DESIGN OF ESCHERICHIA COLI HOST STRAINS FOR IMPROVED RECOMBINANT PROTEIN PURIFICATION: AN APPROACH THAT BRIDGES THE UPSTREAM AND DOWNSTREAM REALMS OF BIOPROCESSING

Escherichia coli is a favored host for rapid, scalable expression of recombinant proteins for academic, commercial or therapeutic use. To maximize its economic advantages, however, it must be coupled with robust downstream processes. These are typically composed of three or fewer chromatography steps that remove the majority of the host proteins to achieve a reasonable degree of purification. Removal of remaining impurities is often very difficult and costly due similarity among physicochemical properties with the target. This work implements a novel approach that overcomes such limitations by bridging upstream and downstream realms, that is, by subjecting lysed cell material to a variety of purification procedures, identifying individual impurities and mitigating their removal by genetic modification. Successful knockout of three prominent contaminants of immobilized metal affinity chromatography (IMAC) without detriment to cell growth or recombinant protein expression was demonstrated. Elution of the recombinant target was strategically manipulated with peptide tags that allowed purification to virtual homogeneity. Additional IMAC studies focused on the most problematic host proteins, those that retained binding affinity under stringent conditions. Two-dimensional difference gel electrophoresis discerned variation in the soluble extract pools loaded in IMAC and the subsequent impurities, with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein were shown to be the most persistent contaminants and had greater prevalence at low target protein expression. Since genetic removal of the transcription activator would negatively impact cellular function, I substituted specific residues to eliminate its IMAC affinity with minimal impact on its activity. I applied this integrative strategy seeking to improve performance of cheaper, non-affinity based processes. Phosphoenolpyruvate carboxykinase and peptidase D were significant contaminants during serial purification of a target by hydrophobic interaction and anion exchange chromatography. Ribosomal protein L25 dominated non-target binding of a polyarginine tagged recombinant on cation exchange resin. With the development of comprehensive genomic manipulation in higher order species, such integrative approaches will be conventional in the development of coupled expression systems for the production of complex biologics

E. coli separatome-based protein expression and purification platform

Provided is a separatome-based peptide, polypeptide, and protein expression and purification platform based on the juxtaposition of the binding properties of host cell genomic peptides, polypeptides, and proteins with the characteristics and location of the corresponding genes on the host cell chromosome of E. coli. The separatome-based protein expression and purification platform quantitatively describes and identifies priority deletions, modifications, or inhibitions of certain gene products to increase chromatographic separation efficiency, defined as an increase in column capacity, column selectivity, or both, with emphasis on the former. Moreover, the separatome-based protein expression and purification platform provides a computerized knowledge tool that, given separatome data, and a target recombinant peptide, polypeptide, or protein, intuitively suggests strategies facilitating efficient product purification. The separatome-based protein expression and purification platform is an efficient bioseparation system that intertwines host cell expression systems and chromatography

Effect of plasmid replication deregulation via inc mutations on E. coli proteome & simple flux model analysis

When the replication of a plasmid based on sucrose selection is deregulated via the inc1 and inc2 mutations, high copy numbers (7,000 or greater) are attained while the growth rate on minimal medium is negligibly affected. Adaptions were assumed to be required in order to sustain the growth rate. Proteomics indicated that indeed a number of adaptations occurred that included increased expression of ribosomal proteins and 2-oxoglutarate dehydrogenase. The operating space prescribed by a basic flux model that maintained phenotypic traits (e.g. growth, byproducts, etc.) within typical bounds of resolution was consistent with the flux implications of the proteomic changes

Separatome-based protein expression and purification platform

Provided is a separatome-based recombinant peptide, polypeptide, and protein expression and purification platform based on the juxtaposition of the binding properties of host cell genomic peptides, polypeptides, and proteins with the characteristics and location of the corresponding genes on the host cell chromosome, such as that of E. coli, yeast, Bacillus subtilis or other prokaryotes, insect cells, mammalian cells, etc. This platform quantitatively describes and identifies priority deletions, modifications, or inhibitions of certain gene products to increase chromatographic separation efficiency, defined as an increase in column capacity, column selectivity, or both, with emphasis on the former. Moreover, the platform provides a computerized knowledge tool that, given separatome data and a target recombinant peptide, polypeptide, or protein, intuitively suggests strategies leading to efficient product purification. The separatome-based protein expression and purification platform is an efficient bioseparation system that intertwines host cell expression systems and chromatography

Lead discovery, chemistry optimization, and biological evaluation studies of novel biamide derivatives as CB2 receptor inverse agonists and osteoclast inhibitors

N,N'-((4-(Dimethylamino)phenyl)methylene)bis(2-phenylacetamide) was discovered by using 3D pharmacophore database searches and was biologically confirmed as a new class of CB(2) inverse agonists. Subsequently, 52 derivatives were designed and synthesized through lead chemistry optimization by modifying the rings A-C and the core structure in further SAR studies. Five compounds were developed and also confirmed as CB(2) inverse agonists with the highest CB(2) binding affinity (CB(2)K(i) of 22-85 nM, EC(50) of 4-28 nM) and best selectivity (CB(1)/CB(2) of 235- to 909-fold). Furthermore, osteoclastogenesis bioassay indicated that PAM compounds showed great inhibition of osteoclast formation. Especially, compound 26 showed 72% inhibition activity even at the low concentration of 0.1 μM. The cytotoxicity assay suggested that the inhibition of PAM compounds on osteoclastogenesis did not result from its cytotoxicity. Therefore, these PAM derivatives could be used as potential leads for the development of a new type of antiosteoporosis agent