Antibodies are immunoglobulins produced by B lymphocytes and can participate in humoral immunity. They are composed of variable domains, light chain variable regions (VL) and heavy chain variable regions (VH). Recombinant antibodies recognize target antigens through their variable fragments. An antibody lacking a light chain was found in the serum of camelids such as camels and alpacas. It has a simple structure and still has antibody activity in the absence of Fc. In the 1980s, BIrd et al. designed a single-chain antibody (scFv) as a genetic fusion of VL and VH, which links VL and VH through a short flexible peptide. scFv is the smallest binding unit with antibody activity, also known as a nanobody. Because it lacks an Fc domain, it is very small, about 25 kDa. Compared with full-length antibodies, it is easier to penetrate into tumor tissues and can be used in targeted cancer therapeutic drugs. At the same time, it also plays an important role in detection reagents.

Recombinant Protein Yeast Expression System:

The structure of recombinant antibodies is complex, and most of them require post-translational modification, so they are suitable for expression in eukaryotic expression systems, but the cost of large-scale antibody preparation is high. Single-chain antibodies are small in size and simple in structure. They can be expressed in a variety of recombinant single-chain antibody expression systems and do not require post-translational modification, making them suitable for large-scale production. Commonly used recombinant antibody expression systems include Escherichia coli expression system, mammalian cell expression system, insect cell expression system, and yeast expression system. Among them, the yeast expression system can perform relatively complete eukaryotic expression and is relatively simple to operate. Expression using Pichia pastoris will not accumulate ethanol and can achieve the same degree of glycosylation as mammalian cells. When using the yeast expression system, the plasmid should be linearized and integrated into the genome so that the exogenous gene can exist stably. Yeast expression vectors are selected according to the purpose of gene expression. Yeast expression system vectors can be divided into yeast intracellular expression vectors and yeast extracellular expression vectors. Among them, yeast intracellular expression vectors include DGAP series, pPIC3, and 5K. Yeast intracellular expression vectors can express the target protein intracellularly to avoid excessive glycosylation of yeast. They are suitable for expressing non-glycosylated proteins in the cytoplasm. Yeast intracellular expression vectors have the characteristics of high expression levels, but the subsequent antibody purification process is relatively complicated. The yeast extracellular expression vector commonly used is pPIC9K, which can secrete the target protein to the extracellular space, and the subsequent antibody purification process is relatively simple.

Pichia pastoris and Saccharomyces cerevisiae are widely used hosts in yeast expression systems. They have the advantages of similar mammalian protein expression systems and the advantages of easy operation of prokaryotic cells. At the same time, the production cost is low and the productivity is high. Pichia pastoris has a fast growth rate, can be cultured at high density, and can achieve intracellular expression and extracellular expression. Since Pichia pastoris can be cultured in protein-free medium, it is easy to carry out subsequent separation and purification. However, the expressed protein will produce mannose residues during the post-translational modification process, resulting in a shorter protein half-life and even immunogenicity. Although the yeast expression system has certain disadvantages, there are still many single-chain antibodies prepared by the yeast recombinant antibody expression system. The antibodies obtained from the yeast recombinant antibody expression system can be used for the development of therapeutic drugs. For example, the single-chain antibody Nb11-59 produced on a large scale in Pichia pastoris can be used for the treatment of new coronaviruses.

Fig. 1 GS115 Pichia yeast expression and identification results of recombinant protein

Design of Single-chain Antibody:

When designing single-chain antibody, VH and VL should be selected according to the specificity of the target antigen, and then the appropriate linker should be selected so that the two can maintain the correct structure. The DNA sequences of VH, VL and Linker are spliced into a complete scFv gene through gene synthesis. The main recombinant antibody expression systems of scFv include prokaryotic expression system and eukaryotic expression system. Different expression vectors, fusion tags, and host bacteria will produce scFv with different activities. The commonly used expression vectors for preparing single-chain antibodies are the PET series with strong promoters and terminators. Phage vectors can also be used as an option, such as pIT2. Affinity purification is performed by adding His tags, and the addition of GST tags can increase the solubility of proteins, but the tags must be removed in subsequent applications. The intracellular localization of scFv can be observed by adding GFP. When preparing antibodies through yeast expression system, the commonly used yeast host cells are Saccharomyces cerevisiae and Pichia pastoris. Among them, Saccharomyces cerevisiae has higher safety, but the protein expression is low, which may cause excessive glycosylation and plasmid loss. It is suitable for some proteins that do not require high glycosylation. Pichia pastoris has strong post-translational modification ability, easy to control expression level, does not produce ethanol, and can produce a large amount of recombinant protein. Therefore, Pichia pastoris is an ideal choice for recombinant single-chain antibody expression and can be widely used in large-scale single-chain antibody preparation.

TekBiotech has rich experience in recombinant protein yeast expression services and can provide customers with protein fermentation services of various scales. With our complete protein purification platform, we can provide customers with high-quality recombinant protein products in a short time. TekBiotech has a complete and mature yeast expression system, providing one-stop services from gene synthesis to yeast protein expression and antibody purification. We have a variety of expression vectors such as pPICZaA, pGAPZaA, pPIC9K, as well as strains such as X33, GS115 and Saccharomyces cerevisiae. Together with large-scale fermentation, we can provide services that meet industrial-grade needs. At the same time, we are equipped with a variety of protein expression and purification methods to meet customers' different protein expression needs and customize complete protein expression and purification solutions for customers.