Optimization And Application Of Yeast Surface Display System For ScFv Screening And Cd²⁺ Accumulation

Yeast surface display and phage display technologies are powerful tools for protein engineering,especially in antibody engineering.Yeast display technology can be combined with flow cytometry to analyze expression,stability and affinity of the displayed protein directly,thereby becoming an efficient library screening system.Yeast surface display technology has been widely used for screening and optimizing single-chain antibodies（scFv）against various antigens.However,there are still some defects in the yeast surface display based scFv library screening,which limits the efficiency of system screening.In this study,we optimized the screening of scFv library from two aspects.First,the construction of the scFv library generally starts with insertions amplification by PCR or insertions mutagenesis by PCR mediated random mutation.The fragments are integrated into the yeast surface display vector.However,in this process it is often inevitable to introduce an internal stop codon in the scFv coding fragment,resulting in truncated scFv expression.If the library contains a large population of such fragments,it will affect the library capacity and screening efficiency.Second,the screened scFv sub-library generally requires high-temperature stability verification to obtain the favorite scFv with higher stability.However,the problem in the traditional system is that it is often necessary to collect the heat-shocked cells to extract the library plasmids and re-transfer them into fresh yeast cells for sorting because the yeast itself is not resistant to high temperature.This process is time consuming and reduces the library diversity during plasmids extraction.In order to solve the problems,in this study,the original yeast display plasmid which based on Aga1-Aga2 was modified by introducing ribosomal skipping peptide（T2A）at the carbon terminus of Aga2.It allows scFv,T2A sequence and auxotrophic marker LEU2 to be transcribed in the same mRNA.The ribosome recognizes the T2A sequence during translation and eventually resulting two independent proteins,scFv and Leu2.If scFv sequence contains an internal stop codon,the ribosome will release,and the following Leu2 protein cannot be expressed,therefore cells contain such plasmids can not survive in leucine-lacking medium.In this study,we demonstrated that the T2A-Leu2 system could exclude sequences containing internal stop codons.Further,we constructed the T2A-Leu2system and compared with the original system,and proved that the T2A-Leu2 system can indeed improve the screening efficiency.At the same time,this study used methyl methanesulfonate（MMS）to mutagenize the yeast strains,and obtained a temperature-resistant mutant without alternation on display profile.Therefore,after heat shock,the yeast can be directly cultured and subjected to flow sorting for high-stable scFv.In addition,we also took advantage of yeast surface display system to improve the ability of yeast to inactivate heavy metals.This strategy is to display metallothionein proteins on the surface of yeast cells for the purpose of directly binding heavy metal ions in the environment,so that heavy metal ions change from free state to bound state,reducing its toxicity.It was detected by atomic absorption spectrometry that yeast displaying metallothionein can effectively enhance its ability to bind heavy metals Cd²⁺.Therefore our work provides an effective strategy for controlling environmental heavy metal pollution.