Overview
Our team reached the following key results after four rounds of DBTL:
- Expression purification and enzyme activity characterisation of SARS-Cov-2 nsp5 with native N-terminal and C-terminal.
- Construct a FlipGFP system that can be activated by nsp5.
- Rational design of nsp5-T21I with enhanced enzyme activity.
Successful expression, purification and characterisation of SARS-CoV-2 nsp5 with native N-terminal and C-terminal.
Through two rounds of DBTL for sequence design and optimisation, we successfully expressed and purified SARS-Cov-2 nsp5 in E. coli BL21.In order to accurately characterise nsp5 we determined the efficiency of nsp5 degradation of substrates using FRET and characterised the kcat/Km of nsp5 based on the Michaelis-Menten equation(Figure 1).
Successful construction of FlipGFP that can be activated by nsp5
To ensure the correct expression of FlipGFP, we used the pRSF-Duet1 and separately inserted the first nine β-strands of FlipGFP and the engineered 10-11 β-strands into two different ORFs. We separately transformed E. coli BL21 with FlipGFP alone and co-transformed E. coli BL21 with FlipGFP and nsp5, then plated them on LB plates containing IPTG at a final concentration of 0.2 mM. The results showed that colonies transformed with FlipGFP alone produced almost no fluorescence, while colonies co-transformed with FlipGFP and nsp5 emitted significant fluorescence. This demonstrates that we successfully constructed a FlipGFP system that can be activated by nsp5 to emit fluorescence(Figure 2).
Successful design of nsp5-T21I with enhanced enzyme activity.
Through rational design (see Model for details), we designed a mutant nsp5-T21I that may have higher enzyme activity than wild type. We first verified that this mutant could be solubilised for expression in E. coli BL21 and purified by nickel affinity chromatography(Figure 3).
We determined the enzyme activity of nsp5-T21I by FRET. The results showed that nsp5-T21I had a higher kcat/Km(35,069 s⁻¹M⁻¹) compared to wild-type nsp5(27,691 s⁻¹M⁻¹), as well as a higher reaction rate at the same substrate concentration(Figure 4). This indicates that we successfully designed the nsp5 mutant with higher enzyme activity compared to the wild type.
