Functional expression of porcine interferon-α using a combinational strategy in Pichia pastoris GS115

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc

“…The P. pastoris GS115 strain was used for the heterologous expression. The plasmid pHBM905M was used as expression vector to carry ChiA gene, which originally constructed in our group [16]. The plasmid pGAPZB from Invitrogen was used to express HAC1, ERV29, SEC16, COG5 and TRM1 constitutively.…”

“…In our previous job, the pHBM905M vector used in this study was constructed based on the pHBM905A plasmid [14] with four major modifications, including the replacement of original AOX1 promoter by d1 + 2 × 201 AOX1 promoter, the replacement of original MFα signal peptide by MF4I-SS signal peptide, and the removal of kanamycin resistance gene by the insertion of heterologous gene during the plasmid construction. In addition, pHBM905M can use the Biobrick method to generate a multi-copy expression cassette in vitro [15,16]. Moreover, there is no exogenous resistance genes in the recombinant P. pastoris, which decreased the spread of antibiotics resistance genes to the environment.…”

A Combinational Strategy for Effective Heterologous Production of Functional Human Lysozyme in Pichia pastoris

“…Different from the conventional method of selecting recombinant strains with multiple genes integrated using antibiotics, such as Zeocin and G418, we adopted the in vitro gene-expression-cassette multimerization method previously reported by us to assemble multiple hLYZ expression cassettes (He et al, 2019). Using this method, we assembled up to 12 gene expression cassettes and integrated them into the P. pastoris genome (Figure 2).…”

“…The optimized human lysozyme gene sequence (opt-hLYZ) was uploaded to the GenBank database (GenBank accession number: MN175974) and synthesized by Sangon Co., Ltd. (Shanghai, China). Then, the opt-hLYZ gene was inserted into the plasmid p905M between CpoI/NotI restriction sites to construct the recombinant p905M-opt-hLYZ-1C plasmid based on a previously reported method (He et al, 2019). Subsequently, plasmids containing 3, 6, and 12 copies of opt-hLYZ (p905M-opt-hLYZ-3C, p905M-opt-hLYZ-6C, p905M-opt-hLYZ-12C) were constructed based on p905M-opt-hLYZ-1C plasmid using XbaI/BamHI and SpeI/BamHI restriction sites.…”

“…Optimizing the gene codon according to the P. pastoris genetic preference and increasing the target gene copies in P. pastoris chromosome have been proven as two efficient methods for heterologous protein production in P. pastoris (Shu et al, 2016;He et al, 2019). Moreover, whether a protein can be well folded determines its secretory efficiency in P. pastoris (Idiris et al, 2010;Young and Robinson, 2014) because misfolded proteins in cell ER could induce an unfolded protein response (UPR) and endoplasmic reticulumassociated degradation (ERAD) (Ahmad et al, 2014).…”