A method for microbial cell surface fingerprinting based on surface plasmon resonance.

A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface.

Process technology for production and recovery of heterologous proteins with Pichia pastoris.

Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes.

RelA1 gene control of Escherichia coli lipid structure and cell performance during glucose limited fed-batch conditions.

At increasing glucose limitation, typical for fed-batch cultivation performance, cultivation of Escherichia coli (relA1) results in development of a lipid structure that radically differs from the wild type and is characterised by accumulation of neutral phospholipids and saturated fatty acids. The mutant can, furthermore, not change the level of cardiolipin, which is generally the hallmark of changes to severe glucose limitation. The result suggests an increased negative control in the mutant with respect to the flux to phosphatidyl glycerol and cardolipin as well as to unsaturated fatty acids.

Interfacing Pichia pastoris cultivation with expanded bed adsorption.

For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density.

Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth.

Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP.

Two-step recovery process for tryptophan tagged cutinase: interfacing aqueous two-phase extraction and hydrophobic interaction chromatography.

In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli).

Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system.

A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 l to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1).

Extraction of endoglucanase I (Ce17B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system.

Endoglucanases (EGI) (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.

Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins.

Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency.

Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins.

A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.

Peptide fusion tags with tryptophan and charged residues for control of protein partitioning in PEG-potassium phosphate aqueous two-phase systems.

A partition study with peptides and recombinant proteins in poly(ethylene glycol)4000-potassium phosphate aqueous two-phase systems has been performed. The aim was to study to what extent the insertion of charged residues could affect protein partition in addition to the already observed effects of tryptophan residues. The model proteins used are based on a staphylococcal protein A derivative, Z, and modified by the insertion of peptide tags close to the C-terminus.

Influence of scale-up on the quality of recombinant human growth hormone.

The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared.

Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems.

The Fusarium solani pisi lipase cutinase has been genetically engineered to investigate the influence of C-terminal peptide extensions on the partitioning of the enzyme in PEG-salt based aqueous two-phase bioseparation systems. Seven different cutinase lipase variants were constructed containing various C-terminal peptide extensions including tryptophan rich peptide tags ((WP)(2) and (WP)(4)), positively ((RP)(4)) and negatively ((DP)(4)) charged tags as well as combined tags with tryptophan together with either positively ((WPR)(4)) or negatively ((WPD)(4)) charged amino acids. The modified cutinase variants were stably produced in Escherichia coli as secreted to the periplasm from which they were efficiently purified by IgG-affinity chromatography employing an introduced N-terminal IgG-binding ZZ affinity fusion partner present in all variants.

Aqueous two-phase systems containing self-associating block copolymers. Partitioning of hydrophilic and hydrophobic biomolecules.

A series of proteins and one membrane-bound peptide have been partitioned in aqueous two-phase systems consisting of micelle-forming block copolymers from the family of Pluronic block copolymers as one polymer component and dextran T500 as the other component. The Pluronic molecule is a triblock copolymer of the type PEO-PPO-PEO, where PEO and PPO are poly(ethylene oxide) and poly(propylene oxide), respectively. Two different Pluronic copolymers were used, P105 and F68, and the phase diagrams were determined at 30 degrees C for these polymer systems.

Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems.

Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspartate residues, were fused near the C-terminus of the recombinant protein ZZT0, where Z is a synthetic IgG-binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70.

Electrostatic and Hydrophobic Effects of Oligopeptide Insertions on Protein Adsorption.

The effects of oligopeptide insertions on the adsorption of the protein ZZ, where Z is the IgG binding domain of staphylococcal Protein A, was investigated by in situ ellipsometry. In particular, the interplay between hydrophobic and electrostatic interactions as driving force for adsorption was investigated by studying the effects of oligopeptide insertions of the type Tn((AlaTrpTrpPro)n), Nn((AlaTrpTrpAspPro)n), and Pn((AlaTrpTrpLysPro)n) on the adsorption at silica, methylated silica, and diaminocyclohexane (DACH) plasma polymer surfaces. For comparison, the adsorption of the inserted peptide stretches was also investigated.

In vitro complex-formation between the molecular chaperone DnaK and staphylococcal protein A derivatives produced in Escherichia coli and its use in the purification of DnaK.

