Development of a Methodology to Adapt an Equilibrium Buffer/Wash Applied to the Purification of hGPN2 Protein Expressed in Escherichia coli Using an IMAC Immobilized Metal Affinity Chromatography System

Abstract

Protein purification is a complex and non-standardized process; the fact that proteins have different structural types making it difficult to create a standard methodology to obtain them in a pure, soluble, and homogeneous form. The present study shows the selective development of a buffer suitable for proteins of interest that allows high concentrations of hGPN2 protein to be obtained with low polydispersion and high homogeneity and purity. By taking the different reagents used in the construction of different buffers as a basis and performing purifications using different additives in different concentrations to determine the optimal amounts, the developed process helps to minimize the bonds, maintain solubility, release the proteins present in inclusion bodies, and provide an adequate environment for obtaining high concentrations of pure protein. GPN proteins are of unknown function, have not been purified in high concentrations, and have been found as part of the RNA polymerase assembly; if they are not expressed, the cell dies, and overexpression of certain GPN proteins has been linked to decreased survival in patients with invasive ductal carcinoma breast cancer types ER+ and HER2+. The results of the present study show that the use of the buffer developed for recombinant hGPN2 protein expressed in Escherichia coli could be manipulated in order to isolate the protein in a totally pure form and without the use of protease inhibitor tablets. The resulting homogeneity and low polydispersion was corroborated by studies carried out using dynamic dispersion analysis. Thanks to these properties, it can be used for crystallography or structural genomics studies.

A New Algorithm for Detecting GPN Protein Expression and Overexpression of IDC and ILC Her2+ Subtypes on Polyacrylamide Gels Associated with Breast Cancer

“…SDS-PAGE images corresponding to samples of different concentrations were obtained to replicate or emulate the GPN protein overexpression in vivo during the involvement of IDC and ILC Her2+ breast cancer [31]. For this activity, the purification methodology shown in [30] was followed. Images of the gels are shown in Figure 2A,B.…”

“…involvement of IDC and ILC Her2+ breast cancer [31]. For this activity, the purification methodology shown in [30] was followed. Images of the gels are shown in Figure 2A,B.…”

“…In this article, the methodology described in reference [30] is used to obtain different concentrations of pure GPN protein added to a bacterial cell extract to create SDS-PAGE gels. The incorrect or excessive expression of some proteins may be associated with an imbalance in health.…”

“…Examples of such proteins are GTPases, such as Rho, Rab27, and GPN, which have been linked to the development of breast cancer [31]. Reference [30], an article by the same authors as the present article, describes a new methodology for obtaining pure GPN protein in high levels in homogeneous form. In the current research, an algorithm was proposed for a new method to identify the lanes and bands of samples present in an SDS-PAGE gel and the molecular weight, to identify the overexpressed protein related to breast cancer.…”