Strategy and implementation of a system for protein engineering.
This paper describes an overall view of an industrial protein engineering project from conception to successful completion. The choice of rational design was determined by the availability of an excellent three-dimensional crystal structure and the availability of information in the literature to define a strategy. The design strategy was refined extensively during the course of the project. The development of methods for mutagenesis, expression, verification, purification, and characterization of mutant enzymes is dictated in part by the enzyme property one chooses to modify and must be rapid yet accurate. Such an approach would be applicable to improve the stability of any other protein or enzyme. Using this approach, we successfully increased the stability of subtilisin BL over 10-fold at 50 degrees C with an overall success rate greater than 60%.
Goddette
D W
DW
COGNIS Inc., Santa Rosa, California 95407.
Christianson
T
T
Ladin
B F
BF
Lau
M
M
Mielenz
J R
JR
Paech
C
C
Reynolds
R B
RB
Yang
S S
SS
Wilson
C R
CR
D001407:Bacillus / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D004795:Enzyme Stability; D007218:Industrial Microbiology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D013381:Subtilisins / Q000378:metabolism
D003902:Detergents; D012697:Serine Endopeptidases; D013381:Subtilisins; C032923:microbial serine proteinases