sepharose and 2-iminobiotin

An affinity purification technique was established that allows the selective isolation of 2-iminobiotinylated peptides from proteolytic digest of proteins in order to identify surface-exposed protein domains. Serving as model systems, two photosystem I subunits, PsaD and PsaE from the cyanobacterium Synechococcus elongatus, were overexpressed in Escherichia coli, modified in vitrowith NHS-2-iminobiotin which incorporates 2-iminobiotin at exposed amino groups, and subjected to proteolytic digestion by Glu-C and Arg-C protease, respectively. 2-Iminobiotin-containing proteolytic peptides were subsequently extracted from the proteolytic digests using avidin agarose in a batch procedure and the extracted peptides were separated by HPLC chromatography. The analysis of the peptide maps by electrospray ionization mass spectrometry or N-terminal sequencing showed that avidin-extracted peptide fractions contain almost exclusively 2-iminobiotinylated proteolytic fragments of PsaE or PsaD. No unmodified peptides of PsaD or PsaE were detected. According to this analysis, PsaE is accessible to biotinylation at all of its 7 lysine residues and at its N-terminus. Similarly, all 11 lysine residues of PsaD can be biotinylated and only the N-terminus of PsaD is not accessible.

Topics: Affinity Labels; Amino Acid Sequence; Avidin; Bacterial Proteins; Binding Sites; Biotin; Biotinylation; Chromatography, High Pressure Liquid; Cyanobacteria; Lysine; Mass Spectrometry; Molecular Sequence Data; Peptide Mapping; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Plant Proteins; Proteins; Recombinant Proteins; Sepharose; Sequence Analysis; Serine Endopeptidases

An efficient lepidopteran insect cell system was established for the expression of a recombinant form of chicken egg-white avidin. The gene product was obtained in both secreted and intracellular forms, and biologically active recombinant avidin was isolated using affinity chromatography on an iminobiotin-agarose column. Similar to the known quaternary structure of the native egg-white protein, the purified recombinant protein was glycosylated and assembled mainly into tetramers. Like native avidin, the recombinant tetramer also exhibited a high level of thermostability, and was further stabilized upon binding biotin. The biotin-binding and structural properties of the recombinant avidin are thus similar to those of the natural egg-white protein, and the insect system is appropriate both for future site-directed mutagenesis studies and for the production of avidin fusion proteins.

Topics: Animals; Avidin; Baculoviridae; Biotin; Chickens; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Genetic Vectors; Glycosylation; Protein Conformation; Protein Denaturation; Recombinant Proteins; Sepharose; Spodoptera

The adhesion force between the tip of an atomic force microscope cantilever derivatized with avidin and agarose beads functionalized with biotin, desthiobiotin, or iminobiotin was measured. Under conditions that allowed only a limited number of molecular pairs to interact, the force required to separate tip and bead was found to be quantized in integer multiples of 160 +/- 20 piconewtons for biotin and 85 +/- 15 piconewtons for iminobiotin. The measured force quanta are interpreted as the unbinding forces of individual molecular pairs.

Topics: Adhesiveness; Avidin; Biotin; Ligands; Microscopy; Microspheres; Receptors, Cell Surface; Sepharose

Topics: Avidin; Biotin; Chromatography, Affinity; Indicators and Reagents; Ovalbumin; Sepharose

Topics: Avidin; Biotin; Carbon Radioisotopes; Chromatography, Affinity; Ovalbumin; Rhodamines; Sepharose; Tritium