Biotechnology Bulletin | Zhejiang University Research Team Summarizes the Application of Yeast Surface Display Technology in Protein Engineering
Yeast cell surface display
Yeast cell surface display technology has become an important tool in protein engineering research. By using this technology, protein interactions can be identified, protein affinity and specificity can be improved, protein stability and expression level can be increased, functional antigen maps can be drawn, and bioactive proteins and enzymes can be immobilized. The application of this technology represents the latest progress in protein engineering research.
Yeast cell surface display technology is a rapidly developing eukaryotic protein expression system in recent years. Its basic principle is to fuse foreign target protein genes (foreign proteins) with specific vector gene sequences and introduce them into yeast cells. The mechanism of transporting yeast intracellular proteins to the membrane surface enables target proteins to be immobilized and expressed on the surface of yeast cells. It has broad application prospects in various industrial fields such as medicine, food, and biofuels. The most common yeast surface display expression systems currently include lectin display expression and flocculent display expression.
Immobilization of proteins
The immobilization technology of proteins is an important aspect of protein engineering. By using yeast surface display and expression technology, the target protein or enzyme is immobilized on the surface of yeast cells, which can improve the stability and reusability of the protein or enzyme without the need for complex separation and purification of the protein. Kuroda et al α- The fusion of lectin fragments is displayed on the surface of yeast cells for chelation and biosorption of toxic heavy metal cadmium ions. The adsorption effect of cadmium ions and the tolerance of yeast engineering bacteria to cadmium depend on the amount of displayed expressed proteins. Shibasaki et al. co cultured the yeast displaying the ZZ domain on the surface with the engineered yeast containing the lipase or green fluorescent protein of the fused Fc fragment, and found that the fusion protein can be effectively recycled by the ZZ domain displayed on the yeast surface. Furukawa et al. used Flo1p to display Streptomyces ovalbumin on the surface of Saccharomyces cerevisiae. The protein showed the ability to bind biotinylation complexes. Therefore, the displayed yeast cells can be widely used as whole cell affinity agents in immunoassay and biosensor research. In addition, yeast surface display antigen technology has also been reported as a preventive or therapeutic vaccine.
Recognition of protein interactions
Exploring the interactions between proteins in organisms is an important aspect of protein engineering and the key to revealing the mechanisms of various diseases and even life phenomena. Yeast display technology provides a research platform for people to study the interactions between natural proteins. Bidlingmaier et al. identified several unreported protein molecules that can interact with EGFR or focal adhesion kinase through tyrosine phosphorylation by displaying a human cDNA library and expressing it on the surface of yeast cells using phosphorylated peptides to screen the protein library on the yeast surface. Research has shown that using a human protein library constructed on the surface of yeast can not only be used for post translational modification analysis, but also for identifying target molecules and identifying unknown proteins that cross react with drugs or small molecules.
YongKuCho et al. recently reported a yeast display fluorescence preparation (YDIP) technique that utilizes scFv to be displayed on the surface of yeast cells as an affinity agent for the separation and identification of soluble and membrane antigens. In addition, some research groups have used yeast display technology to isolate new antibodies that can bind to multiple target proteins from immune or non immune scFV libraries.
Improve protein stability and expression levels
The thermal stability and expression level are important indicators for measuring the practical application of a protein. The stability of the protein determines its storage effectiveness, while the expression level affects the production cost of the protein. Shusta et al. fused several mutant single chain T cell receptors (ScTCR) with lectin (Aga2p) and displayed the expression on the surface of yeast. The results showed that the thermal stability of ScTCR mutants was related to the level of display expression and secretion expression. The yeast display technology could be used as a directed evolution tool to identify the stability and secretion characteristics of mutant proteins.
Mapping protein epitopes
Identifying key amino acids that mediate protein-protein interactions helps people understand biochemical processes and protein design, while yeast display technology can identify these key amino acid residues in a systematic and large-scale manner. Chao et al. constructed a yeast surface display library using EGFR and some anti EGFR antibody antigens, and obtained a high-resolution table map that can recognize amino acids through screening.
Cell surface display technology has become an important tool in protein engineering research, which can be used to selectively modify target proteins and is widely used in protein separation and purification, protein immobilization, protein interactions, and other fields. Yeast is an eukaryotic organism that utilizes yeast as a host to display and express various proteins, making it possible for people to study and manipulate complex eukaryotic proteins. Although there are differences in glycosylation structures between yeast and mammals, it is imperative to develop a human cell surface display system. However, currently, the yeast system is still the most suitable system for eukaryotic protein expression. For important proteins that do not have significant glycosylation differences, using the yeast system for expression can still serve as an alternative method for processing human protein glycosylation processes.
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近日，中国农业科学院深圳农业基因组研究所联合崖州湾国家实验室、中国水稻研究所在Plant Biotechnology Journal(IF：13.8)上在线发表了题为 "Programmable RNA N6-methyladenosine editing with CRISPR/dCas13a in plants" 的研究论文，开发出基于CRISPR/dCas13a的新型植物RNA甲基化编辑工具。
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