Protein expression is a molecular biological technique for expressing foreign gene proteins in model organisms such as bacteria, yeast, animal cells or plant cells. According to the type of host cell, protein expression systems can be broadly classified into: 1. Prokaryotic Expression Systems 2. Yeast expression system 3. Plant Expression Systems 4. Insect expression systems 5. Mammalian expression system
The expression of some proteins in mammalian cells has moved from laboratory research to production or pilot production.
Many important proteins and glycoproteins have been expressed, prepared and produced in large quantities using mammalian cell systems. Some products, such as human tissue type fibrinogen activating factor, coagulation factor VIII, interferon, hepatitis B surface antigen, erythropoietic hormone, human growth hormone, human anticoagulant factor III, colony stimulating factor, etc., have been put into clinical application or trial.
Although the expression level of mammalian cell expression system has been greatly increased after years of efforts, it is still low from the whole level, generally at the lower limit of protein yield of hybridoma monoclonal antibody, i.e. 1-30 μg/l08 cells/24 hours.
Mammalian cell expression systems have become a production platform for a variety of genetically engineered drugs. It also played an extremely important role in the discovery of new genes and the study of protein structure and function.
Mammalian cell expression system
Compared with other systems, mammalian cell expression systems have the advantage of directing the correct folding of proteins, providing a variety of post-translational processing functions such as complex N-glycosylation and accurate O-glycosylation, so that the expression product is closest to native higher biological protein molecules in terms of molecular structure, physicochemical properties and biological functions.
1. Recombinant DNA is easily transfected, genetically stable, and reproducible.
2. Identify and remove introns.
3. To perform post-translational modifications: phosphorylation, glycosylation, disulfide bonds, formation of oligomers, etc., correct folding of higher order structures.
4. The product has a high rate of correct configuration and can be modified to be human-like in nature with good immunogenicity.
5. It can be secreted into the culture medium and the purification process is simple.
6. It can be used for gene therapy.
Compared with other expression systems, mammalian cells have obvious advantages, but their low expression is still the biggest bottleneck, as well as the defects of long production cycle and high production cost.
1. Long production cycle.
2. Low protein production.
3. High production costs.
Commonly used host cells
Commonly used non-lymphoid cells are Chinese hamster ovary (CHO) cells, hamster kidney (BHK) cells, COS cells, mouse NSO thymoma cells and mouse myeloma SP2/0 cells.
The recombinant proteins expressed by different host cells have different stability and protein glycosylation types, so it is necessary to select the best host cell according to the target protein to be expressed.
Until now, Chinese hamster ovary (CHO) cells have been the main recipient cells of higher mammals used for large-scale production of medical products (drugs, antibodies, diagnostic reagents).
CHO cells belong to fibroblast-adherent cells, which have all the characteristics of adherent cells, and because they are fibroblasts, express exogenous proteins, and rarely secrete endogenous proteins, which is conducive to downstream protein isolation and purification.
CHO-k1 is a cell derived from CHO. It is widely used in various experiments. It has simple culture conditions, moderate adherence strength, and is relatively easy to transfect. It is generally used to study mammalian gene functions.
293 cells are relatively easy to transfect and are a common cell line used for expression of exogenous genes, and are mostly used for transient transfection to express recombinant proteins.
Compared with CHO cells, the cells grew faster, had higher growth density, higher transfection efficiency and exogenous protein expression, clear genetic background and stable physiological metabolism.
Adherent culture, microcarrier culture and serum-free suspension culture can be used for large-scale culture of 293 cells. It can grow similarly in Ca2 + -free or Ca2 + -containing media, and also in media with lower serum concentrations. It has high plasmid transfection efficiency and high stability to maintain exogenous genes.
COS cell lines are cell lines derived from African green monkey kidney fibroblasts and transformed with SV40 viral genes. The most commonly used COS cell lines are COS-1 and COS-7.
COS cells are the most versatile host for the transient expression of source genes. Their recombinant vectors are easy to construct, easy to use, and have no limitations on the amount of inserted DNA or the use of genomic DNA sequences. It is convenient to detect the expression to confirm the positive clone of cDNA and facilitate the rapid analysis of mutations introduced into the cloned cDNA sequence.
In recent years, several new cell lines with great application value have also been discovered:
Such as well-differentiated endothelial cell line (MDCK) derived from MadIin-Darby canine kidney. Pei et al. used this cell line to express the secreted matrix metalloproteinase MMPI3 and found that clones with high expression accounted for 5% to 10% of the transfected cells, with one clone expressing 15% to 20% of the total protein in the cell supernatant and up to 10 mg/L in monolayer adherent culture.