Merck
  • Novel membrane-bound reporter molecule for sorting high producer cells by flow cytometry.

Novel membrane-bound reporter molecule for sorting high producer cells by flow cytometry.

Cytometry. Part A : the journal of the International Society for Analytical Cytology (2013-06-15)
Daniel Helman, Mira Toister-Achituv, Meirav Bar-Shimon, Benny Cohen, Issaschar Otmi, Nir Berger, Doron Kalimi, Boaz Kimalov, Tali Medina, Ashi Sapir, Ofer Rotemberg, Natalia Zabavnik, Arie Zauberman, Moshe Smolarsky
ABSTRACT

We developed a membrane bound reporter and selection molecule for sorting by fluorescence activated cell sorting (FACS) of cells producing a protein of interest. This molecule is composed of a transmembrane (TM) domain, fused on its extracellular end to a biotin mimetic peptide (BMP) and on its intracellular side to puromycin N-acetyl transferase (PAC). In this format BMP is displayed on the cell membrane surface and PAC faces the cell cytoplasm. BMP was detected and quantified on the cell surface by fluorescently labelled streptavidin, allowing cell sorting by FACS, according to the reporter expression level. The reporter and a gene of interest (GOI) were connected on the same transcript via an internal ribosomal entry site (IRES). The reporter expression level was found to correlate with that of the GOI, enabling sorting of high producer cells by FACS. Thus, the highest fluorescent cells sorted had also the highest protein of interest (POI) productivity level.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Poloxamer 188 solution, 10%, sterile-filtered, BioReagent, suitable for insect cell culture
Sigma-Aldrich
Endotoxin-Free Ultra Pure Water, Cell Culture