FHL1 (Four and a Half LIM Domains Protein 1) is a protein composed of four and a half LIM domains. Each LIM domain consists of two consecutive zinc finger motifs, coordinated by two Zn$^{2+}$ ions, with each ion coordinating one motif. These domains are involved in protein–protein interactions, which include receptors, transcriptional proteins, signaling pathway proteins, and structural proteins. FHL1 functions as a transcription factor and signaling molecule in many pathways, which is why it is implicated in numerous disease states. Among the most concerning are cardiovascular diseases, leading to increased research into FHL1 and its interactions with other proteins, as these are not well understood.
The aim of our thesis was to determine if FHL1 can be expressed and purified using the same method as for the related protein FHL2, thereby preparing an active form of this protein for subsequent interaction analysis with other proteins. We inserted the FHL1 gene into the pET-32b(+) vector, which contained the His6-sfGFP-TEV-2 × GSS-BamHI insert, using the IVA method. This was followed by expression of the entire insert and purification of FHL1 using nickel affinity chromatography. During purification, the sfGFP and hexahistidine tags were cleaved off using TEV protease.
The results of NaDS-PAGE analysis post-purification indicated that the purification process was not optimal. This was likely due to the insufficient amount of TEV protease added before dialysis, resulting in incomplete cleavage of FHL1 from His6 and sfGFP. FHL1 was well soluble. We did not have any issues with protein aggregation. To achieve better purification results, it would be worthwhile to try adding larger amounts of TEV protease during dialysis.
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