Biochemical Characterization of SARS-CoV-2 N Protein
Please write complete six pages.
In the topic i mentioned the article i choose to write to write my term paper about.
A written term paper (take-home final exam) based on a detailed publication in the recent literature. The publication will be chosen by the student in consultation with the course instructor to contain significant methodology & discussion on protein expression/purification - looking for applying rDNA expression & chromatographic techniques to achieve a high purity (e.g., visualized by Coomassie staining, not by Western blotting) in a large-scale production (> mg-quantity). Suitable papers would utilize both rDNA expression system/vector (like those discussed in modules 2 & 3) and apply chromatographic methods for protein purification (like those discussed throughout the semester), in order to study any scientific topics of students’ interests. Students need to carefully choose their papers by mid-semester, and are advised to seek the input of the instructor as to the appropriateness of a chosen paper. It must be a primary research article or letter from a peer-reviewed scientific journal, such as Nature Methods, J. Biol. Chem., Protein Expression and Purification, or J. Chromatog. etc., and is published within the past 18 months, or research that the students have performed or are currently performing at UML (or elsewhere). FYI, PubMed (https://www(dot)ncbi(dot)nlm(dot)nih(dot)gov/pubmed/advanced) allow ones to set up filter by publication date and keywords etc. example papers chosen by students of previous years are listed in an example folder in the Blackboard. In the written term paper the student will be expected to include a brief review on that topic, analyze, present the required theoretical and experimental approach, and criticize the results obtained within the context of limitations in the methods, as well as discuss the major findings of the article. The report will be evaluated based on the relevance and difficulty of the chosen paper as well as the thoroughness of the discussion.
Biochemical Characterization of SARS-CoV-2 N protein: Article Review
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II. ABSTRACT
The biochemical characterization of the SARS-CoV-2 N protein is essential given its increasing utility in the development of serological assay kits for screening for SARS-COV-2 infections. The popularity of the N-protein is attributed to its high immunogenicity and the relative conservation of the protein among various coronaviruses. Notably, there is a high concern that the high SARS-CoV-2 N protein affinity for nucleic acids could diminish its specificity and sensitivity when used in various serological assays. To this end, Di et al. (2021) conducted a study to establish the efficacy of the SARS-CoV-2 N protein in the development of diagnostic tools. The finding of the research was essential to the acceptability of SARS-CoV-2 N protein as a target molecule for the development of serological assays. The researcher expressed two forms of SARS-CoV-2 N protein; one with bound nucleic acids and one without nucleic acids from bacterial cells. The two forms of SARS-CoV-2 N protein were tested to determine their effectiveness when used in diagnostic tools intended to detect anti-N antibodies. Results from the study showed that both SARS-CoV-2 N proteins with or without nucleic acids had high levels of specificity and sensitivity in the screening of coronaviruses. Nonetheless, the SARS-CoV-2 N protein without Nucleic acids was more reliable in the diagnostic process. More research is required to explore the potential of SARS-CoV-2 N protein in the development of biomarkers and vaccines.
III. INTRODUCTION
The nucleocapsid (N) protein is one of the components of the SARS-CoV-2 RNA virus that encloses the genome of the SARS-CoV-2. It is considered one of the most abundant and conserved proteins in coronaviruses, making it an effective target for antibody development. Consequently, multiple researchers have used it to develop the various serological tools used to diagnose COVID-19. In addition, the protein has been identified as a reliable target for vaccine development although there is limited evidence on the overall protective immunity (Bai et al., 2020). However, a key concern raised with the use of N-protein is its propensity to associate with nucleic acid and the impact on serological assay specificity. In their study, Di et al. (2021) purify the N protein to investigate its propensity to bind to nucleic acids.
