The purification of pepsinogen from the stomach of red perch using aqueous two phase systems (ATPS) formed by polyethylene glycol (PEG) and salt at 4°C was optimized. Salt type, salt concentration, PEG molecular weight and PEG concentration had significant effects on total volume (TV), volume ratio (VR), enzyme activity (AE), protein content (CP), specific activity (SA), purification fold (PF) and recovery yield (RY). (NH4)2SO4 at 15% w/w concentration was selected as the optimum salt type and concentration. PEG 1500 at 18% w/w concentration was selected as the optimum PEG molecular weight and concentration. 15% (NH4)2SO4-18% PEG 1500, the optimal ATPS, was compared with ammonium sulfate fractionation (ASF). ATPS gave better partition of pepsinogen (SA of 5.40 U/mg, PF of 5.20 and RY of 86.6%) than ASF (SA of 2.55 U/mg, PF of 2.46, RY of 70.4%). / This is the electronic copy of partition of pepsinogen in aqueous two phase system method.

Counter current chromatography (CCC) is a form of liquid liquid chromatography, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. In this thesis, its application has been successfully extended to the rapid, scalable purification of monoclonal antibodies (mAb) from mammalian cell culture, using aqueous two-phase systems (ATPS) of inorganic salts and polymer. A polyethylene glycol (PEG) and sodium citrate system was found to be the most appropriate by robotic phase system selection. The search for an economical alternative to protein A HPLC is a substantial bioprocessing concern; in this work CCC has been investigated. Initial studies showed that unpredictably, despite separation from impurities being achieved, some loss in the IgG‘s ability to bind to Protein A was seen, as confirmed by Protein A BiaCore analysis. CCC machines were seen to adversely affect IgG functionality. This led to a systematic investigation of the effect of CCC phase mixing on IgG functionality in a number of different CCC instruments, allowing direct comparisons of modes of CCC (hydrodynamic and hydrostatic CCC) and their associated mixing (wave-like and cascade, respectively). The varying g forces produced within the CCC column were determined using a recently developed model to calculate g force range. The effect of interfacial tension was also studied using a custom built 'g' shaker. The optimum CCC mode was identified to be the non synchronous CCC, operated in a hydrodynamic mode but allowing bobbin to rotor speed (Pr ratio) to be controlled independently. In a normal synchronous J type centrifuge a Pr of 1 is fixed, this is where the bobbin and rotor speed are identical I.e. one bobbin rotation (where mixing occurs) to one rotor revolution (where settling occurs). Constraints were seen with this 1:1 ratio and the separation of mAb using ATPS. This work has shown with the use of the non synchronous CCC at a Pr of 0.33, mixing is reduced and rotor rotations increased. Consequently the associated g force range is decreased. Furthermore, by the extension of settling time, the clear separation of the mAb from impurities has been achieved with retention of biological activity. This thesis demonstrates the importance of settling time for ATPS in phase separation and documents the fundamental requirements for the successful separation of biologics. Purified non synchronous CCC samples have additionally undergone rigorous quality control testing at Lonza Biologics by their purification scientists. This work has ultimately showed that with optimisation, the non synchronous CCC can be used to produce biological samples that are of industry standard.

