Cysteine to serine substitutions in basic fibroblast growth factor: effect on inclusion body formation and proteolytic susceptibility during in vitro refolding.
We have investigated the effect of cysteine to serine substitutions in human basic fibroblast growth factor (bFGF) on the formation of inclusion bodies in Escherichia coli. Using a temperature-sensitive expression, system, about 30% of human bFGF, which contains four cysteines at positions 26, 70, 88, and 93, is deposited into inclusion bodies. A single mutation at position 88 and a double mutation at positions 70 and 88 do not greatly alter the partition of bFGF into soluble and insoluble cell fractions. However, a single substitution of cysteine 70 by serine decreases the fraction of soluble bFGF significantly. When cysteines 26 and 93 (conserved among related growth factors) are replaced by serines, no soluble bFGF is formed in E. coli. Cysteine to serine substitutions also affect proteolytic susceptibility of bFGF during in vitro refolding from crude inclusion bodies. About 60% of human bFGF is lost to proteolytic degradation during in vitro refolding. Replacement of cysteines by serines increases the total recovery of bFGF, although more aggregates are formed during refolding. Ser-88-bFGF was expressed at the highest level, gave the highest soluble fraction in vivo, and exhibited the greatest fractional recovery and was recovered with the largest insoluble fraction after in vitro refolding. Thermal stability experiments at 42 degrees C and 70 degrees C revealed that cysteine to serine substitutions did not cause aggregation of the folded protein in vitro.
Rinas
U
U
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
Tsai
L B
LB
Lyons
D
D
Fox
G M
GM
Stearns
G
G
Fieschko
J
J
Fenton
D
D
Bailey
J E
JE
D001483:Base Sequence; D002458:Cell Fractionation; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation / Q000235:genetics; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D010447:Peptide Hydrolases; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry* / Q000235:genetics; D012995:Solubility
D016222:Fibroblast Growth Factor 2; D012694:Serine; D010447:Peptide Hydrolases; D003545:Cysteine