Team:SCAU-China/improvement
Background
Here, our team is planning to integrate metallothioneins (MTs) and autophagy system to improve the phytoremediation capability of C.reinhardtii. Several MTs from different species were selected. One of MTs, SmtA derived from Synechococcus sp was chosen as a representative for further optimization since it can absorb multiple heavy metals, including copper, zinc and mercury (Shi et al., 1992).
Design
To increase its protein expression in eukaryotic species including Chlamydomonas, we first codon optimized smtA gene based on the sequence of BBA_ K519010. Then we tested its function in zinc tolerance of yeast and examined its protein expression using immunoblotting.
Results
Ectopic expression of wild-type (WT) and codon optimized (Opt) smtA showed that all of them enhanced zinc tolerance of yeast when compared with that transformed with pYES2 vector under 20 mM Zinc treatment (Fig. 1). Moreover, SmtA(Opt) showed much stronger effect than wild-type SmtA on Zinc tolerance.
Figure 1: Increase of zinc tolerance of yeast transformed with codon optimized smtA.
The wild-type yeast strain BY4741 were transformed with EV (pYES2 empty vector), SmtA (Opt) or SmtA (WT), and grown on SD plates with indicated concentrations of ZnCl2 for 3 d. SmtA (Opt): codon optimized SmtA in pYES2 vector fused with a 6 x His tag at its C- terminal; SmtA (WT): wild-type SmtA in pYES2 vector fused with a 6 x His tag at its C- terminal.
Using western blot, we observed apparent higher protein expression in the lane of SmtA (Opt) when compared with yeast transformed with pYES2 or SmtA (WT), indicating that codon optimization of SmtA significantly increase its expression (Fig 2). This observation was also consistent with the better zinc tolerance found in yeast transformed with SmtA (Opt) (Fig 1.)
Figure 2: Expression level of SmtA was increased by codon optimization.
Total yeast cell lysates were prepared using Yeast Protein Extraction Reagent (Takara, Japan) and equal total protein lysates were loaded and separated on 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were then detected with anti-His antibodies and visualized by using ECL detection kits (Beyotime, China)
References
- Jianguo Shi, W.P.L.J. (1992). Cyanobacterial metallothionein gene expressed in Escherichia coli Metal‐binding properties of the expressed protein. FEBS LETT 303, 159-163.
