A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production

Abstract

Background: In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol.

Results: The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA) and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease) of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level.

Conclusion: The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high-throughput screening.

Figure 1

Basic expression vector design. (A) The basic vector design for the pLIC vectors. (B) Shows the nucleotide sequence for the features common to all the vectors. The pLIC-His vector is identical in sequence except it does not contain the NcoI-SpeI restriction enzyme sites between the His tag and the TEV recognition sequence.

Figure 2

Ligation-Independent Cloning (LIC) Methodology. Shows the general procedure for LIC (1) The primer sequences required for amplification of the gene of interest. (2) Both the vector and insert are subject to treatment with T4 DNA polymerase, in the presence of either dTTP (vector) or dATP (insert). (3) The result after successful annealing. (4) After cleavage with TEV protease, the tags are removed and the protein retains 4 amino acids (GAAS).

Figure 3

Small scale expression, purification and cleavage of recombinant proteins. (A) SDS-PAGE analysis of the total cell lysate (T), insoluble (I), soluble (S), and the purified (P) fractions for each of the fusions: His-HHR23a, GST-UIM, MBP-α₁-AT and Nus-UBL (the protein is marked with an asterisk). (B) SDS-PAGE analysis of each fusion during TEV protease digestion, (1) intact fusion (2) TEV digestion (3) purified protein after digestion. The band around 30 kDa observed in lane 2 of each experiment corresponds to the TEV protein.

Figure 4

Parallel expression, purification and cleavage of HHR23A. HHR23A was cloned into all 4 vectors and the constructs were expressed and purified as described in the materials and methods. SDS-PAGE analysis of the total cell lysate (T), insoluble (I), soluble (S), and the purified (P) fractions for each of the fusions are shown.