Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris

Abstract

One of the reasons for limited heterologous protein secretion in Pichia pastoris is the suboptimal folding conditions inside the cell. The Hsp70 and Hsp40 chaperone families in the cytoplasm or the ER regulate the folding and secretion of heterologous proteins. Here, we have studied the effect of chaperones Ydj1p, Ssa1p, Sec63p and Kar2p on the secretory expression of Candida antarctica lipase B (CalB) protein. Expression of CalB in P. pastoris resulted in the induction of Kar2p secretion into the medium surpassing the retrieval capacity of the cell. Individual overexpression of Ydj1p, Ssa1p and Sec63p in recombinant P. pastoris increased CalB expression level by 1.6-, 1.4- and 1.4-fold respectively compared to the control strain harboring only the CalB gene. However, overexpression of Kar2p had a negative effect on the expression of CalB. Moreover, Western blot analysis indicated accumulation and secretion of Kar2p in the ER, Golgi and extracellular medium in the chaperone coexpression strains. When expressed in combinations such as Ydj1p-Ssa1p, Ydj1p-Sec63p, Kar2p-Ssa1p, Kar2p-Sec63p, the expression level of CalB was increased by 2.5-, 1.5-, 1.5- and 1.5-fold respectively. Contrastingly, the Kar2p-Ydj1p combination resulted in decreased CalB secretion in the supernatant. From these results, we conclude that overexpression of Kar2p is not required for the secretion of CalB. Also, our work confirmed the synergistic effect of Ssa1p and Ydj1p chaperones in the expression of CalB.

Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures

“…PDI is responsible for disulfide exchange reactions in the ER helping to rearrange incorrect disulfide pairings [15]. Both proteins as well as Hac1(p) have been co-overproduced in P. pastoris for enhanced secretion of target proteins with mostly unpredictable and varied success [3,12,16-20]. …”

Different construction strategies affected on the physiology of Pichia pastoris strains highly expressed lipase by transcriptional analysis of key genes

“…Some studies have found that overexpression of protein folding-related genes (PDI, ERO1, KAR2, IMH1, SEC31) increases expression levels of foreign proteins [14,28,36]. On the other hand, Samuel et al reported that expression of KAR2 gene in P. pastoris caused a 0.7-fold reduction of Candida antarctica lipase B extracellular expression level [37]. It is also indicated that the restriction factors of different strains are different, and the strategies adopted should also be different.…”

Proteomic Analysis and qRT-PCR Verification of Temperature Response to Arthrospira (Spirulina) platensis

“…Only those proteins that have surpassed the proofreading and quality control check points in the pathway are secreted. The partner proteins bind to nascent peptide chains thereby stabilizing and directing them for translocation into the ER [38]. …”