scholarly journals Identification of a functionally conserved surface region of rat cytochromes P450IA

1991 ◽  
Vol 278 (3) ◽  
pp. 749-757 ◽  
Author(s):  
R J Edwards ◽  
A M Singleton ◽  
B P Murray ◽  
S Murray ◽  
A R Boobis ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Cytochromes P450 ◽  
Surface Region ◽  
Carrier Protein ◽  
Peptide Antigen ◽  
Aryl Hydrocarbon ◽  
C Terminus ◽  
Aryl Hydrocarbon Hydroxylase Activity ◽  
The Difference ◽  
Peptide Antibodies

A region of rat cytochrome P450IA1 at residues 294-301 (Gln-Asp-Arg-Arg-Leu-Asp-Glu-Asn), equivalent to a proinhibitory region of cytochrome P450IA2, was identified by sequence alignment. Anti-peptide antibodies were successfully raised when the peptide was coupled through either its N- or its C-terminus to carrier protein, but no antibodies were produced against the so-called multiple peptide antigen, which consisted of eight copies of the peptide attached through its C-terminus to a synthetic base. Both of the anti-peptide antibodies bound specifically to cytochrome P450IA1 in the rat, as shown by e.l.i.s.a. and immunoblotting. They inhibited microsomal aryl hydrocarbon hydroxylase activity and the mutagenic activation of 2-acetylaminofluorene (these reactions are catalysed by cytochrome P450IA1), but not high-affinity phenacetin O-de-ethylation activity, which is catalysed by cytochrome P450IA2. However, there was differences in the properties of the two antisera in their binding to cytochromes P450IA1 in species other than the rat, their relative binding to the multiple peptide antigen, the yield of antibody following affinity purification using peptide coupled through its N-terminus to CNBr-activated Sepharose, and the binding of the purified preparations to N- and C-terminal-coupled peptide conjugates. These observations indicated that the antibodies were directed to the region of the peptide opposite to the end which was coupled to the carrier protein. Nevertheless, both of the antibody preparations bound equally well to the target cytochrome P450, thus indicating that, in the native protein, the whole of the peptide region is exposed on the surface of cytochrome P450IA1 and is available for binding by the antibodies. The role of this region appears to be the same in both cytochromes P450IA1 and P450IA2, despite the difference in its primary structure in the two cytochromes P450.

scholarly journals Peptides as antigens. Importance of orientation.

1986 ◽  
Vol 164 (4) ◽  
pp. 1344-1349 ◽  
Author(s):  
T Dyrberg ◽  
M B Oldstone
Keyword(s):  
Peptide Sequence ◽  
Carrier Protein ◽  
Binding Pattern ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Terminal Amino ◽  
Peptide Characterization ◽  
A Site ◽  
Peptide Antibodies

Factors known to be important in producing protein-reactive peptide antibodies include the accessibility of the region from which the peptide sequence is derived, the hydrophilic-phobic character of the sequence, and the length of the peptide. The data presented here indicate that the orientation of the peptide coupled to a carrier protein also influences the binding pattern of peptide antibodies. An octapeptide, representing a sequence from the alpha chain of the human acetylcholine receptor, was coupled either through an N- or C-terminal cysteine-glycine-glycine linker to a carrier protein and used to immunize rabbits. The resulting antisera reacted at comparable titers to the uncoupled immunizing peptides, but did not crossreact with the identical but opposite-linked peptide. Characterization of the binding to other homologous peptides showed that immunization with the N-terminal-linked peptide induced antibodies reactive specifically with the C-terminal amino acid(s). Immunization with the C-linked peptide resulted in antibodies reactive with a site of the peptide near the C-terminus.

Affinity Tags for Protein Purification

2020 ◽  
Vol 21 (8) ◽  
pp. 821-830
Author(s):  
Vibhor Mishra
Keyword(s):  
Protein Purification ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein Domain ◽  
Affinity Tag ◽  
Small Peptides ◽  
Affinity Tags ◽  
C Terminus ◽  
Solubility Enhancers ◽  
As Solubility

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. Additionally, some affinity tags also serve a dual purpose as solubility enhancers for challenging protein targets. By applying a combinatorial approach, carefully chosen affinity tags designed in tandem have proven to be very successful in the purification of single proteins or multi-protein complexes. In this mini-review, the key features of the most commonly used affinity tags are discussed. The affinity tags have been classified into two significant categories, epitope tags, and protein/domain tags. The epitope tags are generally small peptides with high affinity towards a chromatography resin. The protein/domain tags often perform double duty as solubility enhancers as well as aid in affinity purification. Finally, protease-based affinity tag removal strategies after purification are discussed.

scholarly journals The Differential Effects of pp120 (Ceacam 1) on the Mitogenic Action of Insulin and Insulin-Like Growth Factor 1 Are Regulated by the Nonconserved Tyrosine 1316 in the Insulin Receptor

2000 ◽  
Vol 20 (11) ◽  
pp. 3896-3905 ◽  
Author(s):  
Payal Soni ◽  
Montaha Lakkis ◽  
Matthew N. Poy ◽  
Mats A. Fernström ◽  
Sonia M. Najjar
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Site Directed Mutagenesis ◽  
Insulin Like Growth Factor ◽  
Β Subunit ◽  
Conserved Domain ◽  
C Terminus ◽  
Mitogenic Action ◽  
The Difference ◽  
Differential Phosphorylation

ABSTRACT pp120 (Ceacam 1) undergoes ligand-stimulated phosphorylation by the insulin receptor, but not by the insulin-like growth factor 1 receptor (IGF-1R). This differential phosphorylation is regulated by the C terminus of the β-subunit of the insulin receptor, the least conserved domain of the two receptors. In the present studies, deletion and site-directed mutagenesis in stably transfected hepatocytes derived from insulin receptor knockout mice (IR−/−) revealed that Tyr1316, which is replaced by the nonphosphorylatable phenylalanine in IGF-1R, regulated the differential phosphorylation of pp120 by the insulin receptor. Similarly, the nonconserved Tyr1316 residue also regulated the differential effect of pp120 on IGF-1 and insulin mitogenesis, with pp120 downregulating the growth-promoting action of insulin, but not that of IGF-1. Thus, it appears that pp120 phosphorylation by the insulin receptor is required and sufficient to mediate its downregulatory effect on the mitogenic action of insulin. Furthermore, the current studies revealed that the C terminus of the β-subunit of the insulin receptor contains elements that suppress the mitogenic action of insulin. Because IR−/− hepatocytes are derived from liver, an insulin-targeted tissue, our observations have finally resolved the controversy about the role of the least-conserved domain of insulin and IGF-1Rs in mediating the difference in the mitogenic action of their ligands, with IGF-1 being more mitogenic than insulin.

Aryl Hydrocarbon Hydroxylase Activity “Of Mice and Humans”

1983 ◽  
pp. 179-197 ◽  
Author(s):  
R. E. Kouri ◽  
R. A. Lubet ◽  
C. E. McKinney ◽  
G. M. Connolly ◽  
D. W. Nebert ◽  
...  
Keyword(s):  
Aryl Hydrocarbon Hydroxylase ◽  
Hydroxylase Activity ◽  
Aryl Hydrocarbon ◽  
Aryl Hydrocarbon Hydroxylase Activity
Sign in / Sign up

Export Citation Format

Share Document