Studies On Optimization Of PAP Codons And The Expression Of PAP In Pichia Pastoris With High Efficiency

Pokeweed antiviral protein (PAP) belongs to Ribosome-inactive Proteins (RIPs). PAP has potent antiviral activity against many plant viruses. Poor expression has been reported as a character of wild type PAP in Pichia pastoris. To increase expressing quantity of PAP gene and obtain high yield of P.pastoris, codon usage of wild type PAP gene is optimized and modified by P.pastoris preferred codons, so that elements of instable sequence is regulated or removed purposefully for efficient expression. The main results are summarized as follows:1. For the purpose of obtaining transgenic P. pastoris, PAP gene (GeneBank:AF338910, encoding a truncated mutant pokeweed antiviral protein. The protein can restrain TMV availably.) is designed and synthesized. Modification and optimization of nucleotide sequence of PAP gene do not alter the amino acid sequence of the PAP. The potential processing sites, which affect levels of transcription, translation and conducing mRNA unstability of PAP, were fully motifided, such as "AT" rich sequence, ATTTA sequence, and so on. We add Snab I restriction enzymes site in 3' tail end and Not I restriction enzymes site and terminator in 5' tail end, respectively.2. In this study, PAP gene is designed according to codon preferring of P. pastoris, and is chemically synthesized by using successive PCR. There are 74.1% of codons in wild type gene unsuitable for expressions in P. pastoris, which are modified to P. pastoris favorable codons without amino acid change. Comparing with the wild type PAP gene, the ratio of G+C content, which is close to P. pastoris', is increased from 38.7% of wild type to 45.1% of the synthetic gene. The synthetic gene is cloned into T-easy vector and is sequenced. Results of sequencing for recombinant plasmid are correct completely. The plasmid was named T-easy-PAPs.3. The newly synthesized gene is cloned into yeast expression vector PIC9k, in which the expression of PAP gene is under the control of AOX1 promoter. The recombinant plasmid is named PIC9k-PAPs. The vector includes a α-Factor secretion signal, a multiple cloning site that contains 5 sites and two screening marker.4. The expression vector pPIC9K-PAPs including the truncated mutant pokeweed antiviral protein (PAP) gene and blank vector pPIC9k are linearized by restriction enzyme Sal I , and then are transformed electrically into cells of strain P.pastoris GS115. Mut~+and Mut~s recombinants are selected by PCR.5. We proved that Mut~+ recombinant has antivirus activity. Western-blot result shows that there is a remarkable hybridize signal between expression produce and PAP antiserum.