Expression of a synthetic gene coding for ostrich egg-white lysozyme in Pichia pastoris and its enzymatic activity.
To investigate the structure-function relationships of goose-type lysozyme, a gene coding for ostrich egg-white lysozyme (OEL) was designed based on the published amino acid sequence and constructed by assembling 32 chemically synthesized oligonucleotides. To obtain the recombinant OEL (rOEL), the synthetic gene was fused to the alpha-factor signal peptide in the expression vector pPIC9K and expressed in the methylotrophic yeast Pichia pastoris. The secreted protein from the transformed yeast was found to be processed at three different sites, including the correct site. The correctly processed rOEL was purified to homogeneity and shown to be indistinguishable from the authentic form in terms of circular dichroism (CD) spectrum and enzyme activity. Furthermore, the time-course of the reaction catalyzed by OEL was studied using (GlcNAc)(n) (n = 5 and 6) as the substrate and compared to that of goose egg-white lysozyme (GEL) [Honda and Fukamizo (1998) BIOCHIM: Biophys. Acta 1388, 53-65]. OEL hydrolyzed (GlcNAc)(6) in an endo-splitting manner producing mainly (GlcNAc)(2), (GlcNAc)(3), and (GlcNAc)(4), and cleavage to (GlcNAc)(3) + (GlcNAc)(3) predominated over that to (GlcNAc)(2) + (GlcNAc)(4). This indicates that OEL hydrolyzes preferentially the third glycosidic linkage from the nonreducing end of (GlcNAc)(6) as in the case of GEL. The cleavage pattern seen for (GlcNAc)(5) was similar to that seen for (GlcNAc)(6). Theoretical analysis of the reaction time-course for OEL revealed that the binding free energy values for subsites B, E, and G were different between OEL and GEL, although these lysozymes were estimated to have the same type of subsite structure.
Kawamura
Shunsuke
S
Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404. skawa@ktmail.ktokai-u.ac.jp
Fukamizo
Tamo
T
Araki
Tomohiro
T
Torikata
Takao
T
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis; D020310:Struthioniformes
D004527:Egg Proteins; D011994:Recombinant Proteins; D009113:Muramidase; D000117:Acetylglucosamine