Short Communication - Journal of Systems Biology & Proteome Research (2022) Volume 3, Issue 4
Protein quantification using several short chimeric standards without purification
Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou 515041, China
- *Corresponding Author:
- Hung Chai
Department of Biochemistry and Molecular Biology
Shantou University Medical College
Shantou 515041, China
E-mail: [email protected]
Received: 27-Jun-2022, Manuscript No. AASBPR -22-70877; Editor assigned: 28-Jun-2022, PreQC No. AASBPR -22-70877 (PQ); Reviewed: 12-Jul-2022, QC No. AASBPR -22-70877; Revised: 18-Jul-2022, Manuscript No. AASBPR -22-70877 (R); Published: 26-Jul-2022, DOI:10.35841/aacmt-3.4.120
Citation: Chai H. Protein quantification using several short chimeric standards without purification. J Syst Bio Proteome Res. 2022;3(4):120
The post-weaning multi-systemic wasting syndrome is brought on by porcine circovirus. Despite the availability of commercial vaccines, the development of vaccines that are more cost-efficient and effective is anticipated. Serological distinction between naturally infected and vaccineexposed animals is made possible by the use of chimeric antigens. In this study, chimeric fusion proteins derived from the multimerizing subunit of the mouse polyomavirus capsid protein VP1 and a circovirus capsid antigen Cap were purified and characterised using asymmetric flow field-flow fractionation (AF4) in conjunction with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Different elution profiles, such as decreasing cross-flow and constant cross-flow, were examined (linearly and exponentially). The comparison of the hydrodynamic radius measured by online DLS allowed for the evaluation of the ideal sample retention, separation effectiveness, and resolution. According to the findings, using dual elution profiles (exponential and constant cross-flow rates) speeds up separation time, avoids unfavourable sample-membrane interaction, and produces superior resolution. Additionally, the data reveal no sample deterioration during the AF4 separation and no self-associations of the individual pentameric particles into bigger clusters. Transmission electron microscopy morphological evaluations and the Rg/Rh ratios for various fractions are well correlated. The individual fractions were also submitted to offline analysis, such as batch DLS, TEM, and SDSPAGE, followed by Western blot, in addition to the online examination.
Despite the availability of commercial vaccinations, there is still a significant effort being made to create additional efficient vaccines for animal usage that would be more affordable while maintaining or even improving efficacy. The ability of BEVS technology to quickly create vast quantities of different proteins, including chimeric proteins like the PCV2b Cap described here, has significant commercial potential. This protein generates a pentamer chimeric protein called VP1-Cap when fused with the C-terminus of the mouse polyomavirus capsid protein VP1. This allows serological distinction between infected (only Cap-reacting) and immunised (both VP1- and Cap-reacting) mice. The most important stages in the research and commercial manufacture of vaccines are antigen purification and complex characterisation. Proteins and protein particles can be identified and purified using a range of analytical methods, including size exclusion chromatography, electrophoresis, and ultracentrifugation .
These methods may have drawbacks with regard to large recombinant antigen complexes like VLPs or multimeric protein antigens, such as limited selectivity, alteration or aggregation of the sample during analysis, and reduced sample recovery because of the sample's interaction with the stationary phases. The field-flow fractionation separation method is an addition to the ones mentioned above .
Contrary to the methods previously stated, AF4 has no stationary phase or packing material, which reduces sample-stationary phase contact and sample shear degradation. AF4 is compatible with a wide range of solvents and has a size separation range of 1 nm to micrometres. Another benefit, in addition to separation and characterisation, is the capability to analyse complicated materials in conditions that are similar to those found in nature, including the use of suitable buffers of a specific ionic strength. Narrow fractions of the sample's individual eluates can be collected for further "offline" examination using various techniques, such as electron microscopy for morphological analysis and SDS-PAGE/Western blot for the verification of the identity and purity of each separated protein .
Protein absolute (molar) quantitation plays several different roles. The abundance of non-proteinous substances, such as enzyme cofactors, lipids, or metabolites, is related to the stoichiometric ratios seen in molecular assemblies and metabolic pathways. The reference values and physiological fluctuation ranges for proteins that are crucial for diagnosis in liquid and solid biopsies are also provided. The AF4 theory is supported by the presence of low molecular weight proteins, whose early elution time at a certain cross-flow is seen . Particularly noticeable are the visible band in fractions F3 and F4. The fraction of His-Tag groups that contains the most particles, F2, does not exhibit this percentage. The structure of bigger clusters of pentameric protein particles, whose size expressed as z-average in F3 is roughly double that of F2, is what leads to the band disproportion. The particles' volume, which will differ in size by two, will be eight times bigger. As a result, bigger particles, especially those in fractions F3 and F4, have a significantly higher .
The separation and characterisation of pentameric protein particles and their aggregates were carried out using an asymmetric flow field-flow fractionation system with UV, MALS, and online DLS detection. The ideal separation parameters were established after testing several elution profiles. Since the exponential decrease shortens total separation time while preserving separation effectiveness, it has been proven to be the best combination for optimal separation. Additional offline DLS, TEM, and SDS-PAGE studies were performed on each fraction. SDS-PAGE/Western blot analysis was used to evaluate the general purity and quality of each fraction. In particular for the elution of individual protein pentamers, the observed particle size, given as the hydrodynamic radius, matches the hydrodynamic radius values obtained from the simplified retention equation.
- Gillespie J, Opriessnig T, Meng XJ, et al. . Porcine circovirus type 2 and porcine circovirus?associated disease . J Vet Intern Med. 2009;23(6):1151-63.
- Gillespie J, Opriessnig T, Meng XJ, et al. . A very small porcine virus with circular single-stranded DNA . Nature. 1982;295(5844):64-6.
- Meehan BM, McNeilly F, Todd D, et al. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs . J Gen Virol. 1998;79(9):2171-9.
- Fachinger V, Bischoff R, Jedidia SB, et al. The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex . Vaccine. 2008;26(11):1488-99.
- Wahlund KG, Giddings JC. Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall . Anal Chem. 1987;59(9):1332-9.