Dataset Information

Control of total GFP expression by alterations to the 3' region nucleotide sequence.

ABSTRACT:

Background

Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression.

Results

The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3' region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5' poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5' poly(A) coding sequence interacted with the reconstructed 3' region nucleotide sequence to create a new mRNA tertiary structure between the 5' and 3' regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3' nucleotide sequence, after replacing selected C-terminal 5' codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3'-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3' region nucleotides.

Conclusions

We found that the hydrophilic C-terminal end of GFP increased Tat pathway specificity and that the 3' nucleotide sequence played an important role in total protein expression through translational and transcriptional regulation. These findings may be useful for efficiently producing recombinant proteins as well as for potentially controlling the expression level of specific genes in the body for therapeutic purposes.

SUBMITTER: Lee SJ

PROVIDER: S-EPMC3726318 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Publications

Control of total GFP expression by alterations to the 3' region nucleotide sequence.

Lee Sang Jun SJ Park Eun Hee EH Kim Young Ok YO Nam Bo Hye BH Kim Dong Gyun DG

Microbial cell factories 20130708

<h4>Background</h4>Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression.<h4>Resu ...[more]

PMID: 23834827

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Project description:In the last three decades, researchers have utilized genome engineering to alter the DNA sequence in the living cells of a plethora of organisms, ranging from plants, fishes, mice, to even humans. This has been conventionally achieved by using methodologies such as single nucleotide insertion/deletion in coding sequences, exon(s) deletion, mutations in the promoter region, introducing stop codon for protein truncation, and addition of foreign DNA for functional elucidation of genes. However, recent years have witnessed the advent of novel techniques that use programmable site-specific nucleases like CRISPR/Cas9, TALENs, ZFNs, Cre/loxP system, and gene trapping. These have revolutionized the field of experimental transgenesis as well as contributed to the existing knowledge base of classical genetics and gene mapping. Yet there are certain experimental/technological barriers that we have been unable to cross while creating genetically modified organisms. Firstly, while interfering with coding strands, we inadvertently change introns, antisense strands, and other non-coding elements of the gene and genome that play integral roles in the determination of cellular phenotype. These unintended modifications become critical because introns and other non-coding elements, although traditionally regarded as "junk DNA," have been found to play a major regulatory role in genetic pathways of several crucial cellular processes, development, homeostasis, and diseases. Secondly, post-insertion of transgene, non-coding RNAs are generated by host organism against the inserted foreign DNA or from the inserted transgene/construct against the host genes. The potential contribution of these non-coding RNAs to the resulting phenotype has not been considered. We aim to draw attention to these inherent flaws in the transgenic technology being employed to generate mutant mice and other model organisms. By overlooking these aspects of the whole gene and genetic makeup, perhaps our current understanding of gene function remains incomplete. Thus, it becomes important that, while using genetic engineering techniques to generate a mutant organism for a particular gene, we should carefully consider all the possible elements that may play a potential role in the resulting phenotype. This perspective highlights the commonly used mouse strains and the most probable associated complexities that have not been considered previously, resulting in possible limitations in the currently utilized transgenic technology. This work also warrants the use of already established mouse lines in further research.

Dataset Information

Control of total GFP expression by alterations to the 3' region nucleotide sequence.

Background

Results

Conclusions

Publications

Control of total GFP expression by alterations to the 3' region nucleotide sequence.

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