Abstract
BackgroundHeterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form.ResultsWhen screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins.ConclusionsWe present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.
Highlights
Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host
Upon examination of signal sequences that mediate optimal targeting of recombinant proteins to the E. coli periplasm, we serendipitously found that the 39-amino acid long signal sequence of E. coli TMAO-reductase promoted high-level expression of heterologous proteins in inclusion body (IB), instead of facilitating translocation of these proteins across the cytoplasmic membrane
We aimed to identify the most effective strategy to mediate the periplasmic localization of the recombinant human epidermal growth factor
Summary
Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Overexpression of recombinant proteins in the cytosol and sometimes even in the periplasm leads to the formation of aggregates that consist almost exclusively of the recombinant protein [6] Using lightmicroscopy, these aggregates or inclusion bodies (IBs) can be observed as large refractive bodies that are predominantly located at one or both cell poles [7, 8]. Proteins in IBs are largely resistant against degradation by host cell proteases and less likely to exert toxic effects. Due to their high density, IBs are easy to isolate from cell lysates by differential centrifugation, providing fast, robust and cost-efficient [11] protocols to obtain large amounts of relatively pure protein [12,13,14]. Rather than being seen as unwanted byproducts of protein production, IBs are nowadays regarded as functional nanoparticles with potential applications in for example biocatalysis, diagnostics, tissue engineering and drug delivery [17]
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