Part:BBa_K3916001

S1

S1

Properties: Used for protein expression

Usage and Biology

The coronavirus genome is comprised of ∼30000 nucleotides. It encodes four structural proteins, Nucleocapsid (N) protein, Membrane (M) protein, Spike (S) protein and Envelop (E) protein and several non-structural proteins. COVID-19 is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection may be asymptomatic or it may cause a wide spectrum of symptoms, such as mild symptoms of upper respiratory tract infection and life-threatening sepsis. The S glycoprotein is the immunodominant target for previous NAbs, and comprises an N-terminal domain (NTD), a receptor-binding domain (RBD/S1B), and an S2 subunit. The SARS-CoV RBD [amino acids (aa) 338–506] consists of an S1B core domain (S1BCD) (aa 318–424) and a receptor-binding motif (RBM) (aa 438–498) that directly engages the human receptor hACE2. Meanwhile, The RBD is also a significant neutralization determinant in the inactivated SARS-CoV vaccine because it induces potent NAbs that block SARS-CoV entry.

Construct design

We aimed to construct the plasmid to give the protein (S1) synthesized from B138 the ability to perform the protein purification and ELISA testing as well as to mimic the property held by the spike protein of the coronavirus.

Figure 1. Map of pTT5-S1-his-B117 expression plasmids..

Experimental approach

We constructed the plasmid (BBa_K3916001) then used the method of PCR to replicate the fragment of S1. Moreover, we did enzyme cutting then homologous recombination. Finally we could extract the plasmid for subsequent experiment. Purification of protein Remove DH5α receptor cells from -80°C and place on ice to melt. Add 20 μl of recombinant plasmid product to the strain, flick the wall of the tube, place on ice for 30 min, heat excites in a water bath at 42°C for 45 s, and then place on ice for 2 min to cool. Add 1000μl LB medium (without antibiotics), shake at 37°C, 220 rpm, and incubate for 60 min. Centrifuge at 5000 rpm for 5 min at room temperature, discard 900 μl supernatant and mix the remaining medium and cells by blowing. Take 100μl of bacterial solution evenly coated on pre-warmed ampicillin-resistant (Amp+) plates and incubated overnight at 37℃ in an inverted incubator; The next day picks monoclonal colonies in 20μl LB liquid medium and take 2μl for PCR sequencing of the bacterial broth to confirm successful recombination. The remaining 18μl was added to 1ml Amp+ LB and incubated for 6-8 hours at 37℃ in the shaker, 220rpm.

Figure 2. SDS-PAGE Gel..

Proof of function

1.3E8 Blocks The Bindings Of S1-Subunits Or RBD From Multiple Coronaviruses To ACE2 We investigated the abilities of 3E8 to block the ACE2 binding of S1-subunits or RBD from SARS-CoV-2, SARS-CoV-2-D614G, B.1.1.7, B.1.351, B.1.617, P.1, SARS-CoV and HCoV-NL63. These S1-subunits or RBD can all bind to His-tagged human ACE2 molecules (Fig. 5C-G), and the EC50 values to His-tagged recombinant human ACE2 molecule were 11.8, 2.6, 0.8, 6.9, 51.3, 14.9, 1.1 and 24.2 nM, respectively (Fig. 5H).

Figure 3. 3E8 blocked the bindings of recombinant S1 or RBD from multiple coronaviruses and the mutant variants of SARS-CoV-2 to His-tagged recombinant human ACE2 protein: (A-E) Bindings of recombinant S1 or RBD (in D only) from different coronaviruses and SARS-CoV-2 mutated variants to recombinant human ACE2 protein were blocked by 3E8. (F) The IC50 values of 3E8 in blocking S1 or RBD binding to human ACE2 protein..

Incubation with 3E8 effectively blocked all S1-subunits or RBD binding to ACE2 (Fig. 3A-E) and the IC50 values were 7.1, 13.8, 10.0, 3.7, 10.5, 9.3, 13.7 and 5.0 nM, respectively (Fig. 3F). Thus, 3E8 can broadly block the binding of S1-subunits or RBD from multiple coronaviruses, including the fast-spreading SARS-CoV-2 variants, to human ACE2 molecules.

References

1.Assistant Secretary for Public Affairs (ASPA). (2021, June 28). Monoclonal Antibodies for High-Risk COVID-19 Positive Patients. combatCOVID.hhs.gov. 2. Boopathi, S., Poma, A. B., & Kolandaivel, P. (2021, June). Novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment. Journal of biomolecular structure & dynamics. 3. Centers for Disease Control and Prevention. (n.d.). Test for Current Infection. Centers for Disease Control and Prevention. 4. Huang, Y., Sun, H., Yu, H., Li, S., Zheng, Q., & Xia, N. (2020, December 28). Neutralizing antibodies against SARS-CoV-2: current understanding, challenge and perspective. Antibody therapeutics.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 892
  Illegal SapI.rc site found at 288
  Illegal SapI.rc site found at 1371
  Illegal SapI.rc site found at 1584