Cloning and expression of superfolder green fluorescent protein (SFGFP) and anti-gfp nanobody (NBGFP) genes in yeast

• Main Author: Tan, Yu Tong
• Format: Final Year Project / Dissertation / Thesis
• Published: 2024
• Subjects: QR Microbiology; QR180 Immunology
• Online Access: http://eprints.utar.edu.my/6594/1/2101776_Dissertation.pdf; http://eprints.utar.edu.my/6594/
• Institution: Universiti Tunku Abdul Rahman

Membrane protein-protein interactions play pivotal roles in cellular signalling, transport, and homeostasis. Deciphering the intricacies of cellular function and disease pathways requires thorough understandings of these interactions. The yeast two-hybrid system has been a popular choice of screening platform to examine the interactome in cells. It is a fast and convenient first-step analysis of interacting proteins. In this study, a variant of the yeast two-hybrid system, the split-ubiquitin membrane yeast two-hybrid system was used to examine protein-protein interactions involving anchored proteins on the membrane surface. The GatewayTM LR cloning technique was used to clone the target genes, which encode superfolder green fluorescent protein and anti-green fluorescent protein nanobody into yeast expression vectors pXN21-Dest and pMetYC-Dest, respectively. The expression clones were transformed and screened in Escherichia coli and verified by DNA sequencing. The recombinant plasmids were then co-transformed into yeast and analysed by yeast two-hybrid assay. GatewayTM LR cloning allowed successful transfer of genes of interest and was reflected in successfully transformed E. coli and yeast colonies when screened via colony PCR. The observed PCR product sizes were similar to expected band sizes of 544 bp and 542 bp for pMetYC-NbGFP and pXN21- sfGFP, respectively. Productive yeast growth was observed on synthetic defined medium lacking leucine, tryptophan and histidine. The yeast cells were subjected to two-fold serial dilutions up to 7 times, but growth was still observed, which signifies the strong intensity of interaction. Hence, the screening platform is functional and may be used to detect interactions between other proteins of interest in the future.