Recombinant Severe acute respiratory syndrome coronavirus 2 Replicase polyprotein 1ab (rep), partial

Overview

Recombinant Severe acute respiratory syndrome coronavirus 2 Replicase polyprotein 1ab (rep), partial is a recombinant protein preparation from Severe acute respiratory syndrome coronavirus 2 (2019-nCoV) (SARS-CoV-2) designed for use in assay development, binding studies, and functional characterization. Key attributes such as expression system, expressed region, and affinity tag(s) help researchers match the reagent to specific experimental readouts.

Key elements and design rationale

• Expression system: Yeast expression is commonly used for rapid, scalable production. For targets that require glycosylation or other post-translational modifications, consider how a prokaryotic system may affect folding or activity.
• Expression region: The expressed fragment (5325-5925aa) focuses the reagent on a defined domain/segment, which can influence binding interfaces and epitope availability.
• Tag(s)/format: GST tags can support purification and detection in pull-down or binding assays; confirm that the tag position does not interfere with the interaction of interest.
• Purity: ≥85% (SDS-PAGE) provides a quick checkpoint for reagent quality in downstream analytical workflows.
• Form: Supplied as Liquid or Lyophilized powder; select the format that best fits your lab’s handling and aliquoting preferences.

Recombinant design choices (expression host, fragment boundaries, and tag configuration) help balance yield, solubility, and assay compatibility. Choose conditions and controls that match the recombinant format to your experimental question.

