Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris

A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter. In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared. After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems. Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.     

Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli

Significant leakage of host-cell proteins into the culture medium occurred during high-cell-density cultivation of E. coli. Identification of these medium proteins revealed almost exclusively a periplasmic origin. Release of periplasmic proteins into the culture medium was observed throughout the entire cultivation of recombinant or non-recombinant cells. Leakage was intensified, however, in the final part of high-cell-density cultures (>100 g L(-)(1) dry cell mass) or when a temperature upshift was used for induction of recombinant protein synthesis. After temperature upshift, formation rates and residual cellular concentrations of periplasmic proteins declined with individual rates; e.g., the cellular content of the large periplasmic dipeptide binding protein DppA (57.4 kDa) started to decline about 4 h after the temperature upshift, whereas the smaller periplasmic d-galactose/d-glucose binding protein MglB (33.4 kDa) was already lost during the first hour after the upshift. In addition to periplasmic proteins, the osmotic-shock-sensitive heat-shock protein DnaK was found in significantly higher proportion in the cell-free medium of the temperature-challenged culture than other cytoplasmic proteins. Cell lysis was not observed even after prolonged cultivation. Thus, loss of a subset of cellular proteins of mainly periplasmic origin ordinarily occurs during cultivation and is intensified through stressful conditions in high-cell-density cultures. The selective release of cellular proteins of periplasmic origin offers the opportunity to simplify the downstream processing of recombinant proteins directed to the periplasm of E. coli.     

Small heat shock proteins from extremophiles: a review

Many microorganisms from extreme environments have been well characterized, and increasing access to genomic sequence data has recently allowed the analysis of the protein families related to stress responses. Heat shock proteins appear to be ubiquitous in extremophiles. In this review, we focus on the family of small heat shock proteins (sHSPs) from extremophiles, which are -crystallin homologues. Like the -crystallin eye lens proteins, sHSPs act as molecular chaperones and prevent aggregation of denatured proteins under heat and desiccation stress. Many putative sHSP homologues have been identified in the genomic sequences of all classes of extremophiles. Current studies of shsp gene expression have revealed mechanisms of regulation and activity distinct from other known hsp gene regulation systems. Biochemical studies on sHSPs are limited to thermophilic and hyperthermophilic organisms, and the only two available crystal structures of sHSPs from Methanocaldococcus jannaschii, a hyperthermophilic archaeon and a mesophilic eukaryote, have contributed significantly to an understanding of the mechanisms of action of sHSPs, although many aspects remain unclear.Communicated by D.A. Cowan     

A catalog of human cDNA expression clones and its application to structural genomics

We describe here a systematic approach to the identification of human proteins and protein fragments that can be expressed as soluble proteins in Escherichia coli. A cDNA expression library of 10,825 clones was screened by small-scale expression and purification and 2,746 clones were identified. Sequence and protein-expression data were entered into a public database. A set of 163 clones was selected for structural analysis and 17 proteins were prepared for crystallization, leading to three new structures.     

In control: systematic assessment of microarray performance

Expression profiling using DNA microarrays is a powerful technique that is widely used in the life sciences. How reliable are microarray-derived measurements? The assessment of performance is challenging because of the complicated nature of microarray experiments and the many different technology platforms. There is a mounting call for standards to be introduced, and this review addresses some of the issues that are involved. Two important characteristics of performance are accuracy and precision. The assessment of these factors can be either for the purpose of technology optimization or for the evaluation of individual microarray hybridizations. Microarray performance has been evaluated by at least four approaches in the past. Here, we argue that external RNA controls offer the most versatile system for determining performance and describe how such standards could be implemented. Other uses of external controls are discussed, along with the importance of probe sequence availability and the quantification of labelled material.     

TETRA: a web-service and a stand-alone program for the analysis and comparison of tetranucleotide usage patterns in DNA sequences

Background  

In the emerging field of environmental genomics, direct cloning and sequencing of genomic fragments from complex microbial communities has proven to be a valuable source of new enzymes, expanding the knowledge of basic biological processes. The central problem of this so called metagenome-approach is that the cloned fragments often lack suitable phylogenetic marker genes, rendering the identification of clones that are likely to originate from the same genome difficult or impossible. In such cases, the analysis of intrinsic DNA-signatures like tetranucleotide frequencies can provide valuable hints on fragment affiliation. With this application in mind, the TETRA web-service and the TETRA stand-alone program have been developed, both of which automate the task of comparative tetranucleotide frequency analysis.