Efficient Preparation Platform Of Recombinant Protein Antigen

1. Technical platform introduction
According to the type of host cell, gene expression systems can be broadly classified into prokaryotic, yeast, plant, insect and mammalian cell expression systems. Compared with other systems, the advantage of mammalian cell expression system is that it can guide the correct folding of proteins and provide a variety of post-translational processing functions such as complex N-type glycosylation and accurate O-type glycosylation. Therefore, the expression products are close to natural higher biological protein molecules in molecular structure, physical and chemical properties and biological functions. Mammalian cell expression systems are widely used in the development and production of recombinant protein drugs, especially therapeutic recombinant monoclonal antibodies.

SGE BIOTECH has established computer-aided analysis, designed and developed self-optimized eukaryotic serum-free mammalian recombinant protein expression systems, including CHO cell expression system and HEK293 expression system, which can provide customers with high-quality and competitive protein expression and Purification services.

2. Technical features
1) Self-optimized mammalian cell expression platform with high expression;
2) Computer-aided design to solve the expression problem of complex difficult-to-express proteins.
3) protein expression to ensure the success rate,Not successful no charge!
 

3. Experimental technical process

➢ Plasmid construction
The target protein was cloned into the expression vector optimized by SGE BIOTECH biology, and the high-quality transient expression plasmid was obtained by plasmid pumping, and the sequence was verified by sequencing.

➢ Cell blink and culture
SGE BIOTECH has two effective transient expression systems of CHO and 293F. Appropriate protein expression systems are selected for different proteins. High concentration protein supernatant can be obtained through 7 days of transient expression.

➢ Protein purification and recovery
The target protein was purified by AKTA, and the purity of the protein was identified by SDS-PAGE and SEC-HPLC.

4. Case sharing
Case 1:M01 (code) protein expression
The M01 protein is a protein molecule with biological activity, consisting of two subunits, and no recombinant protein of the protein is currently available on the market. After receiving the order from the customer, we carried out the secondary structure analysis and tertiary structure prediction of the protein, which has the risk of inactivation of the hydrophobic structure domain and the biological activity of conventional expression. We have designed a variety of protein expression strategies, including: unstable site amino acid mutation, double subunit coupling, increasing chaperone stable protein and selecting suitable purification tags. After 4 weeks of trial and expression, the recombinant protein was successfully expressed. After customer verification, the protein has good biological activity and fully meets the requirements of subsequent experiments.
 

Case 2:NT2 (code name) protein expression
NT2 protein is a nuclear protein with a molecular weight of nearly 200kd, and the nuclear localization sequence analysis and structure prediction of the protein were carried out. In order to realize the extracellular secretion expression of the protein, the nuclear localization peptide masking strategy was used to construct the expression vector. After the special reagent stimulation treatment in the process of expression, the expression of NT2 protein in the extracellular was successfully realized, but the expression amount could not meet the demand of dosage. Therefore, on the basis of previous experiments, we optimized the expression system, provided the extracellular secretory expression of the protein, and successfully prepared high-purity recombinant protein, which also met the needs of customers.
 

Case 3:SARS-CoV-2-related protein expression
After the outbreak of the new coronavirus in SARS-CoV-2, SGE BIOTECH quickly developed new coronavirus S recombination, ACE2 recombinant protein and other scientific research tools, for the majority of researchers in the new coronavirus research and therapeutic antibodies, vaccine research and development to provide more support.

5. Article Citation
 1. Luo, Y., Liu, S., Xue, J. et al. High-throughput screening of spike variants uncovers the key residues that alter the affinity and antigenicity of SARS-CoV-2. Cell Discov （IF=38）9, 40 (2023).https://doi.org/10.1038/s41421-023-00534-2
(The ACE2 protein used in the experiment provided preparation for SGE BIOTECH  in the article of nature published by Peking University)