VGF fusion polypeptides
a technology of fusion polypeptides and polypeptides, applied in the field of vgf polypeptides and selective binding agents, can solve problems such as the imbalance of energy intak
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example 1
Production of Anti-VGF Polypeptide Antibodies
[0262] Antibodies against VGF polypeptides were produced by injecting rabbits with VGF polypeptides synthesized by the Amgen Boulder Peptide Technology group using standard peptide synthesis protocols. Following peptide synthesis, VGF polypeptides from different batches were pooled and lyophilized. The peptide content for each VGF polypeptide was determined by hydrolysis with HCl.
[0263] To prepare polyclonal anti-VGF polypeptide antibodies from VGF polypeptides (VGF-1; SEQ ID NO: 1 or VGF-2; SEQ ID NO: 4; Table I) lacking cysteine residues, 5 mg of a VGF polypeptide and 0.5 ml of Imject® EDC Conjugation Buffer (Pierce, Rockford, Ill.) were transferred to a 2 ml vial, and the mixture was vortexed. A 20 mg vial of Imject® Keyhole Limpet Hemocyanin (Pierce) was diluted in 2 ml sterilized water, and 0.5 ml of this solution was added to the VGF polypeptide solution. Following the addition of Keyhole Limpet Hemocyanin, 0.1 ml of EDC (10 mg of...
example 2
Biological Activity of VGF Polypeptide in Knockout Mice
[0269] The biological activity of VGF polypeptides in knockout mice was analyzed using VGF polypeptides synthesized by the Amgen Boulder Peptide Technology group using standard peptide synthesis protocols. Following peptide synthesis, VGF polypeptides from different batches were pooled and lyophilized. The peptide content for each VGF polypeptide was determined by hydrolysis with HCl.
[0270] VGF gene knockout mice were obtained from Steve Salton of Mt. Sinai School of Medicine, New York, N.Y. Prior to administration of VGF polypeptide, knockout mice were housed under LD 12:12 conditions (i.e., 12 hours of light and 12 hours of dark). Twenty-four hours before VGF polypeptide injection, knockout mice were individually caged and moved to a temporary housing room where the mice were kept for the remainder of the experiment under LD 12:12 conditions.
[0271] VGF polypeptide was administered to knockout mice twice daily (at 9:00 a.m. ...
example 3
VGF Polypeptide-Fc Fusion Proteins
[0273] VGF polypeptides are expressed as fusion proteins with a human immunoglobulin IgG heavy chain Fc region at their carboxyl- or amino-terminus. Using appropriate PCR primers and standard PCR amplification procedures, VGF polypeptide-Fc fusion constructs are prepared by fusing in-frame a DNA sequence coding for the Fc region of human IgGI with that encoding VGF-1 (SEQ ID NO: 1), VGF-1b (SEQ ID NO: 3), or VGF-2 (SEQ ID NO: 4) polypeptide.
[0274] VGF polypeptide-Fc fusion products generated by PCR are digested with the restriction endonucleases Nde I and Bam HI, ligated into the vector pAMG21, and then transformed into competent E. coli strain 2596 cells using standard techniques. Clones are screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence.
[0275] Bacterial cultures containing the Fc fusion constructs in the E. coli strain GM221 are grown at 37° C. in Luria B...
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