Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors

Abstract

Background: The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC).

Results: A single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods.

Conclusion: The experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression.

Figure 1

Maps of the pETM vectors. The vectors pETM11, 44, and 55 are shown as representative examples of the different variables used to combine tags and protease recognition sites.

Figure 2

Comparison of the wt and I48A mutant EYFP soluble yields using the different pETM vectors. A) Small-scale affinity purification of two EYFPs (wild type and mutant I48A) expressed in BL21(DE3) bacterial cells and using the following vectors: pETM10, pETM14, pETM30, pETM11, pETM20, pETM22, pETM33, pETM44, pETM50, pETM52, pETM55, pETM66, pETM80, pETM54, pETM60, pETM70, pETM82. B) Small scale affinity purification of constructs expressed by pETM20 under different growth conditions and the alternative bacterial strain BL21(DE3) pLysS. C) Small-scale purification of wt EYFP expressed in pRSET and pETM10 vectors. D) Large-scale purification pattern of wt EYFP expressed in pETM11.

Figure 3

Comparison of the protease efficiency of TEV and C3. A) The Trx-EYFP fusion protein recovered using the pETM20 and pETM22 vectors was digested in the presence of 1 μg (TEV) and 0.2 μg (C3) protease for 100 μg of fusion protein. B) The Trx-EYFP constructs resuspended in different buffers were digested at 30°C for 3 h in the presence of 1:100 (TEV) and 1:500 (3C) diluted proteases.