I. Introduction to Mammalian Expression Systems

    Another unique advantage of mammalian cell expression systems lies in their ability to undergo rapid translation or post-translational modifications, resulting in correctly folded proteins. These proteins can be engineered to be human-like in origin, exhibiting favorable immunogenicity, and are generally similar to natural human proteins in terms of molecular structure, physicochemical and tissue properties, and biological functions.

II. Types of Mammalian Cell Expression Vectors

    Based on the method of entry into host cells, expression vectors can be classified into viral vectors and plasmid vectors.

1. Plasmid Vectors: These consist of a eukaryotic replication signal, a promoter, a transcriptional unit, and a plasmid fragment. The commonly used pcDNA3.1 vector has the following characteristics: it contains a forward (+) or reverse (–) large-molecule polyclonal site; a cytomegalovirus (CMV) enhancer promoter for high-level protein expression; a bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability; and an ampicillin resistance gene and pUC replication region sequence for selection and maintenance in E. coli.

2. Viral vectors: In the form of viral particles, these vectors induce the direct entry of the exogenous gene vector from the original host into the new host cell through mechanisms such as cross-linking with viral envelope protein complexes and their receptors, as well as proteins produced on the surface of the original host cell membrane. Several widely used integrative viral vectors are as follows:

III. Commonly Used Host Cells in Mammalian Expression Systems

1. CHO Cell Lines: CHO cell lines are the primary cells used for mammalian protein production. They are highly adaptable, can grow at high densities in suspension culture, and are easily adapted to serum-free conditions; however, they have poor clonal stability. CHO cells have different lineages: CHO-K1, CHO-S, CHO-DG44, and CHO-DXB11.

2. HEK293 Cell Line: This is another cell line derived from human embryonic kidney cells. It is characterized by relatively high transfectation efficiency and ease of repeated culture and propagation; One of the derivatives of the 293 cell line—293T/17—exhibits even higher transfection efficiency. A potential drawback of 293 cells is their generally weak adhesion to culture surfaces during growth and proliferation, which can lead to significant loss of nutrients throughout the experimental process, thereby affecting the overall results.

IV. Methods for Introducing Exogenous Genes into Host Cells

1. Infectious viral particles infect host cells.

2. Several specialized techniques—such as the liposome method, microinjection, calcium phosphate precipitation, and the DEAE-dextran method—are used to rapidly introduce and fuse various viral gene vectors into host receptor cells.

V. Common Issues with Expression Systems

1. Selection of Expression Systems: 1. The required yield of the protein expression product; 2. The actual application of the expressed protein; 3. The duration of the experimental process; 4. Toxicity to host cells.

2. Other Factors Affecting Normal Protein Expression Levels: Many biological factors—such as a lack of nutrient ions and certain growth-inducing factors in the culture environment, hypoxia, viral infection, excessive accumulation of toxic metabolites, mechanical damage from agitation, and a continuous increase in pressure within the culture vessel—can induce cell apoptosis. Mass cell death in culture also severely impacts normal protein expression yields.

3. Measures to Enhance Protein Expression: 1. When considering how to improve productivity per cell—such as through host cell structural modification—it is typically necessary to comprehensively evaluate five key aspects: protein folding, transport, modification, and secretion; 2. Optimizing in vitro cell culture and protein preparation and purification processes reduces the accumulation of various harmful substances—such as free deaminated compounds and free protein lactate—during in vivo host cell culture. This improves the environment for normal in vitro growth, development, and reproduction of host cells, increases the effective cell density, and ultimately boosts the total yield of recombinant proteins produced by host cells; 3. Common in vitro cell culture techniques used in industrial production include batch culture, continuous feed culture, and perfusion culture; 4. There are three main types of cell death: tissue necrosis, apoptosis, and two other forms of programmed cell death—namely, the apoptotic response and in vitro tissue autophagy. Currently, both domestically and internationally, the following two technical strategies are primarily used to construct cells resistant to induced apoptosis: increasing the expression of anti-apoptotic genes in vitro and inhibiting the expression of pro-apoptotic genes.

VI. Services Provided by Kamede

1. Codon optimization, vector construction, and subcloning of target genes. Based on the codon preferences of HEK293 and CHO cells, we can directly optimize the codon sequence of the target sequence. This involves systematic research and optimization that comprehensively considers modifications to specific functional sequences, GC content, repetitive sequences, and modifications to the secondary structure of the mRNA sequence.

2. Mammalian cell transfection and stable cell line panning. We can employ transient transfection methods to rapidly obtain small amounts of protein, as well as establish stable cell line platforms for long-term, stable production.

3. Small-scale expression of recombinant proteins to evaluate protein yield and properties.

With extensive experience in protein purification, we are capable of large-scale protein expression and purification.