An efficient expression tag library based on self-assembling amphipathic peptides.
Authors of this article are:
Zhao W, Liu S, Du G, Zhou J.
A summary of the article is shown below:
BACKGROUND: Self-assembling amphipathic peptides (SAPs) may improve protein production or induce the formation of inclusion bodies by fusing them to the N-terminus of proteins. However, they do not function uniformly well with all target enzymes and systematic research on how the composition of SAPs influence the production of fusion protein is still limited.RESULTS: To improve the efficiency of SAPs, we studied factors that might be involved in SAP-mediated protein production using S1 (AEAEAKAK)2 as the original SAP and green fluorescent protein (GFP) as the reporter. The results indicate that hydrophobicity and net charges of SAPs play a key role in protein expression. As hydrophobicity regulation tend to cause the formation of insoluble inclusion bodies of protein, an expression tag library composed of SAPs, which varied in net charge (from + 1 to + 20), was constructed based on the random amplification of S1nv1 (ANANARAR)10. The efficiency of the library was validated by polygalacturonate lyase (PGL), lipoxygenase (LOX), L-asparaginase (ASN) and transglutaminase (MTG). To accelerate preliminary screening, each enzyme was fused at the C-terminus with GFP. Among the four enzyme fusions, the SAPs with + 2 - + 6 net charges were optimal for protein expression. Finally, application of the library improved the expression of PGL, LOX, ASN, and MTG by 8.3, 3.5, 2.64, and 3.68-fold relative to that of the corresponding wild-type enzyme, respectively.CONCLUSIONS: This is the first report to study key factors of SAPs as an expression tag to enhance recombinant enzyme production. The SAP library could be used as a novel plug-and-play protein-engineering method to screen for enzymes or proteins with enhanced production.
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This article is a good source of information and a good way to become familiar with topics such as: Expression tags; High-throughput screening; Hydrophobicity; Positive charge; Self-assembling amphipathic peptides.
