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Line Cantin, Christian Salesse; Usefulness of Recoverin to express and purify visual proteins . Invest. Ophthalmol. Vis. Sci. 2019;60(9):2355.
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Recoverin is involved in the regulation of the activity of rhodopsin kinase during the phototransduction cascade. In addition, Recoverin has properties typical of solubility-enhancing and purification tags. Indeed, it is a highly soluble protein and it can be purified with high purity in a single step on the basis of the properties of its calcium-myristoyl switch. Given that only a few tags are available and that proteins can hardly be produced and purified on the basis of their intrinsic properties, this work was aiming to find out whether visual Recoverin could be used as a solubility-enhancing and purification tag to express and purify photoreceptor and visual cycle proteins.
Recoverin and 2XRecoverin were individually cloned in fusion with Retinitis Pigmentosa 2 (RP2), RGS9-1-Anchor-Protein (R9AP) and truncated Lecithin retinol acyltransferase (tLRAT). These fusion proteins have then been expressed in E. coli. Their level of expression and solubility has been evaluated by polyacrylamide gel electrophoresis and compared to that of two well-known solubility-enhancing and purification tags: Maltose-Binding protein (MBP) and Glutathione-S-Transferase (GST). The enzymatic activity of tLRAT has been measured as previously described.
The expression level of the Recoverin-RP2 and 2XRecoverin-RP2 fusion proteins was a little smaller to that of MBP and GST. However, the solubility of RP2 in fusion with the Recoverin tags was much higher than when RP2 was in fusion with MBP and GST. In addition, the level of expression of the R9AP fusion proteins was as follows: GST-R9AP > Recoverin-R9AP > 2XRecoverin-R9AP = MBP-R9AP. R9AP was however most soluble in fusion with MBP and 2XRecoverin. In contrast, although a larger level of expression was observed for GST-tLRAT and MBP-tLRAT, these proteins were poorly soluble. High solubility was however achieved with Recoverin-tLRAT and 2XRecoverin-tLRAT. The enzymatic activity of tLRAT from the Recoverin-tLRAT fusion protein was found to be similar to that previously reported.
Recoverin can be successfully used to express and solubilize the photoreceptor and visual cycle proteins assayed as efficiently as well-known solubility-enhancing and purification tags. Other photoreceptor and visual cycle proteins will then be assayed to further demonstrate the efficiency of the Recoverin tags.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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