A procedure for the preparation of spinach leaf mitochondria was developed. The procedure combines differential centrifugation, partition in dextran- polyethyleneglycol two-phase system and Percoli density gradient centri- fugation. The different steps separate the material mainly according to size, surface properties and density, respectively. No chlorophyll was present in the final mitochondrial preparation and the mitochondria were also markedly enriched relative to peroxisomes and microsomes as esti­mated from the recovery of marker enzymes. The latency of enzyme activities was used to study the apparent intactness of the mitochondrial membranes. These measurements showed that both the inner and outer mitochondrial membranes were more than 90 % intact. The mitochondria were also functionally intact since the coupling between respiration and oxidative phosphorylation was retained. The purity of the preparation made it possible to study cytochromes from leaf mitochondria. The cytochrome content of stalk and leaf mitochondria was measured in order to compare mitochondria from photosynthesizing and non-photosynthesizing tissue. The measurements were performed by difference spectroscopy both at room temperature and at liquid nitrogen temperature. Qualitatively the cytochrome content in mitochondria from stalks and leaves was identical. Quantiatively leaf mitochondria contained,on a protein basis, only half the amount of the different cytochromes as compared to stalk mitochondria. The relative content of the different cytochromes was, however, similar suggesting that the composition of the respiratory chain was the same. The photorespiratory conversion of glycine to serine takes place in the mitochondria and involves oxidative decarboxylation of glycine. The ability to oxidize glycine via the respiratory chain was present in spinach leaf mitochondria, but absent in mitochondria prepared from roots, stalks and leaf veins from the same plants. This confirmed the specific localization of the glycine oxidizing activity to photosyntheticaliy active tissue, as suggested by studies with other plant material. The conversion of glycine to serine is a complex reaction depending on the combined action of two enzymes: glycine decarboxylase and serine hydroxymethyltransferase. The effect of inhibitors on the serine hydroxy­methyl transferase activity and the rate of the glycine bicarbonate exchange reaction associated with glycine decarboxylase was studied. These reactions represent partial steps in the conversion of glycine to serine and the aim was to investigate the site of inhibition for the different inhibitors, namely, isonicotinyl hydrazide (a pyridoxa!phosphate antagonist), amino- acetonitrile, glycinehydroxamate (glycine analogues) and cyanide. The results showed that these inhibitors had a complex pattern of inhibition. The same inhibitor affected more than one site and often with an apparently different mechanism. It was, however, found that aminoacetonitrile at low concentrations specifically inhibited glycine decarboxylase and that cyanide specifically inhibited serine hydroxymethyltransferase. / digitalisering@umu

Submitted by Raquel Vergara Gondran (raquelvergara38@yahoo.com.br) on 2016-04-22T03:53:34Z No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) / Rejected by Gilmar Barros (gilmargomesdebarros@gmail.com), reason: Faltou colocar o nome do co-orientador. on 2016-04-22T14:51:38Z (GMT) / Submitted by Raquel Vergara Gondran (raquelvergara38@yahoo.com.br) on 2016-04-25T19:34:12Z No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) / Rejected by dayse paz (daysepaz@hotmail.com), reason: Tens que incluir co-orientador ele segue sendo o segundo. Atenciosamente Equipe RI-FURG on 2016-04-26T14:45:06Z (GMT) / Submitted by Raquel Vergara Gondran (raquelvergara38@yahoo.com.br) on 2016-04-26T15:32:29Z No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) / Approved for entry into archive by dayse paz (daysepaz@hotmail.com) on 2016-04-26T15:37:03Z (GMT) No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) / Made available in DSpace on 2016-04-26T15:37:03Z (GMT). No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) Previous issue date: 2011 / A purificação de proteínas consiste em um dos desafios da área de Bioprocessos. O grau de pureza requerido para enzimas está diretamente relacionado com a aplicação na qual estas serão empregadas. Os sistemas aquosos bifásicos (SAB) e o processo de separação por membranas (UF) vêm sendo amplamente estudados na purificação de diversas proteínas, por apresentarem algumas vantagens sobre os processos cromatográficos, como facilidade de escalonamento e baixo custo. No entanto, a purificação de lipases por SAB ainda é pouco reportada. Neste sentido, o foco deste