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 derived from Severe acute respiratory syndrome coronavirus 2 (2019-nCoV) (SARS-CoV-2). It is commonly used as a defined reagent for assay development, binding studies, and analytical controls where consistent protein specifications are required.

Key elements and design rationale

• Expressed region: 5325-5925aa.
• Expression system: Baculovirus (may influence folding and post-translational modifications).
• Tag/format: C-terminal 6xHis-tagged; Liquid or Lyophilized powder.
• Expected size: 70.0 kDa (as provided).
• Purity: Greater than 85% as determined by SDS-PAGE.

Region choice, expression system, and tag/format can influence folding, post-translational modifications, and interaction behavior in downstream assays.

Biological background

[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.

Research relevance and current trends

• Antigen design and domain selection to better capture neutralizing versus non-neutralizing antibody responses.
• High-throughput serology and antibody screening using standardized antigens and plate-based formats.
• Integrating binding kinetics with epitope mapping to support variant-aware immunology studies.

Common research applications

• Coat plates with rep antigen for ELISA antibody titration (serum/plasma).
• Screen anti-rep antibodies by indirect ELISA and immunoblot readouts.
• Map antigenic epitopes using rep fragments/domains (in vitro binding assays).
• Develop antigen-capture assays using rep as a standard (spike-in controls).

Interpret results in the context of the biological system, assay format, and any known domain/isoform constraints for the target.

Notes for experimental interpretation

• Antigenic proteins may contain immunodominant regions; domain choice can affect assay readouts and cross-reactivity.
• Include relevant negative controls (e.g., unrelated antigens) and dilution series to support interpretation of binding signals.

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