| Name: | Recombinant SARS-CoV-2 binding domain for ACE2 |
| HGNC Name: | NA |
| RRID: | Pending |
| Format: | Supplied as 1mg/mL in 6M urea, 10mM phosphate buffer pH=7.5 |
| Applications: | Antibody generation, ELISA |
| Storage: | Stable at 4°C for several months. For longer term store at -20°C, minimize freeze/thaw cycles |
| Uniprot: | P0DTC2 |
SDS-PAGE gel of recombinant human SARS-CoV-2 cell binding region. Lane 1 shows protein standards of apparent molecular weight as indicated in kiloDaltons. Lanes 2 and 3 are two loadings of the recombinant SARS-CoV-2 binding domain construct, running at about 32kDa as expected. Lanes 4 to 6 are 5.6μg,2.83μg and 1.4μg of BSA.
Recombinant SARS-CoV2 S-Protein ACE2 Binding Domain
$300.00 – $2,000.00
There has been an enormous amount of interest in the novel virus SARS-CoV-2 for very obvious reasons. It is a corona virus, a member of a large family of spherical viruses with prominent spikes which give them the appearance of a cartoon sun, which is why they are called corona viruses. The SARS-CoV-2 virus was first identified in Wuhan, China, at the end of 2019 and almost certainly originated from bats, possibly infecting humans indirectly from another animal host (1,2). The virus transmits very readily and has a significant mortality with particular risk to individuals with comorbidities such as diabetes, hypertension, immune compromise and heart problems. While no age group seems to be safe the majority of patients who have serious symptoms or succumb are of advanced age. Much research has shown that a part of the “spike” or S protein on the surface of the virus has a high affinity binding site for angiotensin converting enzyme 2 (ACE2) a protein on the surface of human cells in the lung epitheliun and other tissues (3). This specific binding is required for internalization of the virus and is the first step in viral infection. Accordingly antibodies or other reagents which bind to the ACE2/SARS-CoV-2 interaction site may interfere with viral internalization and hence infection. Recent cryoEM studies have characterized the exact interface (4). We have therefore made two recombinant constructs, this one including the segment of the SARS-CoV-2 spike protein containing the ACE2 binding site, and also Prot-ACE2-bd, containing the extracellular domain of ACE2 including the SARS-CoV-2 binding domain.
The sequence below is that of our recombinant construct of the SARS-CoV-2 spike or S-protein which includes the entire region which interacts with ACE2. The specific binding to ACE2 is essential for viral internalization and infection. We designed this construct based on amino acids 308-541 in the S-protein sequence in Isolate Wuhan-Hu-1, complete genome. This is a defined globular domain recently shown to include all of the amino acids necessary for ACE2 binding. The construct was expressed in and purified from E. coli and includes an N-terminal His-tag and other vector derived sequence shown underlined below. Amino acids which interact directly with the ACE2 protein are printed in bold, data based on the cryoEM study of Walls et al. (3).
MHHHHHHSSG LVPRGSGMKE TAAAKFERQH MDSPDLGTDD DDKAMADIGS EFVEKGIYQT 60
SNFRVQPTES IVRFPNITNL CPFGEVFNAT RFASVYAWNR KRISNCVADY SVLYNSASFS 120
TFKCYGVSPT KLNDLCFTNV YADSFVIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA 180
WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPCNGVEG FNCYFPLQSY 240
GFQPTNGVGY QPYRVVVLSF ELLHAPATVC GPKKSTNLVK NKCVNF 286
Number of amino acids: 286
Molecular weight: 32074.01
Theoretical pI: 8.01
Amino acid composition:
Ala (A) 17 5.9%
Arg (R) 13 4.5%
Asn (N) 22 7.7%
Asp (D) 16 5.6%
Cys (C) 9 3.1%
Gln (Q) 9 3.1%
Glu (E) 11 3.8%
Gly (G) 21 7.3%
His (H) 8 2.8%
Ile (I) 11 3.8%
Leu (L) 16 5.6%
Lys (K) 16 5.6%
Met (M) 4 1.4%
Phe (F) 19 6.6%
Pro (P) 15 5.2%
Ser (S) 23 8.0%
Thr (T) 16 5.6%
Trp (W) 2 0.7%
Tyr (Y) 16 5.6%
Val (V) 22 7.7%
Total number of negatively charged residues (Asp + Glu): 27
Total number of positively charged residues (Arg + Lys): 29
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Extinction coefficient 35340
Abs 0.1% (=1 g/l) 1.102, assuming all pairs of Cys residues form cystines
Extinction coefficient 34840
Abs 0.1% (=1 g/l) 1.086, assuming all Cys residues are reduced
1. Wu, F et al. A new coronavirus associated with human respiratory disease in China. Nature doi:10.1038/s41586-020-2008-3.2020 579:265-269 (2020).
2. Ren, L-L et al. Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study. Chin Med J (Engl) doi:10.1097/CM9.0000000000000722 133:1015-24 (2020).
3. Walls, A C et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell doi: 10.1016/j.cell.2020.02.058 180:1-12 (2020)
4. Yan, R et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science doi:10.1126/science.abb2762 367:1444–8 (2020).