Biological background

rep has been reported to be involved in [Replicase polyprotein 1ab]: Multifunctional protein involved in the transcription and replication of viral RNAs. Contains the proteinases responsible for the cleavages of the polyprotein. ; [Host translation inhibitor nsp1]: Inhibits host translation by associating with the open head conformation of the 40S subunit. The C-terminus binds to and obstructs ribosomal mRNA entry tunnel. Thereby inhibits antiviral response triggered by innate immunity or interferons. The nsp1-40S ribosome complex further induces an endonucleolytic cleavage near the 5'UTR of host mRNAs, targeting them for degradation. Viral mRNAs less susceptible to nsp1-mediated inhibition of translation, because of their 5'-end leader sequence. ; [Non-structural protein 2]: May play a role in the modulation of host cell survival signaling pathway by interacting with host PHB and PHB2. Indeed, these two proteins play a role in maintaining the functional integrity of the mitochondria and protecting cells from various stresses. ; [Papain-like protease nsp3]: Responsible for the cleavages located at the N-terminus of the replicase polyprotein. Participates together with nsp4 in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication. Forms a molecular pore spanning the double membrane of the coronavirus replication organelle. Antagonizes innate immune induction of type I interferon by blocking the phosphorylation, dimerization and subsequent nuclear translocation of host IRF3. Prevents also host NF-kappa-B signaling. In addition, PL-PRO possesses a deubiquitinating/deISGylating activity and processes both 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains from cellular substrates. Cleaves preferentially ISG15 from antiviral protein IFIH1 (MDA5), but not DDX58 (RIG-I). Can play a role in host ADP-ribosylation by binding ADP-ribose. ; [Non-structural protein 4]: Participates in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication. ; [3C-like proteinase nsp5]: Cleaves the C-terminus of replicase polyprotein at 11 sites. Recognizes substrates containing the core sequence [ILMVF]-Q-|-[SGACN]. Also able to bind an ADP-ribose-1''-phosphate (ADRP). ; [Non-structural protein 6]: Plays a role in the initial induction of autophagosomes from host reticulum endoplasmic. Later, limits the expansion of these phagosomes that are no longer able to deliver viral components to lysosomes. Binds to host TBK1 without affecting TBK1 phosphorylation; the interaction with TBK1 decreases IRF3 phosphorylation, which leads to reduced IFN-beta production. ; [Non-structural protein 7]: Plays a role in viral RNA synthesis. Forms a hexadecamer with nsp8 (8 subunits of each) that may participate in viral replication by acting as a primase. Alternatively, may synthesize substantially longer products than oligonucleotide primers. ; [Non-structural protein 8]: Plays a role in viral RNA synthesis. Forms a hexadecamer with nsp7 (8 subunits of each) that may participate in viral replication by acting as a primase. Alternatively, may synthesize substantially longer products than oligonucleotide primers. Interacts with ribosome signal recognition particle RNA (SRP). Together with NSP9, suppress protein integration into the cell membrane, thereby disrupting host immune defenses. ; [Non-structural protein 9]: May participate in viral replication by acting as a ssRNA-binding protein. Interacts with ribosome signal recognition particle RNA (SRP). Together with NSP9, suppress protein integration into the cell membrane, thereby disrupting host immune defenses. ; [Non-structural protein 10]: Plays a pivotal role in viral transcription by stimulating both nsp14 3'-5' exoribonuclease and nsp16 2'-O-methyltransferase activities. Therefore plays an essential role in viral mRNAs cap methylation. ; [RNA-directed RNA polymerase nsp12]: Responsible for replication and transcription of the viral RNA genome. ; [Helicase nsp13]: Multi-functional protein with a zinc-binding domain in N-terminus displaying RNA and DNA duplex-unwinding activities with 5' to 3' polarity. Activity of helicase is dependent on magnesium. Binds to host TBK1 and inhibits TBK1 phosphorylation; the interaction with TBK1 decreases IRF3 phosphorylation, which leads to reduced IFN-beta production. ; [Proofreading exoribonuclease nsp14]: Enzyme possessing two different activities: an exoribonuclease activity acting on both ssRNA and dsRNA in a 3' to 5' direction and a N7-guanine methyltransferase activity. Acts as a proofreading exoribonuclease for RNA replication, thereby lowering The sensitivity of the virus to RNA mutagens. ; [Uridylate-specific endoribonuclease nsp15]: Plays a role in viral transcription/replication and prevents the simultaneous activation of host cell dsRNA sensors, such as MDA5/IFIH1, OAS, and PKR. Acts by degrading the 5'-polyuridines generated during replication of the poly(A) region of viral genomic and subgenomic RNAs. Catalyzes a two-step reaction in which a 2'3'-cyclic phosphate (2'3'-cP) is first generated by 2'-O transesterification, which is then hydrolyzed to a 3'-phosphate (3'-P). If not degraded, poly(U) RNA would hybridize with poly(A) RNA tails and activate host dsRNA sensors. ; [2'-O-methyltransferase nsp16]: Methyltransferase that mediates mRNA cap 2'-O-ribose methylation to the 5'-cap structure of viral mRNAs. N7-methyl guanosine cap is a prerequisite for binding of nsp16. Therefore plays an essential role in viral mRNAs cap methylation which is essential to evade immune system. May disrupt host mRNA splicing in nucleus by interacting with pre-mRNA Recognition Domains ofthe U1 and U2 snRNAs.. When interpreting results, consider species context, domain architecture, and whether the recombinant format represents full-length or a defined region.

Research relevance and current trends

• Antigen and virulence-factor studies that compare strain- or domain-specific binding and immune recognition.
• Use of recombinant proteins as standards for quantitative assays and serology-oriented method development.

Common research applications

• Binding and interaction assays: quantify partner binding and rank conditions using plate-based formats or biophysical methods (SPR/BLI).
• Enzymology: assess catalytic activity and compare substrate preferences or inhibitor effects using appropriate controls.
• Assay development: use as a standard, spike-in control, or positive control where consistent specifications are required.

Interpretation typically relies on relative comparisons (treated vs control, mutant vs wild-type, or dose/time series) using consistent sample handling and appropriate normalization.

Notes for experimental interpretation

• Post-translational modifications: expression system can affect glycosylation and processing; interpret differences cautiously when comparing to native protein.
• Isoforms and domains: expressed regions may not capture all isoform-specific features; match fragment boundaries to your assay’s binding site.
• Controls: include blank matrix controls, tag-only controls (where relevant), and orthogonal readouts (e.g., WB/qPCR/ELISA) to support interpretation.

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