Recombinant Expression And Fermentation Of A Novel Human Lysozyme Homologous Protein LYC1 In Pichia Pastoris

Lysozyme(full name:1,4-β-N-acetylmuramide glycanohydrolase or muramidase) distributes widely in nature. As a defensive effector in innate immunity, lysozyme plays an important role in anti-pathogen mechanism during infection. Lysozymes can be classified in to six major types:chicken-type lysozyme(c-type), goose-type lysozyme(g-type), invertebrate lysozyme(i-type), plant lysozyme, bacterial lysozyme, and phage lysozyme(p-type), according to their species source, structure and molecular weight. As the most widely distributed type and the only type found both in vertebrates and invertebrates, c-type lysozyme is the most studied type.LYC1 (human lysozyme C1), or LYZL6(lysozyme like 6) is a novel gene discovered and cloned by our laboratory belonging to the c-type lysozyme/alpha-lactalbumin family. The overall identity of LYC1 with H-LYZ in amino acid sequence is 44%, and the featured eight cysteine residues in all members of c-type lysozyme and the two essential catalytic residues at position 35(Glu) and 52(Asp) are conserved in LYC1.In this study, we clone synthetic gene sequence of LYC1 with its own signal peptides removed according to its amino acid sequence and the condon preference of Pichia pastoris into yeast vector pPiC9k, leaded by yeast a-factor signal peptides for secreted expression of LYC1 with native N terminus. The linearized recombinant plasmid was transducted into Pichia pastoris strain GS115 and SMD1168 and integrated into Pichia genome in specific locus. The transformants underwent screening for Mut+and Muts phenotype and screening of multiple inserts. Transformants with higher recombinant protein ability were then screened through culture and induction in baffled flask. Selected recombinant strains were then fermented in 30L fermenter. Fermentation parameters such as temperature, dissolved oxygen, agitation, anti-foam and carbon source were monitor and controlled throughout the fermentation process and optimized in order to get higher recombinant protein expression level.SDS-PAGE of the fermentation supernatant revealed inconformity of the expressed protein, which might be due to degradation, glycosylation or aggregation. We performed MS analysis and determined the presence of expressed LYC1. Besides, the expression profiles of SMD1168 recombinant strains are better than GS115, possibly because GS115 suffered more severe recombinant protein degradation. Optimizing fermentation parameters increased expression to some extent.Lysozyme activity was detected in the fermentation supernatant of recombinant SMD1168. However, the activity is weak compared to H-LYZ. This might be resulted from the relatively low expression level of LYC1, degradation or glycosylation, and that LYC1 have different substrate specificity and optimal reaction condition from H-LYZ considering their difference in pI. It is also possible that LYC1 plays other potentially more important roles except for antibacterial activity, which will be further studied.