Introduction

Protein expression refers to a molecular biology technique for the expression of exogenous gene proteins by patterned organisms such as bacteria, yeast, animal cells, or plant cells. According to the type of host cell, protein expression system can be roughly divided into:

1. Primary nuclear expression

2. yeast expression

3. Insect expression

4. mammalian cell expression

Recent Situation of the Development

The expression of some proteins in mammalian cells has moved from laboratory research to production or pilot production. There are many important proteins and glycoproteins that use mammalian cell system expression and large-scale preparation and production. Such as human tissue type hemolytic protease activation factor, clotting factor VIII, interferon, hepatitis B surface antigen, red blood cell production hormone, human growth hormone, human anticoagulant III, set stimulation factors, some products have been put into clinical application or trial.

Application Areas

Mammal cell expression system has become the production platform of a variety of gene engineering drugs. Mammal cell expression systems also play an important role in the discovery of new genes, the structure and function of proteins.

Advantages of Mammalian Cell Expression

1. Recombinant DNA is susceptible to transfection, genetic stability, repeatable.

2. Identify and remove inclusions.

3. After translation modification: phosphorylation, glycolysis, disulfur bonds, oligomers, etc. , the correct folding of advanced structure.

4. The product's configuration accuracy rate is high, can be transformed into human-like originality, good immunogenicity.

5. Can be secreted to the medium, purification process is simple.

6. Can be used for gene therapy.

Biological Properties of Mammalian Cell Expression

1.Can achieve post-translation modification and correct protein folding complex
Mammals are eukaryotic cells, and the organelle can fold proteins correctly.

2.High expression level
Vectors can express recombinant proteins in mammalian cells with expressions up to 1-3 g/L

3.Protein expression Efficient and stable
Efficient promoter control, stable expression, and a variety of expression system options:instantaneous expression, stable cell line expression, nutritional defect cell line expression (DHFR/GS), large-scale suspended cell expression, etc.

4.Suitable for large-scale protein expression:
The most commonly used mammalian expression host cells (HEK293 and CHO) can be used to culture suspended cells produced within one month number of grams protein. 

Conditions of Mammalian Host Cells

1. Cell line characteristics: loss of cell contact inhibition and anchor dependence characteristics, easy to large-scale culture.

2. Genetic stability: foreign genes are not lost after multiple generations, easy to long-term preservation.

3. Suitable marking: easy to transform the screening and maintenance of the plant.

4. Growth fast and consistent: the split cycle is short, the growth is equal, easy to control.

5. Good safety performance: do not synthesize the secretion of pathogenic substances, not carcinogenic.

Selection of Expression System

To conduct an expression experiment, the choice of which expression system should be determined according to actual needs:

1. Demand for expression protein

2. The use of expression protein

3. The experimental time

4. Toxicity to cells

After selecting the expression system, the reasonable combination of expression vector and host cell should also be considered. In mammalian cell expression systems, the level of recombinant protein expression is associated with many factors, so if a gene is required to express high, it is best to try several different vectors and host cells.

Factors Affecting Protein Expression

Recombinant protein expression depends on total live cell density and unit cell productivity. Many solids, such as lack of nutrient and growth factors in the culture environment, hypoxia, viral infection, accumulation of toxic metabolites, mechanical agitation and increased culture pressure, can induce apoptosis, and the death of a large number of cells seriously affects the expression yield of proteins. During the mass cultivation of cells, the continuous proliferation of cells causes protein expression to quickly enter the decline period, so it is necessary to control the cell's cessation of proliferation during the period of reaching the most appropriate expression product.

Measures to Increase the Output of Protein Expression

1.Anti-apoptosis engineering: There are two main strategies to build anti-apoptosis cells, increase the expression of anti-apoptosis genes and inhibit the expression of apoptosis genes.

2.Metabolic engineering regulation: Optimize the culture process, reduce the accumulation of metabolites such as ammonia and lactic acid in cell culture, improve the cell growth environment, improve cell growth density, and thus increase the yield of recombinant proteins.

3.Cell cycle engineering: By regulating the process of cell cycle G1/G0, G2/M or G1/S (of which G1/S is most common), the proliferation rate is reduced, the cell stats at a static state, enhances metabolic activity, and improves protein expression levels.

4.Secretion engineering: Endosome net intrinsic protein plays an important role in the protein folding and secretion process, improves the expression of the protein in the process of intracellular translation modification, and helps to improve the secretion of recombinant protein.