Despite its utility and application, the full-length structure of the N-proteins in SARS-CoV and SARS-CoV-2 remains widely unavailable owing to the technical challenges involved in the purification process. Timani et al. (2004) and Yue et al. (2021) managed to purify the full length of the protein for application in serology but neither of the studies effectively could determine the extent to which the purified N protein from SARS-CoV and SARS-CoV-2 retains its ability to bind to the nucleic acid upon purification. Zeng et al. (2020) purified the N protein and discovered its dimeric form and the tendency to non-specifically bind to nucleic acids. Such a protein may have a negative effect when used as a biomarker, making it important to determine the extent to which N-protein can be validly used in serological assay development (Diao et al., 2021).
The objective of the research study was to assess the validity of the serological assay that relies on N proteins of the SARS-CoV-2 when used with either nucleic acid or without. To this end, the paper will present the materials and methods used to investigate the objectives of the article under review as well as the results of the analysis.
IV. MATERIALS AND METHODS
Plasmids: The researchers used pRSF-Duet1 plasmid in the cloning of the SARS-CoV-2 N gene as synthesized by California-based Twist Bioscience. The additional sequence was added to allow for the fusing of N-protein with N-terminal His tag
Protein Expression and Purification: Di et al. (2021) cultured N-protein plasmid pRSF-Duet1 with bacteria cells with 0.1mM of Isopropyl β-d-1-thiogalactopyranoside (IPTG) sourced from Sigma-Aldrich. The BL21 bacterial cells had been grown at 37 degrees and pre-chilled at 4 degrees. The resulting culture was incubated at 16 degrees Celsius for 18 hours. A buffer (20 mM Na2HPO4, pH 7.4, 500 mM NaCl) was used to resuspend the collected bacterial cells and allow their lysis using sonification (Di et al., 2021). The resulting culture was centrifuged for an hour at 23000 x g and the resulting supernatants filtered onto His Trap HP column supplied by GE healthcare using the AKTA pure chromatography system (Di et al., 2021). 20 column of a wash buffer (20 mM Na2HPO4, pH 7.4, 500 mM NaCl, 50 mM imidazole) were applied, following which, the proteins were eluted using 10 columns of elution buffer (20 mM Na2HPO4, pH 7.4, 500 mM NaCl, 500 mM imidazole) (Di et al., 2021). Amicon Ultra-15 centrifugal filter unit (Millipore, Burlington, MA, USA; cat. # UFC900308) was used to pool and concentrate the fractions of recombinant N proteins (Di et al., 2021). OD260/280 ratio as developed by NanoDrop was used to analyze the proteins, with Bradford assay (Bio-Rad, Hercules, CA, USA) assisting in the quantification of the proteins (Di et al., 2021).
Protein Purification: BL21 bacterial cells that were transformed using differing plasmids were grown at 37 degrees overnight using 1mM IPTG. A denaturing buffer (20 mM Na2HPO4, pH 7.4, 500 mM NaCl, and 6 M urea) was used to re-suspend the collected cells for lysis (Di et al., 2021). Following an hour of centrifugation at 2500 x g, His Trap HP column was used to collect bacterial supernatant with the application of the AKTA pure chromatography system. The column was washed away using the denaturing buffer, before a renaturing buffer (20 mM Na2HPO4, pH 7.4, 500 mM NaCl), wash buffer (20 mM Na2HPO4, pH 7.4, 500 mM NaCl, 50 mM imidazole) and elution buffer (20 mM Na2HPO4, pH 7.4, 500 mM NaCl, and 500 mM imidazole) were used (Di et al., 2021). The resulting proteins were pooled and analyzed with the help of PBS buffer. OD260/280 ratio as developed by NanoDrop was used to analyze the proteins, with Bradford assay (Bio-Rad, Hercules, CA, USA) assisting in the quantification of the proteins (Di et al., 2021). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was also used to analyze the protein followed by the COomassie blue staining and gel filtration using GE Healthcare’s Superdex increase 10/300 (Di et al., 2021)
Protein Detection: Commercial COVID-19 N-based human IgG/IgM ELISA kit (MyBioSource, cat. no. MBS3809905) was used to develop an ELISA to detect and quantify the purifi...