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Streptomyces Host-Vector Systems
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Puromycin is an aminoacyl-adenosine antibiotic that is produced by S. alboniger. The antibiotic, a functional analogue of aminoacyl tRNA, inhibits protein synthesis by substituting for the incoming coded aminoacyl tRNA, and serving as acceptor for the nascent peptide chain of ribosome-bound peptidyl tRNA [48]. An enzyme O-demethylpuromycin-O-methyltransferase is present, which converts O-demethylpuromycin to puromycin in extracts of S. alboniger. We and other groups found that the producer organism possesses a puromycin-inactivating enzyme (puromycin 2'-N-acetyltransferase) that may have a role in self-resistance [49-51]. The genes coding for the enzyme and O-demethylpuromycin-O-methyltransferase have been cloned and are present on the same 2.4-kb DNA fragment [52].
Advanced Topics in Molecular Biology
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
A general flowchart for the siRNA experiment is shown in Figure 4.9. B16 cells were seeded onto 10 cm dishes the day before infection at a density that permitted efficient lentiviral infection without cell overgrowth. Four dishes were used per experiment: three were treated with virus cocktail (containing a random mix of genes), and one dish was mock-infected (medium only) as control. Puromycin selection was carried out for 5 days to allow the genes time to express; cells were passaged into 15 cm dishes and treated with Au–Dox at the EC50 for 24 h. The surviving cells were left growing on the same plate under puromycin selection for a week to form colonies. The cells from each colony were then collected separately, and the DNA was extracted using a blood and tissue DNA extraction kit.
Next-Generation Immunoassays
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Valerie Fitzgerald, Paul Leonard
In the case of mRNA display, covalent mRNA–protein complexes are created by ligation of a DNA linker with a small adaptor molecule, puromycin, to create the in vitro–transcribed mRNA, which lacks a stop codon. The mRNA is translated in vitro, and the ribosome stalls at the RNA-DNA junction. Puromycin then binds the ribosomal A-site and the nascent polypeptide is thereby transferred to puromycin, as if it were an aminoacyl-tRNA. The resulting covalently linked mRNA-protein complex is isolated, reverse-transcribed and used in selection procedures. The DNA strand is recovered from target-bound complexes by hydrolysing the complementary mRNA at high pH and then amplified by PCR [24].
Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells
Published in Preparative Biochemistry and Biotechnology, 2019
Omid Mohammadian, Masoumeh Rajabibazl, Es’hagh Pourmaleki, Hadi Bayat, Roshanak Ahani, Azam Rahimpour
CHO-K1 cells were transduced using lentiviral particles. Several studies have suggested application of serially diluted lentiviral particles after concentration using ultracentrifugation. However, our initial analysis with concentrated lentiviral caused massive cell death even when it was highly diluted (1:10,000), therefore we proceeded with un-concentrated supernatant. After virus titration, the maximum MOI of 1 was applied to CHO-K1 cells to avoid cytotoxicity. For development of the stable cell pools, cells were further treated with puromycin-containing medium 72 h post-transduction. PCR analysis was performed on genomic DNA of transduced cell pools and parental CHO cells. The appearance of bands with the expected size of 730 bp for LC (Figure 2(A)) and 1450 bp for HC (Figure 2(B)) confirmed the presence of these genes in the genome of cell pools.
Site-specific integration of light chain and heavy chain genes of antibody into CHO-K1 stable hot spot and detection of antibody and fusion protein expression level
Published in Preparative Biochemistry and Biotechnology, 2019
Songtao Zhou, Yun Chen, Xiaohai Gong, Jian Jin, Huazhong Li
Here, the genes of recombinant antibody (bevacizumab) were to be knocked into the targeting site mentioned above via HDR mediated by CRISPR/Cas9. The knock-in system includes three key elements: Cas9 plasmid, sgRNA plasmid and the donor plasmid. The donor plasmid (Fig. 1) was designed to harbor 600 bp homology arms, which were exactly next to the 23 bp Cas9 cleavable sgRNA sequence. A puromycin-resistant gene cassette together with light chain and heavy chain genes of antibody cassette were placed within homology arms. A cop-GFP gene cassette was placed outside the homology arm to detect any random integration event. Once antibody gene in donor plasmid was precisely integrated into the hot spot locus, cells would lose green fluorescence expression and express light chain and heavy chain genes of antibody together with puromycin resistance gene (Fig. 1). Thus, only cells with no green fluorescence were single cell sorted by FACS and were seeded in the 96-well plates.