The Construction Of Mathematical Model For High-level Expression Of Foreign Genes In PPIC9 Vector And Its Verification

Numerous heterologous proteins have been successfully expressed in the methylotrophic yeast, Pichia pastoris. However, we still cannot predict the expression level of heterologous genes before experiments because many factors are involved in affecting the expression level. Here we report a mathematical model for high-level expression using 40 heterologous genes expressed in pPIC9 vector as the samples, and the results showed the 3'-UTR stable local secondary structure is the necessary condition for high-level expression of heterologous genes in pPIC9 vector. To verify this model, human neutrophil gelatinase-associated lipocalin (NGAL) gene was expressed in pPIC9 in pichia pastoris with high-level expression more than 100 mg/L. The model has the following three potential applications. The first is to predict the expression level of heterologous genes before experiments. The second is to optimize the experiment designs to obtain the high level expression. The third is that the conclusions can be referenced qualitatively for other vectors in yeast expression system. Materials and methods:1. Forty cases of foreign genes expressed in pPIC9 vector in Pichia pastoris were collected as samples, the following information were recorded: the gene name, the expression level, the site of the heterologous gene inserted, and the primers. The 40 heterologous genes are classified into two groups: high-level expression group (HEG) with expression level more than 100 mg/L, and low-level expression group (LEG) with expression level less than 100 mg/L.2. The nucleic acid sequences of the sample genes were retrived from the NCBI, and aligned to the 3' primer to verify the 100bp sequence before and after' translation stop codon, after then a 200bp segment was extracted for each sample.3. Based on the 200bp segment of each sample, we extracted 9,000 intervals [X, Y], where 1≤ X≤100, and 111 ≤Y≤ 200.4. The RNAfold program was used to calculate the secondary structure free energy of each interval with temperature parameter set as 30℃, and a minimum free energy matrix (MFEM, or profile of RNA secondary structure)...