Complex-formation between a truncated staphylococcal Protein A produced in Escherichia coli and a native E coli molecular chaperone, DnaK, can be used for the purification of DnaK by IgG-affinity chromatography. The half-time constant for in vitro formation of the Protein A-DnaK complex is about 14 min. Complex-formation in the presence of ATP is faster, but pre-incubation of DnaK with ATP decreases the final amount of the complex.

Effects of fused tryptophan rich peptides to a recombinant protein. A domain on the partitioning in polyethylene glycol-dextran and Ucon-dextran aqueous two-phase systems.

Genetic engineering has been used to construct fusion proteins with tryptophan containing peptides. The peptides and the fusion proteins have been partitioned in aqueous two-phase systems of poly(ethylene glycol) (PEG)-dextran and Ucon-dextran. The studied model protein was ZZT0, where Z is an engineered domain of domain B of staphylococcal protein A.

Proteolysis of fusion proteins: stabilization and destabilization of staphylococcal protein A and Escherichia coli beta-galactosidase.

The product yield of staphylococcal Protein A reached only 1.8% of the cell dry weight, while the corresponding value was 14% for a fusion protein composed of Protein A and Escherichia coli beta-galactosidase [1], when produced in the same E. coli host strain, with the same promoter and under identical process conditions.

Effects of amino acid insertions on the proteolysis of a staphylococcal protein A derivative in Escherichia coli.

In vivo proteolysis of protein ZZT0, derived from the B domain of staphylococcal protein A, was investigated in Escherichia coli before and after insertion of 1-3 multiples of the tetrapeptide Ala-Trp-Trp-Pro close to the C-terminus of ZZT0. Before insertion, ZZT0 was proteolytically stable as judged from the purity of IgG binding proteins up to 1 h after inhibition of protein synthesis with chloramphenicol. Insertion of 1-3 units of Ala-Trp-Trp-Pro into ZZT0 increased progressively the sensitivity to proteolysis and induced DnaK and GroEL binding to the protein.

A mathematical model to predict the partitioning of peptides and peptide-modified proteins in aqueous two-phase systems.

A mathematical procedure was developed to predict the partition coefficients of the peptides AIIP, AWWP, AIIPAIIP and AWWPAWWP in poly(ethylene glycol) (PEG)/phosphate aqueous two-phase systems from amino acid hydrophobicities. In general, peptides containing tryptophan partition more into the PEG-enriched upper phase than analogous peptides containing isoleucine. Specifically, as the PEG concentration difference between the phases increased in a PEG/potassium phosphate aqueous two-phase system, the peptide AIIP was observed to have a partition coefficient ranging from 1.

Polyethylene glycol-potassium phosphate aqueous two-phase systems. Insertion of short peptide units into a protein and its effects on partitioning.

Two different tetrapeptides, AlaTrpTrpPro and AlaIleIlePro, were inserted near the C-terminus of the protein ZZT0. The Trp-rich peptide unit strongly increased both the partitioning of ZZT0 into the polyethylene glycol (PEG)-rich phase in a PEG-potassium phosphate aqueous two-phase system and its retention on PEG and propyl hydrophobic interaction chromatographic columns with potassium phosphate as eluent. Both the partitioning and the retention increased with increasing number of Trp-rich peptide units inserted into ZZT0.

Recovery of extracellular human insulin-like growth factor-I and II as a fusion protein from Escherichia coli culture broth by aqueous two-phase extraction.

The primary purification of human insulin-like growth factor-I (IGF-I) and IGF-II, produced extracellularly in Escherichia coli as a fusion to two domains (ZZ) derived from staphylococcal protein A, has been studied. First, the partitioning of IgG-affinity purified ZZ-IGF-I and ZZ-IGF-II, respectively, to the top phase in poly(ethylene glycol)/potassium phosphate aqueous two-phase systems were investigated. Thereafter, the extraction of ZZ-IGF-I with a poly(ethylene glycol) 1500/potassium phosphate system was performed directly in the bioreactor after the cultivation.

The molecular basis of partitioning in aqueous two-phase systems.

Protein purification based on partition in aqueous two-phase systems has attracted interest for many years. This approach has been advocated as a primary-stage unit operation in downstream processing. In reality, application has been strictly limited through inadequate understanding of the complex molecular forces involved in partitioning processes.