Improved neurturin molecules
A technology of neurotrophic factors and polynucleotides, applied in the direction of growth factors/inducing factors, neuregulins, nervous system diseases, etc., can solve the problems that the regions are not fully conserved and cannot be accurately inferred
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0268] Example 1: Molecular Modeling
[0269] Preliminary analysis of the primary sequence of neurotrophic factor reveals a segment of residue (RRLRQRRRLRRER) (SEQ.ID.NO 31) in the heel region of neurotrophic factor, and its consensus sequence for binding to heparin is especially consistent with the sequence "BBXB" or "BBBXXB ", where B is a basic amino acid and X is any residue (Hileman et al. (1998) Bioessays. 20(2):156-67). Although the identification of this sequence is consistent with heparin binding, it should be noted that the findings of this consensus sequence in protein primary structure do not imply that this sequence is necessarily involved in heparin binding. In particular, it has been established that i) proteins lacking these consensus sequences can still bind heparin (Delacoux et al. (2000) J. Biol. Chem. 275(38):29377-82), and ii) contain this motif The protein may not bind heparin.
[0270] Indeed, for all proteins, the ability of consecutive arrays of str...
Embodiment 2
[0276] Example 2: Generation of Neurotrophic Factor Variants
[0277] DNA encoding human neurotrophic factor corresponding to accession number BC137399 was purchased from OpenBiosystems. The mature sequence of neurotrophin (excluding its endogenous signal sequence and pro-sequence) was subcloned into vector pSJP-2 whose vector backbone was based on pAAV-MCS (Stratagene ), which encodes an IgG signal sequence (Fjord-Larsen et al., (2005) "Efficient in vivo protection of nigral dopaminergic neurons by lentiviral gene transfer of a modified Neurturin construct (modified neurotrophic factor construct by lentiviral gene transfer Efficient in vivo protection of substantia nigra dopaminergic neurons)." Exp Neurol 195:49-60), followed by the generation of mature neurotrophic factor sequences without the prosequence. E778 was used as template for inverse PCR mutagenesis. Primers for untagged neurturin variant N1 were: F / 5'-ag gcg cgg gcc ctg cgg cgg gag cgggtg cgc-3' (SEQ ID NO: 11...
Embodiment 3
[0279] Example 3: Production and purification of neurotrophic factor variants
[0280] CHO cells were transiently transfected with E436(GFP), V5-tagged wild-type neurturin, or V5-tagged neurturin variants NV1-NV4. Transfection was performed using Turbofect (Fermentas) according to the manufacturer's instructions. After four hours, the medium containing the transfection reagent was replaced with normal medium consisting of DMEM (Sigma), 10% FBS (HyClone), 100 U / ml penicillin (Gibco) and 100 μg / ml streptomycin (Gibco) . After two days, the medium was collected from each plate. To pellet floating cells, the medium was centrifuged (5 minutes, 16,200 xg) before use. Harvested samples were loaded on 15% SDS-PAGE and analyzed by Western blotting with V5-antibody (Invitrogen). All variants were successfully expressed and secreted into the medium, and showed correct predicted molecular weights on SDS-PAGE (data not shown).
PUM
| Property | Measurement | Unit |
|---|---|---|
| Molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More