trabalho foi avaliar a utilização da melhor combinação do SAB formado por polietilenoglicol (PEG) e tampão fosfato de potássio e a combinação com o processo de separação por membrana, na purificação de lipase de Penicillium crustosum produzida por fermentação em estado sólido. O SAB se apresentou como uma técnica potencial para a separação e purificação da lipase obtendo-se fator de purificação 5,8 no sistema composto de 20% PEG 6000, 7,0% fosfato de potássio pH 8. A técnica do planejamento experimental foi utilizada como uma estratégia para a maximização da purificação, no qual foram avaliadas as concentrações de polietilenoglicol (PEG), fosfato de potássio e NaCl em sistemas compostos por PEG 6000 fosfato de potássio pH 8. O sistema 20% PEG 6000, 5,8% fosfato de potássio pH 8 com acréscimo de 4,8% de NaCl apresentou recuperação da atividade de hidrólise de 53,8% e o maior fator de purificação 10,5. O sistema composto de 20% PEG 6000, 5,8% de fosfato de potássio sem o acréscimo de NaCl foi o que apresentou maior atividade de transesterificação e fator de purificação, 8,5 U/mg e 28,3 vezes, respectivamente. Membranas comerciais com massa molecular de corte de 30 e 60 kDa foram testadas. Com acréscimo de 0,09 mol/L de NaCl o fator de purificação aumentou de 0,8 para 1,4 vezes. Quando combinou-se o sistema aquoso bifásico e separação por membrana, o fator de purificação em termos de atividade de hidrólise diminuiu, mas com relação à de atividade de transesterificação aumentou, alcançando valores em torno de 39 vezes para os sistemas compostos de 11,6% PEG 6000, 10% fosfato de potássio e 1,2% NaCl e 38,3 para o sistema formado por 16% PEG 6000, 8% fosfato de potássio e 4,8% NaCl. / The purification of proteins is one of the challenges in the area of Bioprocesses. The purity degree required for enzymes is directly related to the application in which they are employed. Aqueous two-phase systems (ATPS) and membrane separation process have been investigated in the purification of several proteins, which present some advantages over the chromatographic processes, such as easy scale-up and low cost. However, the purification of lipases by ATPS has been little reported. In this sense, the focus of this study was to evaluate the use of the best combination of the ATPS consisting of polyethylene glycol (PEG) and potassium phosphate buffer and the combination with membrane separation process for the lipases purification from Penicillium crustosum produced by solid state fermentation. The ATPS is a potential technique for the separation and purification of such lipases, resulting in a purification factor 5.8 using the system composed of 20% PEG 6000, 7.0% potassium phosphate, pH 8. The technique of experimental design was used as a strategy for maximizing the purification, in which we assessed the concentrations of polyethylene glycol (PEG), potassium phosphate and sodium chloride in systems composed of PEG 6000 at pH 8. The system 20% PEG 6000, 5.8% potassium phosphate at pH 8, and 4.8% NaCl showed a recovery of the activity (hydrolysis) of 53.8% and yielded the highest purification factor 10.5, based on hydrolytic activity. The system made up of 20% PEG 6000, 5.8% potassium phosphate without the addition of NaCl showed highest activity of transesterification and purification factor, 8.5 U/mg and 28,3, respectively. Commercial membranes with molecular weight cutoff of 30 and 60 kDa were tested. That addition of 0.09 mol/L NaCl to the enzymatic extract in the feed increased the purification factor from 0.8 to 1.4. When the processes of ATPS and UF were combined, the purification factor in terms of hydrolytic activity decreased, but increased in terms of transesterification activity, reaching values around 39 for PF in the systems composed of 11.6% PEG 6000 10% potassium phosphate and 38,3 in the systems composed of 1.2% NaCl and 16% PEG 6000, 8% potassium phosphate and 4.8% NaCl.

No description available.

近年発酵生産が注目されているコハク酸の抽出分離法についてイオン液体を含む液膜法及び水性２相抽出法を用いて研究した．イオン液体を含む含浸型液膜により，受容相に塩酸水溶液を用いることでコハク酸の上り坂輸送を達成できた．次にイオン液体を含む高分子溶媒膜によるコハク酸の透過を検討した．最適な条件は含浸液膜と異なり，透過にはコハク酸の各化学種が関与していた．最後に水溶性有機溶媒やイオン液体を用いた水性２相抽出法によるコハク酸の抽出を行った．塩および糖を相分離剤とすることで，発酵液で想定されるコハク酸の80％以上の抽出が達成できた． / Succinic acid is widely used in many industrial sectors; pharmacy, textile, food, etc. The bio-production of succinic acid from renewable resource was desired because the petrochemical production of succinic acid became costly. In this study, the extraction and separation techniques of succinic acid by liquid membranes (LMs) and aqueous two-phase system (ATPS) containing ionic liquids (ILs) were exploited. The permeation mechanisms and optical conditions were elucidated. / 博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University

No description available.

