemented by LMO2 fusion protein].
Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
Yuan
Wei
W
Laboratory of Molecular Genetics, College of Medicine, Nankai University, Tianjin 300071, China.
Sun
Wei
W
Yang
Shuang
S
Yan
Jidong
J
Zhai
Chunli
C
Du
Jun
J
Wang
Zhaoqi
Z
An
Di
D
Zhu
Tianhui
T
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000737:chemistry*; D011232:Chemical Precipitation; D004268:DNA-Binding Proteins / Q000737:chemistry*; D050982:GATA1 Transcription Factor / Q000737:chemistry*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D020014:K562 Cells; D060588:LIM Domain Proteins; D058619:Maltose-Binding Proteins; D008667:Metalloproteins / Q000737:chemistry*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020673:Protein Renaturation; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D060588:LIM Domain Proteins; C085117:LMO2 protein, human; D058619:Maltose-Binding Proteins; D008667:Metalloproteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase