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An efficient expression tag library based on self-assembling amphipathic peptides.

Weixin Zhao1,2, Song Liu3,4, Guocheng Du2,5

  • 1National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China.

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|May 29, 2019
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Summary
This summary is machine-generated.

Self-assembling amphipathic peptides (SAPs) enhance recombinant protein production by optimizing net charge. A novel SAP library improves expression of enzymes like PGL, LOX, ASN, and MTG.

Keywords:
Expression tagsHigh-throughput screeningHydrophobicityPositive chargeSelf-assembling amphipathic peptides

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Area of Science:

  • Biotechnology
  • Protein Engineering
  • Molecular Biology

Background:

  • Self-assembling amphipathic peptides (SAPs) can enhance protein production or induce inclusion body formation when fused to proteins.
  • Current understanding of how SAP composition influences fusion protein production is limited, with variable efficacy across different enzymes.

Purpose of the Study:

  • To investigate the key factors of SAPs, specifically net charge and hydrophobicity, that influence recombinant protein production.
  • To develop and validate a novel expression tag library of SAPs to enhance the production of target enzymes.

Main Methods:

  • Utilized green fluorescent protein (GFP) as a reporter to study SAP-mediated protein production, starting with S1 (AEAEAKAK)2.
  • Constructed an expression tag library of SAPs with varying net charges ( +1 to +20) based on S1nv1 (ANANARAR)10.
  • Validated the library's efficiency by fusing it to the C-terminus of reporter GFP and four enzymes: polygalacturonate lyase (PGL), lipoxygenase (LOX), L-asparaginase (ASN), and transglutaminase (MTG).

Main Results:

  • Hydrophobicity and net charge of SAPs are critical determinants of protein expression efficiency.
  • SAPs with net charges ranging from +2 to +6 demonstrated optimal protein expression levels.
  • The developed SAP library significantly improved the expression of PGL, LOX, ASN, and MTG by 8.3, 3.5, 2.64, and 3.68-fold, respectively.

Conclusions:

  • This study presents the first systematic investigation into the key factors of SAPs as expression tags for enhancing recombinant enzyme production.
  • The developed SAP library offers a versatile, plug-and-play approach for protein engineering to screen for enzymes with improved production yields.