Generation of a vector suite for protein solubility screening

Abstract

Recombinant protein expression has become an invaluable tool for academic and biotechnological projects. With the use of high-throughput screening technologies for soluble protein production, uncountable target proteins have been produced in a soluble and homogeneous state enabling the realization of further studies. Evaluation of hundreds conditions requires the use of high-throughput cloning and screening methods. Here we describe a new versatile vector suite dedicated to the expression improvement of recombinant proteins (RP) with solubility problems. This vector suite allows the parallel cloning of the same PCR product into the 12 different expression vectors evaluating protein expression under different promoter strength, different fusion tags as well as different solubility enhancer proteins. Additionally, we propose the use of a new fusion protein which appears to be a useful solubility enhancer. Above all we propose in this work an economic and useful vector suite to fast track the solubility of different RP. We also propose a new solubility enhancer protein that can be included in the evaluation of the expression of RP that are insoluble in classical expression conditions.

Cold shock induction of recombinant Arctic environmental genes

“…Only three primers per candidate are required to clone each candidate gene into the vector suite: one forward primer to generate the His, His-TRX, His-MBP and His-TF fusion constructs, a second forward primer for the SUMO fusion constructs required to engineer the immediate transition from SUMO-tag to candidate protein, and a reverse primer which was used for all five (Additional file 1 : Table S4). Complementing a recent report [ 57 ], we demonstrated the application of RF-cloning in building a tailored vector suite and enabling parallel cloning of candidate sequences. All vectors were tested in the E. coli Rosetta2(DE3)pLysS strain for soluble expression of fusion partners without candidate genes, and as expected, the majority of recombinant proteins were soluble (data not shown).…”

High-level expression of Aerococcus viridans pyruvate oxidase in Escherichia coli by optimization of vectors and induction conditions

“…However, the pyruvate oxidase activity of E. coli pET32a‐ pod was lower than that of E. coli pET28a ‐pod because of the fused tag was not universal for heterologous gene expression (Correa et al . ).…”

“…Correa et al . () constructed 12 vectors with different elements for high solubility heterologous protein screening because there was not a conclusion for a vector suitable to a heterologous protein without experiments. The same optimized temperature was used for the three recombinant E. coli strains because high temperature triggered extremely low soluble pyruvate oxidase.…”

“…), it is still difficult to define a ‘universal fusion protein’ (Correa et al . ). In this study, the combined effects of vectors and induction conditions were investigated to obtain high levels of recombinant pyruvate oxidase using recombinant E. coli harbouring different pET vectors.…”

Recombinant protein expression in microbial systems

“…Costa and coauthors give a thorough description of different fusion tags that can be appended to the target protein in order to increase its solubility and/or ease its purification from the cellular milieu (Costa et al, 2014). Along this line, Correa and coauthors present a very promising approach to straightforwardly assess the solubility of a recombinant protein by cloning the corresponding gene in 12 different expression vectors in parallel (Correa et al, 2014). Even though IB formation is mainly regarded as a nuance in the production of recombinant proteins, Ramon and coworkers make the case that this is not always true and focus on the positive side of IBs, highlighting the advantages of producing recombinant proteins as IBs for basic and applied research (Ramon et al, 2014).…”