Download File Saras_1.8.zip
CLICK HERE ->>->>->> https://urloso.com/2tkQgN
PermittedRead, print & downloadRedistribute or republish the final articleText & data mineTranslate the articleReuse portions or extracts from the article in other worksSell or re-use for commercial purposesElsevier's open access license policy
The following recombinant SARS-CoV-2 proteins [18] were procured from the MRC Protein Phosphorylation and Ubiquitination Unit Reagents and Services at the University of Dundee (Dundee, Scotland; -covid.bio/), and are listed in Additional file 1. Rabbit polyclonal antibodies (Kinexus Bioinformatics) directed against synthetic peptides based on SARS-CoV-2 proteins were used in dot blots, and are listed in Additional file 1.
For further work we chose E. coli clones that expressed intact fusion proteins of CBM9 and fragments of SARS-CoV-2 proteins as the dominant recombinant products. The expression of clones CBM9-(PT)4P, CBM9-ID-H1 and CBM9-N are shown in Fig. 1F and the purified products in Fig. 1G; for comparison, the carrier protein module CBM9 is also shown. By comparing the staining intensity of the protein band in the cell extracts to the band of purified CBM9-N, we estimated that the clones expressed recombinant product at levels of at least 100 mg/L upon IPTG-induction, and this is consistent with the 200 mg/L estimates of Kavoosi et al. [26]. We examined the purification yield of clone CBM9-ID-H1 (Table 2 and Additional file 1: Fig. S3). Because the CBM9-ID-H1 lacks any enzymatic activity that can be used to quantify its specific activity we used the inherent fluorescence of Coomassie blue stained proteins with 700 nm excitation to measure the relative amount of CBM9-ID-H1 in a protein mixture of known protein content. After 6 h of IPTG induction this clone yielded recombinant protein at about 27% of the total soluble protein, or 756 mg/L. The final recombinant protein yield of the specific protein band confirmed by mass spectroscopy to be the complete, intact CBM9-ID-H1 fusion protein was 16%, or 122 mg/L. A slightly smaller protein co-purified with CBM9-ID-H1, and presumably this is a proteolytic fragment of CBM9-ID-H1. This lesser band represents 33% of the total protein of the final purified protein preparation, and, thus, the CBM 9-ID-H1 is about 67% pure. That is, the final purified protein preparation yielded 122 mg/L of CBM9-ID-H1, and 60 mg/L of a fragment of CBM9-ID-H1. The contaminating band could be removed using a further purification step, but this may be unnecessary in an antibody detection assay as the contaminating protein is unlikely to interfere in the assay any more than the CBM9 portion of the CBM9-ID-H1 fusion protein. These experiments were performed using standard research growth flasks at an A600 of less than 10, and it is likely that the levels of recombinant protein produced could be significantly increased using an optimized fed-batch bioreactor protocol.
All of the data relevant to the conclusion are presented in the manuscript or in the material presented in the additional file. The accession numbers for the nucleotide sequences of the recombinant clones and the source for obtaining clones is described in Additional file 1. 59ce067264
https://www.dialogosemeducacaoespecial.com.br/forum/deficiencia-auditiva/ben-hur-season-1-episode-1