1. Expression and characterization of recombinant nerve growth factor
- Author
-
Luo, Yuling
- Subjects
- Chemistry, Biochemistry, Recombinant nerve growth factor (NGF)
- Abstract
Recombinant mouse β-nerve growth factor was expressed in bacterial and baculovirus systems. Expression of the recombinant NGF in a bacterial gene fusion system reached a high level of over 4 mg from one liter of bacterial cell culture. The recombinant NGF had the same molecular weight and shared the same N-terminal amino acid sequences as that of the native mouse β-NGF (mNGF). The recombinant NGF was immunoreactive to anti-NGF antiserum and was biologically active. However, the protein did not have the same specific biological activity as that of the native mNGF, indicating that the recombinant NGF did not completely fold into a native conformation. The problems of folding of the recombinant NGF were at least partially attributed to interchain disulfide formation between NGF monomers. Recombinant NGF was also expressed in a baculovirus system to a level of 1 mg from one liter of insect cell culture. Isolation of the recombinant NGF (rNGF) indicated that it was present in three stable, yet distinct forms. The three forms of rNGF exhibited distinct biological activities and differed in their abilities to compete with mNGF for high affinity NGF receptors. However, they were chemically and structurally indistinguishable from each other. All three forms of rNGF differed from mature mNGF from mouse submaxillary gland in that the C-terminal Arg-Gly dipeptide had not been proteolytically removed. Removal of the C-terminal dipeptide by γ-NGF peptidase treatment converted the three forms into a single form identical to mature mNGF. The above results demonstrate that a single polypeptide of rNGF, due to the presence of a C-terminal dipeptide, exhibits three stable dimeric protein conformations with distinct biological activities. The apparent lack of γ-NGF peptidase in the nervous system raises the possibility that the biologically significant form of NGF may differ from mature mNGF; such a difference may be of physiological relevance. Several NGF mutants from bacterial and baculovirus systems were expressed, purified, and partially characterized. The mutant NGFs (W21L, W21R, G33V, NGF-NT3) were all biologically active, indicating that the residues investigated are neither essential for the biological activity nor for the neuronal specificity of NGF
- Published
- 1992