Design of a yeast SUMO tag to eliminate internal translation initiation.

Protein Sci

Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, USA.

Published: January 2025

After overexpression in a suitable host, recombinant protein purification often relies on affinity (e.g., poly-histidine) and solubility-enhancing (e.g., small ubiquitin-like-modifier [SUMO]) tags. Following purification, these tags are removed to avoid their interference with target protein structure and function. The wide use of N-terminal His-SUMO fusions is partly due to efficient cleavage of the SUMO tag's C-terminal Gly-Gly motif by the Ulp1 SUMO protease and generation of the native N-terminus of the target protein. While adopting this system to purify the Salmonella homodimeric FraB deglycase, we discovered that Shine-Dalgarno (SD) sequences in the eukaryotic SUMO tag resulted in truncated proteins. This finding has precedents for synthesis of partial proteins in Escherichia coli from cryptic ribosome-binding sites within eukaryotic coding sequences. The SUMO open reading frame has two "GGNGGN" motifs that resemble SD sequences, one of which encodes the Gly-Gly motif required for Ulp1 cleavage. By mutating these SD sequences, we generated SUMO (no internal translation), a variant that eliminated production of the truncated proteins without affecting the levels of full-length His-SUMO-FraB or Ulp1 cleavage. SUMO should be part of the toolkit for enhancing SUMO fusion protein yield, purity, and homogeneity (especially for homo-oligomers). Moreover, we showcase the value of native mass spectrometry in revealing the complications that arise from generation of truncated proteins, as well as oxidation events and protease inhibitor adducts, which are indiscernible by commonly employed lower resolution methods.

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.5256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11653089PMC

Publication Analysis

Top Keywords

truncated proteins
12
sumo
8
sumo tag
8
internal translation
8
target protein
8
cleavage sumo
8
gly-gly motif
8
ulp1 cleavage
8
design yeast
4
yeast sumo
4

Similar Publications

[Donor DNA Modification with Cas9 Targeting Sites Improves the Efficiency of MTC34 Knock-in into the CXCR4 Locus].

Mol Biol (Mosk)

December 2024

Center of Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334 Russia.

To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA.

View Article and Find Full Text PDF

Neuron navigators (NAVs) are cytoskeleton-associated proteins well known for their role in axonal guidance, neuronal migration, and neurite growth necessary for neurodevelopment. Neuron navigator 3 (NAV3) is one of the three NAV proteins highly expressed in the embryonic and adult brain. However, the role of the NAV3 gene in human disease is not well-studied.

View Article and Find Full Text PDF

Background: the protein phosphatase 3 catalytic subunit alpha (PPP3CA) gene encodes for the alpha isoform of the calcineurin catalytic subunit, which controls the phosphorylation status of many targets. Currently, 23 pathogenic variants of PPP3CA are known, with clinical manifestations varying by mutation type and domain.

Results: through whole exome sequencing, we found two de novo variants in PPP3CA: a frameshift variant predicted leading to a truncated protein in Pt.

View Article and Find Full Text PDF

Clinical and functional characterization of p.Lys322stop variant in the SERPINC1 gene causing severe thrombophilia.

Orphanet J Rare Dis

December 2024

Thrombosis Research Center, Beijing Jishuitan Hospital, Capital Medical University, Xicheng District, Beijing, 100035, China.

Background: Identification of mutations in the SERPINC1 has illuminated the intricate pathways underlying antithrombin (AT) deficiency. Our group identified a variation in the SERPINC1 gene (c.964 A > T, p.

View Article and Find Full Text PDF

Bacterial intragenic inversions: a new layer of diversity.

Trends Genet

December 2024

National Library of Medicine, National Institutes of Health, Bethesda, MD, USA. Electronic address:

DNA inversions in bacteria were known to create diversity through intergenic or partial intergenic changes. Now, Chanin, West, et al. reveal intragenic inversions, enabling single genes to encode multiple protein variants via sequence recoding or truncation - an unexpected mechanism for expanding protein diversity without increasing genome size.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!