Selection System for Yeast Clones with the Highest Production of Recombinant Proteins

Yeast expression systems have been used for the commercial production of a number of recombinant pharmaceuticals — insulin, interferon-alpha-2a, hepatitis B virus vaccine, and human papillomavirus — demonstrating the importance of yeast as a platform for the production of recombinant proteins for the pharmaceutical industry. Currently, the main approach to obtaining high-performance producing strains of the yeast Pichia pastoris is to use combinatorial enumeration of elements of the genetic environment and screening a large number of clones. Among screening methods, screening using 96-well plates remains the most universal; however, this method is labor-intensive, which significantly complicates the study of all possible combinations of genetic elements. The goal of this study was to create a selection system that allows the selection of P. pastoris yeast cells with the maximum level of production of the recombinant target protein from the clone repertoire by selecting cells with the maximum concentration of target protein mRNA. The proposed selection system is based on creating a relationship between the level of expression of the marker fluorescent protein and the level of mRNA of the target protein in the producing cell. This relationship is based on the competitive binding of tetracycline by aptamer sequences to tetracycline 32sh, integrated into the promoter region of the fluorescent marker and into the sequence of the target protein as part of the intron. Binding of tetracycline by aptamers in the promoter region of the GFP marker protein leads to a decrease in its expression level. A high level of aptamer-containing introns from the pre-mRNA of the target protein creates competition for tetracycline binding. Tetracycline binding occurs primarily with an aptamer in the intron, which releases aptamers in the promoter region of the GFP marker protein and allows its translation. Accordingly, with high expression of the target protein gene, high GFP production is observed. Thus, cells with the maximum level of green fluorescence, at the same time, provide the maximum level of expression of the target protein, and can be selected by cytofluorimetry. To achieve this goal, a recipient strain was obtained that produces GFP into the cell cytoplasm under the control of a promoter containing three tandemly located tetracycline aptamer sequences. The system was tested using a library of three signal sequences for red fluorescent protein (RFP) as a target and a library of promoter, terminator and signal sequences for human transferrin protein (TFR) as a target. From the experimental data it follows that the system functions imperfectly; despite this, it has the potential to select highly expressing clones when screening a significant sample of transformants. A more promising application can be considered the use of the system for the purpose of screening combinations of regulatory elements of the genetic environment of the target recombinant protein gene for the further creation of a producer strain based on the selected optimal combination of genetic elements.