Influences on gene expression <i>in vivo</i> by a Shine–Dalgarno sequence

Jin, Haining; Zhao, Qiuyan; Valdivia, Ernesto González de; Ardell, David H.; Stenström, Magnus; Isaksson, Leif A.

doi:10.1111/j.1365-2958.2006.05110.x

Cited by 32 publications

(29 citation statements)

References 76 publications

(108 reference statements)

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“…Low protein expression using pBAD24 vector has been reported (Golich et al 2006;Oku et al 2004;Nelson et al 2006;Marolda et al 2004;Jin et al 2006) but no reason for this has been given. This prompted us to look into the reasons for such an experimental finding.…”

Section: Discussionmentioning

confidence: 91%

“…Jacob et al (1987) and Jin et al (2006) report that a single base change in the SD sequence causes a dramatic change in the synthesis of many proteins. We, therefore, modified the existing SD sequence of the pBAD24T7g10 vector to create pBAD24M where the optimized SD sequence from the T7 system was incorporated.…”

Section: Introductionmentioning

confidence: 98%

“…However, low yields of proteins like E. coli aminopeptidase N (pepN), Staphylococcus aureus lysylphosphatidylglycerol synthetase, glutamate transport protein (GltT) and putative O-antigen flippase (Wzx) (Golich et al 2006;Oku et al 2004;Jin et al 2006) have arisen using the pBAD24 vector.…”

Section: Introductionmentioning

confidence: 99%

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Over-expression of proteins using a modified pBAD24 vector in E. coli expression system

et al. 2009

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A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine-Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.

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Section: Discussionmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 98%

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Over-expression of proteins using a modified pBAD24 vector in E. coli expression system

et al. 2009

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confidence: 99%

“…Jin et al (16) reported that when an SD motif is located between two potential start codons, initiation occurs predominantly at the second start site; the first start site is utilized when the distance between this site and the downstream SD motif is increased and/or when the second start site is weakened. In that study, the authors found that the SD motif is systematically avoided in the early coding region of natural genes in Escherichia coli, possibly reflecting a strong evolutionary constraint for codon usage (16). In rare cases, bacterial gene transcripts contain an internal start codon that is preceded by a strong SD motif (e.g., GGAGG); thus, alternative translation initiation leads to the generation of two isoforms of the protein, as evidenced by the heat shock proteins ClpB (17) and ClpA (18), initiation factor 2 (IF2) (19), hemolysin (20), and lycopene cyclase (21).…”

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confidence: 99%

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons

Tang

et al. 2016

J Bacteriol

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Recent studies have shown that haloarchaea employ leaderless and Shine-Dalgarno (SD)-less mechanisms for translation initiation of leaderless transcripts with a 5= untranslated region (5= UTR) of <10 nucleotides (nt) and leadered transcripts with a 5= UTR of >10 nt, respectively. However, whether the two mechanisms can operate on the same naturally occurring haloarchaeal transcript carrying multiple potential start codons is unknown. In this study, the transcript of the sptA gene (encoding an extracellular serine protease of Natrinema sp. strain J7-2) was experimentally determined and found to contain two potential in-frame AUG codons (AUG 1 and AUG 2 ) located 5 and 29 nt, respectively, downstream of the transcription start site. Mutational analysis revealed that both AUGs can function as the translation start codon for production of active SptA, although AUG 1 is more efficient than AUG 2 for translation initiation. Insertion of a stable stem-loop structure between the two AUGs completely abolished initiation at AUG 1 but did not affect initiation at AUG 2 , indicating that AUG 2 -initiated translation does not involve ribosome scanning from the 5= end of the transcript. Furthermore, the efficiency of AUG 2 -initiated translation was not influenced by an upstream SD-like sequence. In addition, both AUG 1 and AUG 2 contribute to transcript stability, probably by recruiting ribosomes to protect the transcript against degradation. These data suggest that depending on which of two in-frame start codons is used, the sptA transcript can act as either a leaderless or a leadered transcript for SptA production in haloarchaea. IMPORTANCEIn eukaryotes and bacteria, alternative translation start sites contribute to proteome complexity and can be used as a functional mechanism to increase translation efficiency. However, little is known about alternative translation initiation in archaea. Our results demonstrate that leaderless and SD-less mechanisms can be used for translation initiation of the sptA transcript from two in-frame start codons, raising the possibility that in haloarchaea, alternative translation initiation on one transcript functions to increase translation efficiency and/or contribute to proteome complexity.

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Design of a yeast SUMO tag to eliminate internal translation initiation

Law,

Gao,

Wysocki

et al. 2024

Protein Science

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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 His6‐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 SUMONIT (no internal translation), a variant that eliminated production of the truncated proteins without affecting the levels of full‐length His6‐SUMO‐FraB or Ulp1 cleavage. SUMONIT 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.

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Influences on gene expression in vivo by a Shine–Dalgarno sequence

Cited by 32 publications

References 76 publications

Over-expression of proteins using a modified pBAD24 vector in E. coli expression system

Over-expression of proteins using a modified pBAD24 vector in E. coli expression system

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons

Design of a yeast SUMO tag to eliminate internal translation initiation

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