Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli

Neves, Fernanda Oliveira; Ho, Paulo Lee; Raw, Isaı́as; Pereira, Carlos Augusto; Moreira, Cristiani; Nascimento, Ana L. T. O.

doi:10.1016/j.pep.2004.02.005

Cited by 37 publications

(38 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Most publications on biological activity of rhIFNα2-b determination were used viral cytophatic inhibition as the observed parameter. (Srivasta et al, 2005;Valente et al, 2005;Neves et al, 2004;Essafi et al, 2007). Antiproliferative activity of our rhIFNα-2b was also studied by using calcein AMEthidium bromide III double staining method.…”

Section: Cdlpqthslgsrrtlmllaqmrkislfsclkdrhdfgfpqeefgnqfqkaetipvl Hemmentioning

confidence: 99%

Antiproliferative Activity of Recombinant Human Interferon Alpha2b on Estrogen Positive Human Breast Cancer McF-7 Cell Line

Ningrum

Wisnuwardhani

Santoso

et al. 2015

Indonesian J. Pharm.

View full text Add to dashboard Cite

Indonesia is the top three countries with hepatitis and moreover has 40.000 cancer mortalities per year. Interferon alpha 2b (IFNα-2b) is a therapeutic standard for cancer and hepatitis B/C treatments. We developed recombinant human interferon alpha 2b (rhIFNα-2b) in methilotropic yeast Pichia pastoris X-33. The protein was produced as extracellular protein with 24 kDa in size. This research was aimed to characterize the protein based on its amino acid sequence and to study its antiproliferative activity on MCF-7 cell line. Amino acid sequencing by using MALDI TOF TOF mass spectrometry with trypsin as proteolytic enzyme identified protein as hIFNα-2b with 33% of amino acid coverage. The antiproliferative activity was determined by 3-[4.5-dimethyltiazol-2il]-2.5-diphenylltetrazolium bromide (MTT) assay. Based on several studies about the synergistic activity of rhIFNα-2b with other anticancer drugs, we combined our rhIFNα-2b with tamoxifen (tmx). The growth percentage of the cells after being treated with 1µM of tmx at various concentrations of rhIFNα-2b was compared with that of untreated cell. This study showed that the antiproliferative activity was dose-dependently. Cell viability assay with calcein and ethidium bromide-based staining by fluorescence microscope confirmed that the rhIFNα-2b had ability to inhibit proliferation of human breast cancer cell line MCF-7.

show abstract

Section: Cdlpqthslgsrrtlmllaqmrkislfsclkdrhdfgfpqeefgnqfqkaetipvl Hemmentioning

confidence: 99%

Antiproliferative Activity of Recombinant Human Interferon Alpha2b on Estrogen Positive Human Breast Cancer McF-7 Cell Line

Ningrum

Wisnuwardhani

Santoso

et al. 2015

Indonesian J. Pharm.

View full text Add to dashboard Cite

show abstract

“…Typically, synthetic genes encode the same product as the gene of interest; extensively recoding affects expression and/or base composition. This technique has been used with great success (Han and Boeke 2004;Neves et al 2004;Tian et al 2004;Patterson et al 2005), often producing codon-optimized (CO) genes capable of elevated expression or expression in nonhost organisms. Furthermore, this strategy has also been utilized on other retroelements, such as HIV-1, to optimize expression and to remove negative cis-acting sequence elements such as the Rev response element (Kotsopoulou et al 2000;Nguyen et al 2004).…”

