Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumine in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial beta-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source

Critical Endpoint and Analytical Phase Diagram of Attractive Hard-Core Yukawa Spheres

We analytically calculate the gas-liquid critical endpoint (cep) for hard spheres with a Yukawa attraction. This cep is a boundary condition for the existence of a liquid. We use an analytical Helmholtz energy expression for the attractive Yukawa (hard) spheres based on the first-order mean spherical approximation to the attractive Yukawa potential by Tang and Lu (J. Chem. Phys. 1993, 99, 9828). This theory and our analytical simplification of it predict the gas-liquid and fluid-solid phase behavior, as found from computer simulations, very accurately as long as the range 1/kappa of attraction is not too short. We find that the cep is situated at kappasigma approximately 6 and at a contact potential around 2 kT. It follows that a liquid state is only possible when the attraction range is longer than (1/6) of the particle diameter sigma, and the attraction strength is smaller than 2 kT. The liquid region does not span more than 0.6 kT in strength, and there is also a relatively narrow window for the attraction range

Phase diagram for a mixture of colloids and polymers with equal size

We present the phase diagram of a colloid-polymer mixture in which the radius a of the colloidal spheres is approximately the same as the radius R of a polymer coil (q=R/a1). A three-phase coexistence region is experimentally observed, previously only reported for colloid-polymer mixtures with smaller polymer chains (q0.6). A recently developed generalized free-volume theory (GFVT) for mixtures of hard spheres and non-adsorbing excluded-volume polymer chains gives a quantitative description of the phase diagram. Monte Carlo simulations also agree well with experimen

Hard colloidal rods near a soft wall: wetting, drying, and symmetry breaking

Within an Onsager-like density functional theory we explore the thermodynamic and structural properties of an isotropic and nematic fluid of hard needle-like colloids in contact with a hard substrate coated with a soft short-ranged attractive or repulsive layer. As a function of the range and the strength of the soft interactions we find wetting and drying transitions, a pre-drying line, and a symmetry-breaking transition from uniaxial to biaxial in the wetting and drying film.Comment: 7 pages, 2 figure

Single polymer adsorption in shear: flattening versus hydrodynamic lift and corrugation effects

The adsorption of a single polymer to a flat surface in shear is investigated using Brownian hydrodynamics simulations and scaling arguments. Competing effects are disentangled: in the absence of hydrodynamic interactions, shear drag flattens the chain and thus enhances adsorption. Hydrodynamic lift on the other hand gives rise to long-ranged repulsion from the surface which preempts the surface-adsorbed state via a discontinuous desorption transition, in agreement with theoretical arguments. Chain flattening is dominated by hydrodynamic lift, so overall, shear flow weakens the adsorption of flexible polymers. Surface friction due to small-wavelength surface potential corrugations is argued to weaken the surface attraction as well.Comment: 6 pages, 4 figure

A Quantitative Theory of Mechanical Unfolding of a Homopolymer Globule

We propose the quantitative mean-field theory of mechanical unfolding of a globule formed by long flexible homopolymer chain collapsed in poor solvent and subjected to extensional deformation. We demonstrate that depending on the degree of polymerization and solvent quality (quantified by the Flory-Huggins $\chi$ parameter) the mechanical unfolding of the collapsed chain may either occur continuously (by passing a sequence of uniformly elongated configurations) or involves intra-molecular micro-phase coexistence of a collapsed and a stretched segment followed by an abrupt unraveling transition. The force-extension curves are obtained and quantitatively compared to our recent results of numerical self-consistent field (SCF) simulations. The phase diagrams for extended homopolymer chains in poor solvent comprising one- and two-phase regions are calculated for different chain length or/and solvent quality.Comment: 24 pages, 18 figure

Elasticity of semiflexible polymers in two dimensions

We study theoretically the entropic elasticity of a semi-flexible polymer, such as DNA, confined to two dimensions. Using the worm-like-chain model we obtain an exact analytical expression for the partition function of the polymer pulled at one end with a constant force. The force-extension relation for the polymer is computed in the long chain limit in terms of Mathieu characteristic functions. We also present applications to the interaction between a semi-flexible polymer and a nematic field, and derive the nematic order parameter and average extension of the polymer in a strong field.Comment: 16 pages, 3 figure

Hydration interactions: aqueous solvent effects in electric double layers

A model for ionic solutions with an attractive short-range pair interaction between the ions is presented. The short-range interaction is accounted for by adding a quadratic non-local term to the Poisson-Boltzmann free energy. The model is used to study solvent effects in a planar electric double layer. The counter-ion density is found to increase near the charged surface, as compared with the Poisson-Boltzmann theory, and to decrease at larger distances. The ion density profile is studied analytically in the case where the ion distribution near the plate is dominated only by counter-ions. Further away from the plate the density distribution can be described using a Poisson-Boltzmann theory with an effective surface charge that is smaller than the actual one.Comment: 11 Figures in 13 files + LaTex file. 20 pages. Accepted to Phys. Rev. E. Corrected typos and reference

Adsorption of Multi-block and Random Copolymer on a Solid Surface: Critical Behavior and Phase Diagram

The adsorption of a single multi-block $AB$-copolymer on a solid planar substrate is investigated by means of computer simulations and scaling analysis. It is shown that the problem can be mapped onto an effective homopolymer adsorption problem. In particular we discuss how the critical adsorption energy and the fraction of adsorbed monomers depend on the block length $M$ of sticking monomers $A$, and on the total length $N$ of the polymer chains. Also the adsorption of the random copolymers is considered and found to be well described within the framework of the annealed approximation. For a better test of our theoretical prediction, two different Monte Carlo (MC) simulation methods were employed: a) off-lattice dynamic bead-spring model, based on the standard Metropolis algorithm (MA), and b) coarse-grained lattice model using the Pruned-enriched Rosenbluth method (PERM) which enables tests for very long chains. The findings of both methods are fully consistent and in good agreement with theoretical predictions.Comment: 27 pages, 12 figure