Pro-sequence assisted folding and disulfide bond formation of human nerve growth factor.
Nerve growth factor (NGF) is a member of the neurotrophin family. These growth factors support neuronal survival and differentiation. Neurotrophins are synthesized as pre-pro-proteins. Whereas the pre-sequences mediate secretion, the function of the pro-peptides is largely unknown. To test the role of the pro-sequence as a folding enhancer, recombinant human pro-NGF (rh-pro-NGF) was produced in Escherichia coli. The oxidative refolding of rh-pro-NGF and rh-NGF was studied using electrospray mass spectrometry (ESIMS) time-course analysis. This analysis permitted both the identification and quantification of intermediates present during the process. The disulfide bonds formed at different times of the refolding processes were characterized by proteolytic digestion followed by matrix assisted laser desorption ionization mass spectrometry (MALDIMS) analysis. Folding yields and kinetics of rh-pro-NGF were significantly enhanced when compared to the in vitro refolding of mature rh-NGF. These results suggest that the pro-sequence of NGF promotes folding of the mature part.
Rattenholl
A
A
Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle/Saale, Germany.
Ruoppolo
M
M
Flagiello
A
A
Monti
M
M
Vinci
F
F
Marino
G
G
Lilie
H
H
Schwarz
E
E
Rudolph
R
R
D000478:Alkylation; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003553:Cystine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D009414:Nerve Growth Factors; D011498:Protein Precursors; D011994:Recombinant Proteins; D003553:Cystine