O ácido clavulânico (AC) é um potente inibidor de β-lactamases utilizado na área médica. Métodos alternativos, econômicos e simples para sua purificação são de grande interesse. Este trabalho objetivou produzir e extrair AC de Streptomyces spp. por fermentação extrativa utilizando sistema de duas fases aquosas (SDFA) - polietileno glicol (PEG)/sais fosfato. Foi selecionado o melhor produtor de AC entre sete linhagens de Streptomyces spp. Avaliou-se a influência de cinco fatores no cultivo do melhor produtor em frascos agitados (pH, temperatura, velocidade de agitação, concentrações das fontes de nitrogênio e de carbono), utilizando planejamento experimental estatístico. Definidas as melhores condições de cultivo, foram estudadas a produção e a extração do AC em fermentação extrativa utilizando SDFA em frascos agitados e em sistema descontínuo utilizando biorreator. Em biorreator também foram realizados o estudo termodinâmico do processo de fermentação nas condições ótimas obtidas nas etapas anteriores e a determinação do coeficiente volumétrico de transferência de massa (kLa), comparando os sistemas de fermentação no meio de cultivo simples (SF) e fermentação extrativa utilizando sistema SDFA PEG/sais fosfato (SFE) sem e com crescimento microbiano. A linhagem de Streptomyces selecionada como a melhor produtora de AC foi a DAUFPE 3060, a qual apresentou a maior produção desse inibidor, 494 mg/L em 48h, em frascos agitados nas condições: pH 6,0, 32°C, 150 rpm, 5 g/L de glicerol e 20 g/L de farinha de soja. Após a etapa de otimização realizada para o estudo da temperatura e da concentração de farinha de soja, variáveis mais significativas no estudo de seleção, a temperatura e a concentração de farinha de soja ótimas, foram 32°C e 40 g/L, respectivamente, com produção de 629 mg/L de AC em 48h. O estudo termodinâmico confirmou que a temperatura de 32°C é a máxima de produção do AC; após esse valor, inicia-se, gradualmente, a degradação do AC. No estudo da determinação do coeficiente de transferência de massa, kLa, sem crescimento microbiano, observaram-se valores maiores de kLa para o SF, devido à viscosidade do PEG utilizado no SFE. A massa molar do PEG e a velocidade de agitação foram as variáveis que mais influenciaram na extração de AC no SFE em frascos agitados, apresentando comportamento semelhante em biorreator. E, finalmente, o estudo da transferência de oxigênio do SFE utilizando SDFA com crescimento microbiano foi avaliado para otimizar a produção e a extração de AC. Os resultados obtidos demonstraram que existe uma faixa ideal de velocidade de agitação e de aeração para evitar o rompimento celular e aumentar a recuperação de AC. / Clavulanic acid (CA) is a potent inhibitor of β-lactamases used in the medical field. Alternative methods, economic and simple purification are of great interest. This PhD project aims to produce and extract clavulanic acid of Streptomyces spp. By extractive fermentation using aqueous two-phase system (ATPS) - Polyethylene glycol (PEG)/phosphate salts. The best producer of clavulanic acid among seven strains of Streptomyces spp was selected. The influence of five factors in the cultivation of the best producer in flasks (pH, temperature, agitation velocity, concentrations of nitrogen and carbon sources) using statistical experimental design was evaluated. Defined the best cultivation conditions, the production and extraction of clavulanic acid by extractive fermentation using ATPS in flasks and in a batch system using a bioreactor was analyzed. In batch system using a bioreactor were also carried out the thermodynamic study of the fermentation process in optimum conditions determined in previous steps and also determined the volumetric mass transfer coefficient (kLa) comparing the fermentation systems in simple culture medium (SF) and in a extractive fermentation using aqueous two-phase system (ATPS) PEG/phosphate salts (SEF) medium with and without microbial growth. A strain of Streptomyces spp. selected as the best producer of AC was DAUFPE 3060, which showed the highest production of this inhibitor, 494 mg/L at 48h, in flasks under the conditions of pH 6.0, 32 °C, 150 rpm, 5 g/L of glycerol and 20 g/L of soybean flour. After the optimization step, the most significant variables in the study selection, temperature and concentration of soybean flour, were studied. The optimal values were 32 °C and 40 g/L of temperature and soybean flour concentration, respectively, with production of 629 mg/L of CA after 48h of cultivation. The thermodynamic study confirmed that 32 °C is the maximum temperature production of CA, after this value, starts gradually, the degradation of CA. In the study of volumetric mass transfer coefficient, kLa, without microbial growth, showed higher values of kLa for the SF, because the high viscosity of the PEG used in the SFE. The PEG molar mas and agitation velocity were the variables that most influenced the extraction of CA in flasks using a SFE, with similar behavior in a bioreactor. Finally, the study of oxygen transfer rate in SFE using ATPS with microbial growth was evaluated to optimize the production and extraction of CA. The results showed that there is an ideal range of agitation and aeration to prevent cell disruption and increase the CA recovery.

