Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems.
Candida antarctica lipase B (CALB) carrying a point mutation, N74S, resulting in a non-glycosylated protein was actively expressed in Pichia pastoris yielding 44 mg/L which was similar to that of the glycosylated CALB wild type expressed in P. pastoris. Hence, the major obstacle in the Escherichia coli expression of CALB is not the lack of glycosylation. To understand and improve the expression of CALB in E. coli, a comprehensive investigation of four different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3) and Origami 2(DE3) as well as co-expression with chaperones groES and groEL in Origami B(DE3), all using the pET-22b(+) vector and the T7lac promoter. Furthermore the E. coli expression was carried out at three different temperatures (16, 25 and 37 degrees C) to optimise the expression. Periplasmic expression resulted in highest amount of active CALB of the four systems, yielding a maximum of 5.2mg/L culture at 16 degrees C, which is an improvement to previous reports. The specific activity of CALB towards tributyrin in E. coli was found to be the same for periplasmic and cytosolic expression. Active site titration showed that the CALB mutant N74S had a lower specific activity in comparison to wild type CALB regardless of expression host. The expected protein identity was confirmed by LC-ESI-MS analysis in E. coli, whereas in P. pastoris produced CALB carried four additional amino acids from an incomplete protein processing.
Larsen
Marianne Wittrup
MW
Department of Biochemistry, School of Biotechnology, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden.
Bornscheuer
Uwe T
UT
Hult
Karl
K
D002175:Candida / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D005656:Fungal Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica