![]() ![]() ![]() The basis for this was the given AA sequence of the corresponding protein, which can be found in the R&D Systems catalog on the page of the respective protein (see catalog numbers in Table 1). We used the SARS-CoV-2 proteins currently available on the market, namely the RBD with His- and Fc-tag, the S1 subunit with His-tag, and the S1/S2 subunits with His-tag.Īs the virus binds to the hACE2 in the human body, this receptor was included in the study (all proteins were provided by R&D Systems, a Bio-Techne brand, Minneapolis, Minnesota, USA).įirst, the theoretical pI values of the proteins were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. Since the isoelectric point of proteins is an important property, we sought to determine it using the Maurice (imaged) CIEF system from ProteinSimple, a Bio-Techne brand. In this case, the imaged CIEF with carrier ampholytes was used, i.e., an on-line imaging detection system that does not require the mobilization of the analytes after focusing. ![]() This technique is still under development, but can bring many advantages. It is also possible in CIEF to use immobilized pH gradients as applied in slab gels. The ampholytic analytes migrate in this pH gradient and then remain in the pH zone that corresponds to their pI value. Using CIEF, a pH gradient is built up in the capillary. The advantages of CIEF over IEF with slab gels are, for example, a smaller sample volume required, shorter analysis times, and higher sensitivity. in the 1980s and can be seen as an improved, new version of the conventional isoelectric focusing in slab gels. The CIEF method was implemented by Hjerten et al. In the CIEF, the isoelectric focusing takes place in a capillary, as a charge-based analysis via capillary electrophoresis. For more information on the physicochemical properties of SARS-CoV-2, please refer to the review of Scheller et al. While the S1 subunit binds to the hACE2 receptor, the S2 subunit is implicated in the merging of the viral and human cell membranes. The spike protein is comprised of two functional subunits: S1 and S2. SARS-CoV-2 uses the receptor-binding domain (RBD) of its surface glycoprotein (spike protein) to enter the human body via the hACE2 receptor. In this short communication, the results of the capillary isoelectric focusing (CIEF) measurements of different sections of the SARS-CoV-2 spike protein and the human angiotensin-converting enzyme 2 (hACE2) are presented. Therefore, our intention was to contribute to the progress of research in this field with the means available to us. For research purposes, there are several significant proteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the market. Since the beginning of the outbreak, research has been conducted into medicines and vaccines against the virus in the field of pharmaceutical research. The Coronavirus Disease 2019 (COVID-19) pandemic is currently shaking up the whole world. severe acute respiratory syndrome coronavirus 2.The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS-CoV-2 proteins. All theoretically derived values were found within these ranges, usually close to the center. The pI range of the S1/S2/His is 4.41–5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19–6.11 (5.60) for all experimental conditions chosen. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor-binding domain (RBD) with His- and Fc-tag, the S1 subunit with His-tag, the S1/S2 subunits with His-tag and the human angiotensin-converting enzyme 2 (hACE2) with His-tag. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |