Large-scale monoclonal antibody purification by continuous chromatography, from process design to scale-up.

The development and optimization of a purification process of monoclonal antibodies based on two continuous chromatography steps for capture and intermediate purification are presented. The two chromatography steps were individually optimized using either batch chromatography or sequential multicolumn chromatography (SMCC). Proprietary simulation software was used to optimize SMCC and to evaluate the potential gains compared with batch chromatography.

The conserved Candida albicans CA3427 gene product defines a new family of proteins exhibiting the generic periplasmic binding protein structural fold.

Nosocomial diseases due to Candida albicans infections are in constant rise in hospitals, where they cause serious complications to already fragile intensive care patients. Antifungal drug resistance is fast becoming a serious issue due to the emergence of strains resistant to currently available antifungal agents. Thus the urgency to identify new potential protein targets, the function and structure of which may guide the development of new antifungal drugs.

Structural and mutational analyses of Deinococcus radiodurans UvrA2 provide insight into DNA binding and damage recognition by UvrAs.

UvrA proteins are key actors in DNA damage repair and play an essential role in prokaryotic nucleotide excision repair (NER), a pathway that is unique in its ability to remove a broad spectrum of DNA lesions. Understanding the DNA binding and damage recognition activities of the UvrA family is a critical component for establishing the molecular basis of this process. Here we report the structure of the class II UvrA2 from Deinococcus radiodurans in two crystal forms.

Structural characterization of CA1462, the Candida albicans thiamine pyrophosphokinase.

Background: In search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. One criterion was the protein sequence conservation between reference pathogenic genomes. A second criterion was that the corresponding gene in Saccharomyces cerevisiae should be essential.

Structure and evolution of the Ivy protein family, unexpected lysozyme inhibitors in Gram-negative bacteria.

Part of an ancestral bactericidal system, vertebrate C-type lysozyme targets the peptidoglycan moiety of bacterial cell walls. We report the crystal structure of a protein inhibitor of C-type lysozyme, the Escherichia coli Ivy protein, alone and in complex with hen egg white lysozyme. Ivy exhibits a novel fold in which a protruding five-residue loop appears essential to its inhibitory effect.

Impact of the excision of an ancient repeat insertion on Rickettsia conorii guanylate kinase activity.

The genomic sequencing of Rickettsia conorii revealed a new family of Rickettsia-specific palindromic elements (RPEs) capable of in-frame insertion in preexisting open reading frames (ORFs). Many of these altered ORFs correspond to proteins with well-characterized or essential functions in other microorganisms. Previous experiments indicated that RPE-containing genes are normally transcribed and that no excision of the repeat occurs at the mRNA level.

In Ssarch of new anti-bacterial target genes: a comparative/structural genomics approach.

We outline a joint academic/industrial (CNRS/AVENTIS) functional genomics project aiming at the discovery of new anti-bacterial gene targets. Starting from all publicly available bacterial genomes, a subset of the most evolutionary conserved protein-coding genes has been identified. We retained genes with clear homolog in E.

Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme.

The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases.

Crystallization and preliminary crystallographic study of b0220, an 'ORFan' protein of unknown function from Escherichia coli.

Newly sequenced microbial genomes continue to reveal up to 50% functionally uncharacterized 'anonymous' genes. A significant fraction of these anonymous ORFs does not exhibit any sequence similarity to any protein in the databases and constitutes a set of unique sequences, denoted 'ORFans'. The structure determination of ORFan proteins is both of evolutionary and functional interest.

Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilization.

Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain. A portion of this domain displays homology to members of the alpha-amylase family (glycoside hydrolase family 13), which all have a (beta/alpha)8 barrel structure. In the glucansucrases, however, the alpha-helix and beta-strand elements are circularly permuted with respect to the order in family 13.

Mutagenesis of asp-569 of glucosyltransferase I glucansucrase modulates glucan and oligosaccharide synthesis.

Glucansucrases of oral streptococci and Leuconostoc mesenteroides are enzymes of medical and biotechnological interest that synthesize alpha-glucans. They can also synthesize oligosaccharides in the presence of a sugar acceptor. Previous reports have identified an amino acid residue that may affect the structure of the glucan product; therefore, random mutagenesis of the corresponding Asp-569 of Streptococcus downei glucosyltransferase I (GTF-I) was used to further understanding of its involvement in the catalytic mechanism and to evaluate how different amino acids can modulate glucan and oligosaccharide synthesis.

Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I.

Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest. Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (beta/alpha)8 barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region. In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain.

Secondary structure of Streptococcus downei GTF-1 glucansucrase.

Multiple sequence alignment and structure prediction of glucansucrases produced by oral streptococci and Leuconostoc mesenteroides showed that all have common structural features, with three major domains. There is no conservation of primary sequence or structure in the N-terminal variable region. Sequence-based structure prediction combined with circular dichroism spectrum analysis of purified truncated forms of Streptococcus downei GTF-I revealed that the core catalytic region has a defined structure consistent with the proposed (alpha/beta)8-barrel structure.

Glucansucrases: mechanism of action and structure-function relationships.

Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides.

Isolation of an active catalytic core of Streptococcus downei MFe28 GTF-I glucosyltransferase.

Truncated variants of GTF-I from Streptococcus downei MFe28 were purified by means of a histidine tag. Sequential deletions showed that the C-terminal domain was not directly involved in the catalytic process but was required for primer activation. A fully active catalytic core of only 100 kDa was isolated.

Effect of Leuconostoc mesenteroides NRRL B-512F dextransucrase carboxy-terminal deletions on dextran and oligosaccharide synthesis.

Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats.

Cloning and sequencing of a gene coding for an extracellular dextransucrase (DSRB) from Leuconostoc mesenteroides NRRL B-1299 synthesizing only a alpha (1-6) glucan.

The coding region for a novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrB) was isolated and sequenced. Using degenerate primers homologous to a conserved region present in dextransucrases from Streptococcus (GTFs) and L. mesenteroides NRRL B-512F (DSRS) and conserved amino acid sequences located in the N-terminal catalytic region of these enzymes, about 60% of the DSRB encoding gene was isolated.

Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity.

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid.

Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only alpha (1-6) and alpha (1-3) linkages.

The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrA) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR).