The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in Escherichia coli

Karimi, Zeinab; Nezafat, Navid; Negahdaripour, Manica; Berenjian, Aydin; Hemmati, Shiva; Ghasemi, Younes

doi:10.1016/j.pep.2015.05.017

Cited by 21 publications

(5 citation statements)

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“…Moreover, replacing rare codons with frequent ones does not necessarily lead to increased yield in protein synthesis. Codon choice might affect expression, solubility, and folding of a protein, − and rare codons can, paradoxically, enhance the translation of a gene via reducing the mRNA secondary structure. ,, Additionally, codon optimization by gene synthesis is not feasible when testing gene libraries. On the other hand, attempts to overexpress rare tRNA species by cloning extra copies of the corresponding tRNA encoding genes into a plasmid have their drawbacks as well.…”

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confidence: 99%

Enhancing the Translational Capacity of E. coli by Resolving the Codon Bias

et al. 2018

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Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.

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confidence: 99%

Enhancing the Translational Capacity of E. coli by Resolving the Codon Bias

et al. 2018

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“…Given the codon usage of E. coli , amino acids encoded by a combination of A and C, including an additional histidine adjacent directly to the 6His tag, were chosen. Codon frequencies were also considered to maximise protein production [ 58 ]. As a result, the site of the original pRSET plasmid (-cggggttct-) was replaced by the site (-accaccccacaacac-) that encodes Thr-Thr-Pro-Gln-His, which also led to replacement of the original 6His-tag to a 7His-tag.…”

Section: Resultsmentioning

confidence: 99%

Using ELP Repeats as a Scaffold for De Novo Construction of Gadolinium-Binding Domains within Multifunctional Recombinant Proteins for Targeted Delivery of Gadolinium to Tumour Cells

Позднякова¹,

Ryabaya²,

Semkina

et al. 2022

IJMS

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Three artificial proteins that bind the gadolinium ion (Gd3+) with tumour-specific ligands were de novo engineered and tested as candidate drugs for binary radiotherapy (BRT) and contrast agents for magnetic resonance imaging (MRI). Gd3+-binding modules were derived from calmodulin. They were joined with elastin-like polypeptide (ELP) repeats from human elastin to form the four-centre Gd3+-binding domain (4MBS-domain) that further was combined with F3 peptide (a ligand of nucleolin, a tumour marker) to form the F3-W4 block. The F3-W4 block was taken alone (E2-13W4 protein), as two repeats (E1-W8) and as three repeats (E1-W12). Each protein was supplemented with three copies of the RGD motif (a ligand of integrin αvβ3) and green fluorescent protein (GFP). In contrast to Magnevist (a Gd-containing contrast agent), the proteins exhibited three to four times higher accumulation in U87MG glioma and A375 melanoma cell lines than in normal fibroblasts. The proteins remained for >24 h in tumours induced by Ca755 adenocarcinoma in C57BL/6 mice. They exhibited stability towards blood proteases and only accumulated in the liver and kidney. The technological advantages of using the engineered proteins as a basis for developing efficient and non-toxic agents for early diagnosis of tumours by MRI as well as part of BRT were demonstrated.

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“…The start codon of TAT‐RsTAL in PET 28a was positioned on the loop, which means that the initiating codon will be more exposed. Therefore, higher levels of translation compared with a start codon positioned on the stem can be expected 36 …”

Section: Discussionmentioning

confidence: 99%

“…Therefore, higher levels of translation compared with a start codon positioned on the stem can be expected. 36 The flexibility of an enzyme active site allows efficient interaction between functional groups of the active site and the substrate. Furthermore, the rigidity and flexibility of ligand binding site residues are usually associated with specificity and tightness of ligand binding and the entrance of ligands into the binding site, respectively.…”

Section: Discussionmentioning

confidence: 99%

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia‐lyase and a TAT‐penetrating peptide

Behzadipour

Sadeghian

Bahraman

et al. 2020

Biotechnology Progress

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Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a trans-activator of transcription (TAT) cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 ± 0.04 and 0.34 ± 0.03 nmol•mg −1 •s −1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7 ± 6.2%, 28.2 ± 5.7%, and 33.9 ± 2.9% compared to the nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 μgml −1 even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.

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The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in Escherichia coli

Cited by 21 publications

References 60 publications

Enhancing the Translational Capacity of E. coli by Resolving the Codon Bias

Enhancing the Translational Capacity of E. coli by Resolving the Codon Bias

Using ELP Repeats as a Scaffold for De Novo Construction of Gadolinium-Binding Domains within Multifunctional Recombinant Proteins for Targeted Delivery of Gadolinium to Tumour Cells

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia‐lyase and a TAT‐penetrating peptide

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