Fast, flexible and non-randomized modification, production and screening of proteins in fully automated system are of high interest in biological research and applications. The conventional methods for protein engineering and screening, especially for mutations of multiple residues. are time consuming and often unreliable. We demonstrate here a new, fast and flexible protein production and screening method which combines linear expression template (LET) based cell free protein synthesis (CFPS) with specific screening methods. This approach is demonstrated using green fluorescence protein, phosphoserine aminotransferase (serC) and aspartokinase III (AKIII) as model systems. The results show that mutants with changes in different protein properties upon multiple point mutations can be produced and screened within 6 to 15 h. This method can be used further to generate mutants of enzymes and multi-enzyme complexes and be implemented within the workflow of a feedback-guided protein optimization and screening system.
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Figure 1.
Conventional protein production and purification method vs LET based cell free protein synthesis system. The absence of cloning and purification steps significantly reduces the time from protein expression to analysis of variants with single and mutliple mutations.
Figure 2.
serC-GDH coupled enzyme assay. The transamination reaction catalyzed by serC produces L-glutamate which is in turn oxidatively deaminated by GDH in the presence of NAD+. The reduction of NAD+ can be observed at 340 nm.
Figure 3.
Ratio of fluorescence signals in FITC to KO channels for GFP and its variants CFP and YFP. YFP contains two mutations – S65T and T203Y while CFP contains three mutations—N146I, S175G and H231L.
Figure 4.
The concentration of protein produced in cell-free system measured using biolayer-interferometry. (a) The protein production at different temperatures was carried out to find the optimal temperature. These reactions were also carried out for 8 h. (b) The protein production was tested for different times at 25 °C. (The error bars indicate 1 % confidence interval n = 3). pAKIII refers to plasmid based CFPS samples; AKIII refers to LET based CFPS samples.
Figure 5.
(a) The fluorescent emission of the ATP sensor of wildtype and different mutants of AKIII at lysine concentration of 200 mM (error bars indicate 1% confidence interval, n = 50,000). (b) The significant difference between various mutants of AKIII from the QUEEN fluorescent signal. There is significant difference between the wildtype and the mutants V339A [21] and T344M [25] (p < 0.01) (p cut off is the red line). There was also mild difference between the wildtype and mutant M-I. (c) The activity of serC mutants measured using the serC-GDH coupled assay. The mutants were produced by using the LET based CFPS method performed at 25 °C for 8 h. (d) Activity of serC mutants produced using LET based CFPS method (black) compared to the same mutants produced conventionally (grey). Activities are measured using the same serC-GDH coupled assay in both cases.
Figure 6.
(a) Fluorescent emission of ATP sensor of wildtype AKIII in comparison with feedback resistant mutant AKIII produced in fully automated system at lysine concentration of 200 mM (error bars indicate 1 % confidence interval n = 50000). (b) Fluorescent signal of wildtype AKIII in comparison with AKIII mutants with different substrate specificity (malate instead of aspartate) (same error bars).
