Multi-Compartment and Multi-Host Vector Suite for Recombinant Protein Expression and Purification

Abstract

Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields.

New tools for recombinant protein production in Escherichia coli: A 5‐year update

“…Choosing one of the many options is not trivial, as fusion tags may work in one case but not in another. To easily screen among different fusion partners, Correa et al designed a T7 vector suite (based on the pET32 plasmid) that includes six histidine‐tagged proteins with solubility‐enhancing properties . Cloning of the coding sequence of interest is achieved by restriction‐free cloning using only a pair of primers for all vectors.…”

“…We strongly recommend the T7 vector suite from Correa et al Cloning into these six different plasmids is quite easy and requires one pair of primers for PCR as employed in restriction‐free cloning. These plasmids will also allow for surveying the effect of five (or six in the latest update) fusion tags on solubility. Other reagents needed in this kit include nickel‐NTA‐agarose beads for purification and the Tobacco Etch Virus (TEV) protease (which can be prepared in house ) for tag removal.…”

Denaturing gradient gel electrophoresis and multi-SIR profiles of soil microbial communities from a karst doline at Aggtelek National Park, Hungary

“…Although the method used in the present study is not suitable for detailed and quantitative analysis of community structure, and hence a direct comparison with the results of Yun et al ( 2016 ) is not possible, DGGE is an excellent tool for comparative analysis of DGGE profiles (Nielsen et al 2013 ), and is often used to indicate changes in the bacterial community structure after soil treatment or different land uses (e.g. Stagniari et al 2014 ; Orlewska et al 2018a , b ). In our study, pH was not different significantly in different parts of doline—slightly acidic through the whole transect—still, there were significant differences in the genetic fingerprints of bacterial communities.…”

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

“…Downstream processes may include the separation of soluble and insoluble cell debris and media components, protein purification, protein formulation (including concentration), bioconjugation, protein-bioconjugate purification, and protein refolding. Only one downstream process is required depending on the protein and its specific requirements [ 8 ]. Proteins are susceptible to denaturation by several factors, such as pH, salt concentration, organic solvents, shearing, surface and interface interaction (including the formation of protein aggregates), lyophilization, moisture levels, protein concentration, and temperature changes.…”

“…The effectiveness of expressing a recombinant protein in E. coli primarily relies on the ability to prevent unfavorable interactions between newly expressed polypeptides. These interactions lead to the aggregation of intermediate folding substances instead of native protein production [ 6 , 7 , 8 , 11 ]. System efficiency can be improved by maintaining conditions that stabilize intermediate folding and promote mature structure formation.…”