Not Limited to E. coli: Versatile Expression Vectors for Mammalian Protein Expression

Abstract

Recombinant protein expression is not limited to E. coli or other prokaryotic systems. It is inevitable to use eukaryotic systems in order to express challenging mammalian proteins. Eukaryotic systems are able to perform complex posttranslational modifications like protein processing, phosphorylation, glycosylation, which are essential for stability and functionality of many proteins. Different eukaryotic protein expression systems employing yeast, insect, or mammalian cell lines are established with each having its own advantages and disadvantages. Often it is quite difficult to decide which will be the most optimal expression system as this depends highly on the protein itself. Expression in stable cell lines requires substantial screening of expressible constructs prior to developing a stable expression cell line. To achieve fast screening by transient expression in multiple hosts, versatile vectors can be applied. In this chapter, we present an overview of the most common multi-host vectors, which allow for fast expression analysis without tedious (re)cloning of the gene of interest in several different protein production systems. The protocols in this chapter describe the latest methods for fast transient expression in insect and mammalian cell lines.

Identifying parameters to improve the reproducibility of transient gene expression in High Five cells

“…To improve the reliability of the transient gene expression in High Five cells using 40 kDa PEI, we identified in a first round of optimization the best DNA:PEI ratio and the optimal DNA amounts for linear 40 kDa PEI Lot#1 (Fig 3), starting from the published protocol for linear 25 kDa PEI [34]. We initially varied the total DNA amount while keeping the DNA:PEI ratio constant at 1:4 and subsequently varied the DNA:PEI ratio while keeping the amount of DNA at 2 μg.…”

“…Instead of 2 μg DNA as used in Karste et al . [34], the highest GFP mean value and transfection efficacy were reached at 1 μg DNA for the constant DNA:PEI ratio of 1:4. However, at a at a constant DNA amount of 2 μg the highest measured values were observed at a DNA:PEI ratio of 1:3.…”

“…Here, at a transfection rate of ~80% the GFP mean value of 40.000 rfu is close to the GFP mean value reached for the best conditions of the 25 kDa PEI, Lot#1 as described in Karste et al . [34] (see Fig 1).…”

“…In this study we tested our plasmid-based method published before [34] in regard of reproducibility and feasible implementation in different laboratories. The replacement of the linear 25 kDa PEI by linear 40 kDa PEI was the most important factor to circumvent the observed batch-to-batch variability in performance of different 25 kDa PEI Lots.…”

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

“…Recombinant protein expression is not limited to E. coli , making the use of eukaryotic hosts inevitable for the expression of many challenging proteins. In addition, depending on the required quantities and quality of the target protein, a single host cell line may be the optimal expression system while others will fail in the expression of the same target ( Karste et al, 2017 ). To tackle this scenario, we extended our vector suite for expression of the target gene in different hosts including D. melanogaster and mammalian cell lines.…”