Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-␣2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-␣2b expression remained poor even when fused to a signal sequence, and an alternative IFN-␣2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter. The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.

In industrial scale recombinant protein production it is often of interest to be able to translocate the product to reduce downstream costs, and heterologous proteins may require the oxidative environment outside of the cytoplasm for correct folding. High-level expression combined with translocation to the periplasm is often toxic to the host, and expression systems that can be used to fine-tune the production levels are therefore important. We previously constructed vector pJB658, which harbors the broad-host-range RK2 minireplicon and the inducible Pm/xylS promoter system, and we here explore the potential of this unique system to manipulate the expression and translocation of a host-toxic single-chain antibody variable fragment with affinity for hapten 2-phenyloxazol-5-one (phOx) (scFv-phOx). Fine-tuning of scFv-phOx levels was achieved by varying the concentrations of inducers and the vector copy number and also different signal sequences. Our data show that periplasmic accumulation of scFv-phOx leads to cell lysis, and we demonstrate the importance of controlled and high expression rates to achieve high product yields. By optimizing such parameters we show that soluble scFv-phOx could be produced to a high volumetric yield (1.2 g/liter) in high-cell-density cultures of Escherichia coli.The application range of antibodies in medicine and biotechnology is broad, and there has been great progress in the design and selection of new variants with novel affinities. In particular, there has been a growing interest in the development of antibody fragments comprising the V H and V L domains connected to each other as a single chain (scFv) (4). The small size of scFv proteins (about 250 amino acids) compared to native antibodies confers certain therapeutic advantages because of their shorter half-life (rapid blood clearance) and faster tissue penetration. scFv molecules can be used as selective carriers for delivering radionucleids, toxins, or cytotoxic drugs to malignant cell populations, as well as providing valuable tools for studying antibody-antigen interactions in detail (14). In addition, this feature makes it possible to construct and screen large scFv libraries by using phage display approaches (for a review, see reference 23).For medical applications scFvs are needed in large amounts, and the ability to produce high yields in Escherichia coli has gained considerable interest (14). Native scFv proteins have two disulfide bonds and require oxidative conditions to fold correctly. Although expression of native scFv proteins without disulfide bridge formation has been reported (9), cytoplasmic production in bacteria typically results in aggregation of scFv polypeptides into insoluble inclusion bodies (14). Therefore, in E. coli it is usually desirable to express scFvs as fusion proteins targeted for translocation to the oxidative periplasm to obtain functional products (3,26). Various vector systems for recombinant scFv expression in E. coli have been reported (11,15,19), but the experiments were typically perfo...

The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.

Alginates from seaweed are used in chronic wound management, though the molecular and cellular effects of various alginate dressings are not well documented. We have developed ultrapure sodium-alginates from Pseudomonas fluorescens with different content and distribution of single guluronic acid (G) residues (0-45% G), and tested their biological activities on human primary keratinocytes (KCs). The alginates inhibited KC migration and induced expression of differentiation markers. The potency of the alginates correlated with the increasing percentage of single G residues. These findings were explained by different binding and release of ionic calcium (Ca++) from the alginates which subsequently triggered differentiation. Ca-free alginates had no effect on KC migration and differentiation, but the chemokine receptor CXCR7 was upregulated. Q-PCR revealed that also CXCL12/SDF-1, one of two known CXCR7-ligands, was induced by the alginates. Both CXCR7 and CXCL12-induction was dependent on the alginate G-content, and highest upregulation was induced by an alginate with 19% single G residues. In the epidermis, CXCR7 expression was restricted to the basal layer. This study defines two biological effects of ultrapure alginates on KCs, both being dependent on the alginate structure, and being either dependent or independent of Ca.