Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS.
Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.
Ezure
Toru
T
Life Science Laboratory, Analytical and Measuring Instruments Division, Shimadzu Corporation, Kyoto, Japan.
Suzuki
Takashi
T
Shikata
Masamitsu
M
Ito
Masaaki
M
Ando
Eiji
E
Nishimura
Osamu
O
Tsunasawa
Susumu
S
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007313:Insecta / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin
D004220:Disulfides; D000469:Alkaline Phosphatase; D009113:Muramidase; D014357:Trypsin