Part:BBa_K5184022

G1M5-SUMO-tag

Abstract

Since both SVPs and MVPs are both cysteine-rich proteins that are easily folded incorrectly while using E. coli as chassis, we aim to find out a method that can solve this problem. We discovered that through utilizing the G1M5-SUMO tag, solubility of the proteins expressed can be increased and successful expression is enabled. This investigation hence has reference value in E. coli tag selection for expression of cysteine-rich peptides for the iGEM community.

Usage and Biology

G1M5 is the mutated, less hydrophobic version of the secretion signal peptide of the G1 cyclomaltodextrin glucanotransferase (CGtase) of Bacillus sp., which allows the extracellular secretion of the bacterial enzyme. Conduction of proteins attached by G1M5 out of the cytosol is achieved by the Sec pathway, a very common secretion system seen in all three major domains of life: arachaea, prokaryote, and eukaryotes. Once the signal peptide, in this case G1M5 is synthesized, the protein chaperon SecB binds to the preprotein (that is attached to G1M5), and transfers the preprotein to the protein translocase SecA, of which binds to the membrane bound protein conducting channel SecYEG. Once bound to the membrane, SecA binds to a molecule of ATP, of which is hydrolyzed to conduct the protein through heterotrimer complex of SecYEG. A membrane bound SPaseI then, once enough of the preprotein had been conducted through the channel, will remove the SP and allow the preprotein to fold properly into the correct protein.[1]
The in vivo SUMO tags, or small ubiquitin-like modifiers, are a diverse class of ubiquitin-related proteins that allow a versatile range of operations to be carried on a tagged protein. Amongst many of these is the selective proteolysis that removes the SUMO tag from the attached protein. Its ability to be detected and removed with high precision leads to the creation of the recombinant SUMO tag: a SUMO tag that, while different enough to the wildtype SUMO tag to not be degraded within eukaryotic hosts, can be detected and have the SUMO removed by special recombinant SUMO proteases. The recombinant SUMO therefore presents themselves as precise markers for points for proteolytic digestion that can remove attached (to SUMO tag) protein domains. SUMO tags also share high level of homology with the ubiquitin, and therefore, once synthesized, folds rapidly to hide its hydrophobic cores. It is proposed that this initial folding assists in the correct folding of the attached fusion protein by supplying a great initial "kinetic push". While this can explain SUMO tag's ability to facilitate protein folding and enhance soluble expression, there are also theories proposing the SUMO tag to work as a chaperone to assists in the attached protein's folding.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The G1M5 secretion system was incorporated along with the SUMO tag in an attempt to achieve both extracellular expression and correct folding of cysteine-rich venom peptides. This combination is used for both the expression of MVPs and SVPs. Induced expression and SDS-PAGE analysis were carried out respectively after the construction of vectors, including pET-GNA-His, pET-His-GNA ,pET-PelB, pET-MalE, pET-PelB-SUMO, pET-MalE-SUMO, and pET-G1M5-SUMO. In which, only pET-G1M5-SUMO exhibit correct folding of venom peptides in the supernatant portion. Others are observed in the precipitate (P) after lysis, indicating inclusion bodies. Thus, the results suggest that only expressions of proteins including the usage of G1M5-SUMO tag are capable of demonstrating correct expressions.[figure 1]

Figure 1: SDS-PAGE of S: supernatant and P: precipitate (A) SP-rCtx4-GNA-His and SP-SUMO-rCtx4-GNA-His, (B) rCtx4-GNA-His, (C) G1M5-SUMO-rCtx4-GNA-His (D) His-rCtx4-GNA; all with complete proteins of BL21(DE3) as control

Reference

[1]Liu, Changjun, et al. ‘A Secretory System for Extracellular Production of Spider Neurotoxin Huwentoxin-I in Escherichia Coli’. Preparative Biochemistry & Biotechnology, vol. 53, no. 8, Sept. 2023, pp. 914–22. DOI.org (Crossref), https://doi.org/10.1080/10826068.2022.2158473

[2]Jürgen Dohmen, R. ‘SUMO Protein Modification’. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, vol. 1695, no. 1–3, Nov. 2004, pp. 113–31. DOI.org (Crossref), https://doi.org/10.1016/j.bbamcr.2004.09.021