PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 7765237

The affinity technology in downstream processing.

N Labrou·Y D Clonis
View original source ↗Export metadata ↓

Abstract

The quality criteria imposed on several biochemicals are stringent, thus, high-separation purification technology is important to downstream processing. Affinity-based purification technologies are regarded as the finest available, and each one differs in its purifying ability, economy, processing speed and capacity. The most widely used affinity technology is classical affinity chromatography, however, other chromatography-based approaches have also been developed, for example, perfusion affinity chromatography, hyperdiffusion affinity chromatography, high-performance affinity chromatography, centrifugal affinity chromatography, affinity repulsion chromatography, heterobifunctional ligand affinity chromatography and the various chromatographic applications of 'affinity tails'. On the other hand, non-chromatographic affinity technologies aim at high throughput and seek to circumvent problems associated with diffusion limitations experienced with most chromatographic packings. Continuous affinity recycle extraction, aqueous two-phase affinity partitioning, membrane affinity filtration, affinity cross-flow ultrafiltration, reversible soluble affinity polymer separation and affinity precipitation are all non-chromatographic technologies. Several types of affinity ligands are used to different extents; antibodies and their fragments, receptors and their binding substances, avidin/biotin systems, textile and biomimetic dyes, (oligo)peptides, antisense peptides, chelated metal cations, lectins and phenylboronates, protein A and G, calmodulin, DNA, sequence-specific DNA, (oligo)nucleotides and heparin. Likewise, there are several support types developed and used; natural, synthetic, inorganic and composite materials.

Explore related subjects

Affinity LabelsBase SequenceBiotechnologyChromatography, AffinityDNALigandsMolecular Sequence DataJournal ArticleReview

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

N Labrou, Y D Clonis. 1994-08-15. The affinity technology in downstream processing.. https://doi.org/10.1016/0168-1656(94)90047-7

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed1998

Expression, characterization, and purification of C-terminally hexahistidine-tagged thromboxane A2 receptors.

Thromboxane A2 (TxA2) receptors belong to the class of G-protein-coupled receptors. Knowledge of the relationship of structure to function for TxA2 receptors is limited because of their low levels of expression, lengthy purification procedures and poor recoveries. A C-terminal hexahistidine-tag (C-His) was ligated to the alpha-isoform of TxA2 receptors and expressed in COS-7 and Chinese hamster ovary cells. The C-His-TxA2 receptors bound the radioligands 125I-7-[(1R,2S,3S,5R)-6, 6-dimethyl-3-(4-benzenesulfonylamino)bicyclo[3.1. 1]hept-2-yl]-5(Z)-heptenoic acid, an antagonist, and 125I-[1S-1alpha, 2beta(5Z),3alpha(1E,3S*), 4alpha]-7-[3[(3-hydroxy-4-(4'-phenoxy)-1butenyl)-7-oxabicycl o-[2.2. 1]heptan-2-yl]-5-heptanoic acid, an agonist, with affinities not significantly different from those of the wild type (wt)-TxA2 receptors. LipofectAMINE transfection of the cDNAs resulted in high levels of expression (Bmax = 95 +/- 6 pmol/mg) of the C-His-TxA2 receptors. In competition binding studies the IC50 values of five different ligands were not significantly different between C-His-TxA2 and wt-TxA2 receptors. Agonist-induced stimulation of cAMP and total inositol phosphate formation were not significantly different between the two receptors. Purification on a Ni2+-NTA column resulted in a rapid (within 4 h) purification with a 36 +/- 2% recovery and a 30 +/- 6-fold purification (n = 5). The partially purified receptors were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, dissolved in acetone/trifluoroacetic acid/hexafluoroisopropanol/sinapinic acid, and successfully subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results suggest that the combination of a high level of expression of C-His-TxA2 receptors and a rapid purification procedure followed by SDS- polyacrylamide gel electrophoresis may provide a useful approach for mass-spectrometry based structure-function and other studies of TxA2 receptors.

Affinity Labels↗
PubMed1998

Chimeric constructs between human and rat equilibrative nucleoside transporters (hENT1 and rENT1) reveal hENT1 structural domains interacting with coronary vasoactive drugs.

We have recently isolated cDNAs from human placenta and rat jejunum encoding the prototypic human and rat equilibrative nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporters hENT1 and rENT1. The two proteins (456 and 457 residues, Mr 50,000) are 78% identical in amino acid sequence and contain 11 potential transmembrane segments (TMs) with a large putative extracellular loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. When expressed in Xenopus oocytes, recombinant hENT1 and rENT1 transport both purine and pyrimidine nucleosides, including adenosine, and are inhibited by nanomolar concentrations of NBMPR. hENT1 is also potently inhibited by coronary vasodilator drugs (dipyridamole, dilazep, and draflazine), whereas rENT1 is insensitive to inhibition by these compounds (dipyridamole IC50 values 190 nM (hENT1) and >/=10 microM (rENT1) at 10 microM uridine). In the present study, we have generated reciprocal chimeras between hENT1 and rENT1, using splice sites at residues 99 (end of TM 2) and 231 (end of TM 6), to identify structural domains of hENT1 responsible for transport inhibition by vasoactive compounds. Transplanting the amino-terminal half of hENT1 into rENT1 converted rENT1 into a dipyridamole/dilazep-sensitive transporter, whereas the amino-terminal half of rENT1 rendered hENT1 dipyridamole/dilazep-insensitive. Domain swaps within the amino-terminal halves of hENT1 and rENT1 identified residues 100-231 (incorporating TMs 3-6) of hENT1 as the major site of vasodilator interaction. Since these drugs function as competitive inhibitors of nucleoside transport and NBMPR binding, TMs 3-6 are likely to form part of the substrate-binding site.

Affinity Labels↗
PubMed1998

Evidence for a regulatory binding site for arginine-rich peptides on protein kinase C.

The peptides N-biotinyl-RRRCLRRL and N-biotinyl-RKRCLRRL covalently modify protein kinase C (PKC) through reaction of the Cys sulfhydryl group with the active site of the enzyme. The labeling of PKC occurs only in the presence of the cofactors phosphatidylserine, diacylglycerol, and Ca2+ but not in their absence. Low concentrations of the Arg-rich substrate, R4YGSR6Y greatly increase the extent of the reaction of these biotinylated peptides with PKC in the presence of lipid cofactors but in the absence of calcium. This effect can be observed at 50 nM R4YGSR6Y and suggests the presence of a high-affinity binding site for Arg-rich peptides which is separate from the active site but which enhances accessibility of the active site. The study also demonstrates the utility of the biotinylated peptides as active site labels which can detect the conformational change accompanying the activation of PKC.

Affinity Labels↗