TAILIEUCHUNG - Báo cáo khoa học: Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris

We report the secreted expression byPichia pastorisof two human lyso-zyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. | ềFEBS Journal Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris Janet R. Kumita1 Russell J. K. Johnson1 Marcos J. C. Alcocer2 Mireille Dumoulin1 Fredrik Holmqvist3 Margaret G. McCammon1 Carol V. Robinson1 David B. Archer3 and Christopher M. Dobson1 1 Department of Chemistry University of Cambridge UK 2 Schoolof Biosciences University of Nottingham Loughborough UK 3 Schoolof Biology University of Nottingham UK Keywords amyloidosis lysozyme protein degradation protein folding protein secretion Correspondence C. M. Dobson Department of Chemistry Lensfield Road University of Cambridge Cambridge CB2 1EW UK Fax 44 1223 763418 Tel 44 1223 763070 E-mail cmd44@ Received 4 November 2005 revised 9 December 2005 accepted 12 December 2005 doi We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R associated with systemic amyloid disease and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower respectively than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins under controlled conditions can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly our results show that the human .

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