QC Report Eukaryotic Proteins Assemble Proteins Mammalian Expression System

Introduction

Mammalian cells can correctly and effectively express eukaryotic proteins, including the synthesis, processing, and secretion of proteins. The mammalian system has the excellent function of post-translational modification of proteins and can assemble proteins correctly. This is because mammalian systems have endoplasmic reticulum, Golgi apparatus and other organelles that are key elements in cell transport and post-translational modification.

Most post translational modifications occur during or shortly after translation. Excreted proteins are made in the endoplasmic reticulum where there are enzymes that facilitate proper folding, disulfide bonding, and N-linked glycosylation. Additionally, before it is excreted, a protein would pass through the golgi apparatus, where O-linked glycosylation may occur as well. All of these modifications occur in many proteins expressed by mammalian cells, including antibodies.

There are two ways for protein expression of mammalian cells: transient transfection and stable transfection. HEK293 or CHO cells are often used in transient transfection. A small amount of protein can be quickly obtained within one week after the transient transfection. Meanwhile, there are dozens of cells that can be stably transfected, which can be used to produce cell lines after stable transfection and screening. Stable cell lines can grow continuously over a long period of time without significant change in the expression level of the target gene.

Experimental Process

1. Analysis of gene sequences provided by customers: including codon optimization, protein transmembrane structure, hydrophobicity, whether it’s secretory protein or not.
2. Gene synthesis and plasmid construction: the target gene is connected to CELLFREE’s proprietary vector by the method of double enzyme digestion, and its correctness is tested by enzyme digestion and sequencing.
3. Plasmid transformation and max-prep: plasmid transformation competent cells & transfected plasmid max-preparation.
4. Cell transfection: method of expression transfection for the mammalian cell are transient transfection and stable transfection expressions.
5. Cell culture and protein expression.
6. Protein purification & QC

Transient VS. Stable Transfection

Transient Transfection

1. Quickly transform cloned genes into large amounts of protein in 3-7 days.

2. Obtain fully post-translational modified and active mammalian protein.

3. Easily purify secreted protein from serum-free cell culture medium.

4. Expand culture volume from that of 50 multi-well plates to that of 25L and more

Stable Transfection

1. Research assay: transmembrane, ion channels, enzymes, GPCRs and many others
2. Cell-based assay and other functional studies
3. Recombinant protein/antibody production (industrial)