StayGold variants for molecular fusion and membrane-targeting applications

Ando R, Shimozono S, Ago H, Takagi M, Sugiyama M, Kurokawa H, Hirano M, Niino Y, Ueno G, Ishidate F, Fujiwara T, Okada Y, Yamamoto M, Miyawaki A

(2023). Nature Methods, , . doi: 10.1038/s41592-023-02085-6. Article   Pubmed

    Primary Proteins:
  1. mStayGold
  2. mStayGold2
  3. QC2-6
  4. td5oxStayGold
  5. td5StayGold
  6. td8ox2StayGold
  7. td8oxStayGold
    Secondary Proteins:
  1. oxStayGold,
  2. tdoxStayGold
Add photostability measurements

Excerpts

The oxStayGold variant of StayGold was engineered to efficiently label the ER from the inside. Because oxStayGold was found to label all subcellular components, including the cytoplasm, very brightly, it has replaced StayGold in many experiments; however, oxStayGold remains a dimer.

We comprehensively prepared several libraries of (n1)oxStayGold variants that carried different partial mutations at the interface. In one attempt, we focused on threonine substitution at Pro151 and Leu155 and screened candidates in a library of (n1)oxStayGold P151T/L155T iteratively with multiple cycles of random mutagenesis to produce QC2-6. This variant contained four mutations (P151T, L155T, N132D and K162E) relative to (n1)oxStayGold. When observing the fluorescence in the cytosolic and nuclear compartments of transfected cells under intense illumination, we confirmed that QC2-6 was highly photostable and bright.

We noticed, that QC2-6 yielded a substantial number of puncta in a Fluoppi assay (score, 55.6%). With this unacceptable result, we decided to drive the monomerization further on the basis of QC2-6. Performing combinatorial saturation mutagenesis at Tyr187, Arg144 and Thr155 and screening for brightness, photostability and monomericity yielded QC2-6 FIQ, which contained Y187F, R144I and T155Q mutations relative to QC2-6. QC2-6 FIQ gave high scores for both OSER (92.3%) and Fluoppi (100%) assays.

In one experiment to amplify the full length of QC2-6, we accidentally used a defective reverse primer to introduce a mutation at the termination codon. The translation readthrough led to the addition of a short C-terminal tail (PT) composed of YSRTKLE. Notably, this protein product QC2-6(PT) showed high scores in OSER (82.6%) and Fluoppi (100%) assays.

All things considered, we designated QC2-6 FIQ as the most useful monomeric version and named it mStayGold. We also kept QC2-6(PT) as a reserve monomer and named it mStayGold2.

Subsequently, oxStayGold was substituted in the td construct to create tdoxStayGold, which consistently achieved brighter molecular fusions than tdStayGold; however, we found that CytERM-tdoxStayGold showed an undesirable score (58.3%).

To improve the dispersibility of tdoxStayGold, we re-examined the polypeptide linker between the two copies of (ox)StayGold. First, we trimmed the EV linker (116 residues) into three polypeptides that spanned 34, 66 and 97 amino acids; however, none of the td constructs with these shortened EV linkers (td2oxStayGold, td3oxStayGold and td4oxStayGold, respectively) showed improved OSER or Fluoppi scores. We subsequently harnessed the linker used for generating tdTomato. The use of a 21-residue linker resulted in the generation of td5StayGold and td5oxStayGold, both of which gave high OSER scores (91.5% and 88.5%, respectively) and high Fluoppi scores (96.4% and 94.1%, respectively).

We found that PT can be used as another adaptor for fusion at the StayGold C terminus. By replacing c4 with PT in td5oxStayGold, we generated td8oxStayGold... Both td8oxStayGold and td8ox2StayGold exhibited excellent dispersibility.

We found that PT can be used as another adaptor for fusion at the StayGold C terminus. By replacing c4 with PT in td5oxStayGold, we generated td8oxStayGold. In addition, we performed saturation mutagenesis on td8oxStayGold for Tyr169 and generated td8ox2StayGold with a Y169F mutation. Both td8oxStayGold and td8ox2StayGold exhibited excellent dispersibility.