title: 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.
authors.affiliation: Centre for Biotechnology, Anna University, Chennai 600 025, India.
authors.forename: Premsingh
authors.initials: P
authors.lastname: Samuel
authors.forename: Ashok Kumar
authors.initials: AK
authors.lastname: Prasanna Vadhana
authors.forename: Ramakrishnan
authors.initials: R
authors.lastname: Kamatchi
authors.forename: Aju
authors.initials: A
authors.lastname: Antony
authors.forename: Sankaranarayanan
authors.initials: S
authors.lastname: Meenakshisundaram
mesh_terms: D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica
keywords: CalB; Chaperones; Kar2p; P. pastoris; Ydj1p