The effect of target gene copy number on the efficiency of expressing foreign protein in yeast system

Yeast is a single-celled lower eukaryote with ordinary culture conditions, rapid growth and reproduction, and can tolerate high hydrostatic pressure. It can be mass-produced when used to express genetic engineering products, effectively reducing production costs. 

The vector of the yeast expression system is generally a shuttle plasmid, which belongs to the E. coli-yeast shuttle vector, that is, it is screened and amplified in E. coli and expressed in yeast. In the application, it is necessary to consider the different uses of the E. coli-yeast shuttle vector, the replication mode in yeast and the expression mode of the target gene carried are also different. The copy number of the target gene is an important factor that affects the efficiency of the yeast expression system to express foreign proteins.

Generally, the higher the copy number of the target gene, the higher the expression efficiency. Appropriately increasing the copy number of the target gene integrated into the yeast chromosome can effectively increase the expression of foreign genes. For example, human tumor necrosis factor (TNF), tetanus toxin fragment C and mouse epidermal growth factor (EGF), etc. At present, many researchers regard increasing copy number as an important aspect of increasing expression. The copy number of the foreign gene in the yeast expression system affects the expression level of the foreign gene. 

At present, the commonly used methods to increase the integrated copy number of the foreign gene are: (1) Increase the integrated copy number through different transformation methods; (2) In Insert the target gene fragment multiple times into the in vitro vector; (3) Connect both ends of the gene in the vector to the gene fragment from the host or other unnecessary high repetitions, and achieve the purpose of high copy integration through homologous recombination.

For example, researchers construct multi-copy recombinant expression plasmid of hepatitis E virus (HEV) ORF2128-660 in vitro to increase the expression level of HEVORF2128-660 in Pichia pastoris [1]. The researchers used PCR technology to amplify the target gene ORF2128-660(E), and then the ORF2128-660(E) synonymous point mutation was changed to ORF2128-660 to construct a recombinant expression plasmid ORF2128-660/pAO815 (single copy), BamH and Bgl The target gene expression cassette (AOX-ORF2128-660) obtained by double enzyme digestion was inserted into the dephosphorylated plasmid ORF2128-660/pAO815 to obtain the 2(AOX-ORF2128-660)/pAO815 (2 copies) plasmid and so on. Finally, the researcher successfully constructed the HEVORF2128-660 multi-copy expression plasmid, which improved its expression level in Pichia pastoris.

Yeast expression system is a powerful tool for studying eukaryotic protein expression and analysis. It has post-transcriptional processing and modification functions and is suitable for stable expression of functional foreign proteins. Medicilon researchers have established a mature yeast expression and purification service platform to provide high-quality recombinant protein expression and purification services in Pichia pastoris and Saccharomyces cerevisiae.

However, integrating too many copies of foreign genes will cause the instability of recombinant DNA and sometimes have negative effects. Studies have also found that the target protein expression of some multi-copy genes is not linearly positively correlated with its copy number. Some researchers have analyzed the expression of the multi-copy recombinant strain of Plasmodium falciparum pfs25 in Pichia pastoris, and studied the effect of gene copy number on the expression of Pfs25 protein. They plan to increase the secretion of pfs25 recombinant protein in the Pichia pastoris expression system. Expression [2]. 

The researchers constructed a recombinant plasmid pAO815-αpfs25, using the characteristics of the Bgll-BamHI homotailase, inserted the gene expression cassette AOX1-αpf25-AOX1 (TT) into a single-copy expression plasmid, repeated in turn, and constructed a multi-copy recombinant plasmid pAO815 -(αpfs25 ) n. After linearization, Pichia pastoris GS115 was electroporated, screened with MD plate and analyzed for expression. The researchers successfully obtained a multi-copy expression recombinant strain of the pfs25 gene. After analysis, the target protein expression of the multi-copy pfs25 gene is not linearly positively correlated with its copy number.

Although the copy number of the target gene is one of the important factors affecting the expression level of the foreign protein in the yeast expression system, the effect of the copy number of the target gene on the expression level of the foreign protein will vary depending on the characteristics of the foreign protein, and may be positive. The dose-effect relationship will also have a negative dose-effect relationship. Therefore, in actual research, it is not yet possible to predict the effect of target gene copy number on the expression of foreign protein from a theoretical level, and it needs to be studied through specific experimental work.

[1] Construction of multi-copy recombinant plasmid of hepatitis E virus and its expression in Pichia pastoris [J].

[2] Expression analysis of multiple copies of Plasmodium falciparum pfs25 gene in Pichia pastoris[J].