mentioning

confidence: 99%

Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

et al. 2012

View full text Add to dashboard Cite

Extensive mutagenesis via massive recoding of retrotransposon Ty1 produced a synthetic codon-optimized retrotransposon (CO-Ty1). CO-Ty1 is defective for retrotransposition, suggesting a sequence capable of down-regulating retrotransposition. We mapped this sequence to a critical 20-bp region within CO-Ty1 reverse transcriptase (RT) and confirmed that it reduced Ty1 transposition, protein, and RNA levels. Repression was not Ty1 specific; when introduced immediately downstream of the green fluorescent protein (GFP) stop codon, GFP expression was similarly reduced. Rap1p mediated this down-regulation, as shown by mutagenesis and chromatin immunoprecipitation. A regular threefold drop is observed in different contexts, suggesting utility for synthetic circuits. A large reduction of RNAP II occupancy on the CO-Ty1 construct was observed 39 to the identified Rap1p site and a novel 39 truncated RNA species was observed. We propose a novel mechanism of transcriptional regulation by Rap1p whereby it serves as a transcriptional roadblock when bound to transcription unit sequences. SACCHAROMYCES cerevisiae Ty1 is the best-characterized and most abundant of five retrotransposon families present in the genome. Ty1 shares many similarities with retroviruses, such as HIV-1 and makes similar use of an RNA intermediate in its propagation. Unsurprisingly, this Ty1 RNA intermediate is essential for Ty1 retrotransposition. Not only does this "mRNA" code for the GAG and POL proteins required for retrotransposition, but it also serves a critical function as the genetic material for replication. Both Ty1 mRNA and its cDNA product contain cis-acting nucleotide determinants required for retrotransposition.Mutagenesis of Ty1 and screening for cis-acting nucleotide determinants has been previously performed with various strategies, including in-frame linker insertion mutagenesis Devine and Boeke 1994;Monokian et al. 1994), analysis of synthetic Ty1 DNA fragments to determine minimal requirements for the integration reaction (Eichinger and Boeke 1990;Braiterman et al. 1994;Devine and Boeke 1994;Sharon et al. 1994), deletion analysis and PCR-mediated mutagenesis of mini-Ty1 elements (Xu and Boeke 1990;Bolton et al. 2005), and comparative sequence analysis followed by targeted mutagenesis and/or generation of complementary mutations in RNA stem regions (Chapman et al. 1992;Heyman et al. 1995;Lauermann et al. 1995;Friant et al. 1998;Cristofari et al. 2002;Bolton et al. 2005). These studies provided insights into mechanisms underlying Ty1 retrotransposition and revealed several cis-acting sequences, including the Met i -tRNA:primer binding site (PBS) interaction (Chapman et al. 1992; Friant et al. 1998), LTRs (Eichinger andBraiterman et al. 1994;Devine and Boeke 1994;Sharon et al. 1994), polypurine tracts (PPTs) (Heyman et al. 1995;Lauermann et al. 1995), the GAG-POL frameshift region (Belcourt and Farabaugh 1990; Lawler et al. 2001), CYC5 and CYC3 (Cristofari et al. 2002), and an extensive 59 RNA structure required for efficient ini...

show abstract

“…Interferon alpha 2b (IFN-␣2b) belongs to the IFN family of cytokines, which can induce antiproliferative, immunomodulatory, and potent antiviral activities against a wide range of mammalian viruses (7). IFN-␣2b is used to treat several diseases, including some types of cancer and hepatitis, in particular hepatitis C. Since it can increase the intensity of antigen expression on certain tumors, IFN-␣2b has a potential for use in diagnostics and therapeutics (20). As for GM-CSF, IFN-␣2b has also been expressed recombinantly into active form in E. coli (28).…”

mentioning

confidence: 99%

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

Sletta

Tøndervik

Hakvåg

et al. 2007

Appl Environ Microbiol

View full text Add to dashboard Cite

Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-␣2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-␣2b expression remained poor even when fused to a signal sequence, and an alternative IFN-␣2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

show abstract

Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli

Cited by 37 publications

References 20 publications

Antiproliferative Activity of Recombinant Human Interferon Alpha2b on Estrogen Positive Human Breast Cancer McF-7 Cell Line

Antiproliferative Activity of Recombinant Human Interferon Alpha2b on Estrogen Positive Human Breast Cancer McF-7 Cell Line

Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

Contact Info

Product

Resources

About