Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system.
Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion. Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.
Koganesawa
N
N
Division of Biological Sciences, Graduate School of Science, Faculty of Agriculture, Hokkaido University, Sapporo 060-0810, Japan.
Aizawa
T
T
Masaki
K
K
Matsuura
A
A
Nimori
T
T
Bando
H
H
Kawano
K
K
Nitta
K
K
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D015870:Gene Expression; D005822:Genetic Vectors; D005838:Genotype; D006358:Hot Temperature; D006801:Humans; D000072235:Mating Factor; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010641:Phenotype; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000072235:Mating Factor; D009113:Muramidase