An optimized yeast display strategy for efficient scFv antibody selection using ribosomal skipping system and thermo resistant yeast
- 34 Downloads
Establish a method to restrict unexpected fragments including stop codons in scFv library and generate a thermo resistant strain for screening of thermal stable scFv sequences.
Here, we have constructed a T2A–Leu2 system for selection of yeast surface display libraries that blocks amplification of “stop codon” plasmids within the library, thereby increasing the quality of the library and efficiency of the selection screen. Also, we generated a temperature-resistant yeast strain, TR1, and validated its combined use with T2A–Leu2 for efficient screening. Thus, we developed a general approach for a fast and efficient screening of scFv libraries using a ribosomal skipping system and thermo-resistant yeast.
The method highlights the utility of the T2A–Leu2-based ribosomal skipping strategy for increasing the quality of the input library for selection, along with an optimized selection protocol based on thermo-resistant yeast cells.
KeywordsYeast surface display scFv antibody Ribosomal skipping T2A Antibody screen
Supplementary Table 1—Nter-SfiI and scFv DNA sequences used in this study.
Supplementary Table 2—Oligos used in this study.
Supplementary Figure 1—Map of pCTcon2-derived plasmid pDJ21.
Supplementary Figure 2—Map of T2A-Leu2 plasmid pDJ22.
This work was supported by a Starting Grant from Hebei Agricultural University (to Fanli Zeng; Grant No. 2018KYYJ01) and by a grant from the National Natural Science Foundation of China (to Fanli Zeng; Grant No. 31801039).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- Dusseaux MMF (2018) Cells for immunotherapy engineered for targeting cd38 antigen and for cd38 gene inactivation. United States Patent, 20180236053Google Scholar
- Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Yeung YA, Cochran JR, Heinzelman P, Colby D, Swers J, Graff C, Wiley HS, Wittrup KD (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21:163–170CrossRefGoogle Scholar
- Hu Y, Jia Y, Zhao X, Yang Z, Hao Z, Dong J, Zeng F (2019) A new strategy for seamless gene editing and marker recycling in Saccharomyces cerevisiae using lethal effect of Cwp1. Microbiologyopen 8:e750Google Scholar
- Yuan X, Chen X, Yang M, Hu J, Yang W, Chen T, Wang Q, Zhang X, Lin R, Zhao A (2016) Efficient construct of a large and functional scFv yeast display library derived from the ascites B cells of ovarian cancer patients by three-fragment transformation-associated recombination. Appl Microbiol Biotechnol 100:4051–4061CrossRefGoogle Scholar
- Zeng F, Hua Y, Liu X, Liu S, Lao K, Zhang Z, Kong D (2018) Gpn2 and rba50 directly participate in the assembly of the rpb3 subcomplex in the biogenesis of RNA polymerase II. Mol Cell Biol 38:18–91Google Scholar