Boronic acid functionalized fibrous cellulose for the selective enrichment of glycopeptides.

Enrichment of glycoproteins has been important because of their dynamicity and role in biological systems. Study of glycoproteins is complex because of the simultaneous glycosylation and deglycosylation inside the body. Often employed affinities for glycopeptides are hydrazide, boronic acid, or physiosorbed lectin on support materials.

Escherichia coli Signal Peptidase Recognizes and Cleaves Archaeal Signal Sequence.

Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction.

Characterization and bioassay of post-translationally modified interferon α-2b expressed in Escherichia coli.

Examples of N-terminal acetylation are rare in prokaryotic systems, but in this study, we report one such example in which N-terminal Cys residue of recombinant human interferon α-2b produced in Escherichia coli is a favourite site for N(α)-acetylation. The recombinant protein following Q-sepharose chromatography gave a single band on PAGE analysis. However, on reverse phase HPLC the material separated into three peaks.

Studies on the expression and processing of human proinsulin derivatives encoded by different DNA constructs.

A synthetic gene encoding human proinsulin, containing Escherichia coli preferred codons, with an additional N-terminal methionine, was used for the expression, of M-proinsulin and construction of nine derivatives. No improvement in expression was noted, relative to that of M-proinsulin, when the 5'- of the gene was appended to codons for seven amino acids of a well expressed E. coli protein (threonine dehydrogenase), or the constructs contained multiple copies of the proinsulin gene.

TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH.

Seven nicotinamide adenine dinucleotide oxidase homologs have been found in the genome of Thermococcus kodakaraensis. The gene encoding one of them, TK1299, consisted of 1326 nucleotides, corresponding to a polypeptide of 442 amino acids. To examine the molecular properties of TK1299, the structural gene was cloned, expressed in Escherichia coli and the gene product was characterized.

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry.

Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself.

Mechanistic and stereochemical studies of glycine oxidase from Bacillus subtilis strain R5.

Glycine oxidase gene from a strain of Bacillus subtilis was cloned and expressed in Escherichia coli. The purified enzyme was found, by mass spectrometry, to have a protein M(r) of 40763 (value of 40761.6 predicted from DNA sequence) and a FAD prosthetic group M(r) of 785.

Inventory of 'slow exchanging' hydrogen atoms in human proinsulin and its derivatives: observations on the mass spectrometric analysis of deuterio-proteins in D(2)O.

Secondary structure elements of human proinsulin and of its tryptic products were compared by H/D exchange, in a single-pot, using mass spectrometry. Human proinsulin containing an N-terminal methionine, M-proinsulin, was engineered and converted into a perdeuterio derivative, which using an optimized mass spectrometric protocol and manual calculations gave a mass of 9669.6 (+/-1) Da showing the replacement, with deuterium of 146.