O ácido clavulânico (AC) é um potente inibidor de β-lactamases utilizado na área médica. Métodos alternativos, econômicos e simples para sua purificação são de grande interesse. Este trabalho objetivou produzir e extrair AC de Streptomyces spp. por fermentação extrativa utilizando sistema de duas fases aquosas (SDFA) - polietileno glicol (PEG)/sais fosfato. Foi selecionado o melhor produtor de AC entre sete linhagens de Streptomyces spp. Avaliou-se a influência de cinco fatores no cultivo do melhor produtor em frascos agitados (pH, temperatura, velocidade de agitação, concentrações das fontes de nitrogênio e de carbono), utilizando planejamento experimental estatístico. Definidas as melhores condições de cultivo, foram estudadas a produção e a extração do AC em fermentação extrativa utilizando SDFA em frascos agitados e em sistema descontínuo utilizando biorreator. Em biorreator também foram realizados o estudo termodinâmico do processo de fermentação nas condições ótimas obtidas nas etapas anteriores e a determinação do coeficiente volumétrico de transferência de massa (kLa), comparando os sistemas de fermentação no meio de cultivo simples (SF) e fermentação extrativa utilizando sistema SDFA PEG/sais fosfato (SFE) sem e com crescimento microbiano. A linhagem de Streptomyces selecionada como a melhor produtora de AC foi a DAUFPE 3060, a qual apresentou a maior produção desse inibidor, 494 mg/L em 48h, em frascos agitados nas condições: pH 6,0, 32°C, 150 rpm, 5 g/L de glicerol e 20 g/L de farinha de soja. Após a etapa de otimização realizada para o estudo da temperatura e da concentração de farinha de soja, variáveis mais significativas no estudo de seleção, a temperatura e a concentração de farinha de soja ótimas, foram 32°C e 40 g/L, respectivamente, com produção de 629 mg/L de AC em 48h. O estudo termodinâmico confirmou que a temperatura de 32°C é a máxima de produção do AC; após esse valor, inicia-se, gradualmente, a degradação do AC. No estudo da determinação do coeficiente de transferência de massa, kLa, sem crescimento microbiano, observaram-se valores maiores de kLa para o SF, devido à viscosidade do PEG utilizado no SFE. A massa molar do PEG e a velocidade de agitação foram as variáveis que mais influenciaram na extração de AC no SFE em frascos agitados, apresentando comportamento semelhante em biorreator. E, finalmente, o estudo da transferência de oxigênio do SFE utilizando SDFA com crescimento microbiano foi avaliado para otimizar a produção e a extração de AC. Os resultados obtidos demonstraram que existe uma faixa ideal de velocidade de agitação e de aeração para evitar o rompimento celular e aumentar a recuperação de AC. / Clavulanic acid (CA) is a potent inhibitor of β-lactamases used in the medical field. Alternative methods, economic and simple purification are of great interest. This PhD project aims to produce and extract clavulanic acid of Streptomyces spp. By extractive fermentation using aqueous two-phase system (ATPS) - Polyethylene glycol (PEG)/phosphate salts. The best producer of clavulanic acid among seven strains of Streptomyces spp was selected. The influence of five factors in the cultivation of the best producer in flasks (pH, temperature, agitation velocity, concentrations of nitrogen and carbon sources) using statistical experimental design was evaluated. Defined the best cultivation conditions, the production and extraction of clavulanic acid by extractive fermentation using ATPS in flasks and in a batch system using a bioreactor was analyzed. In batch system using a bioreactor were also carried out the thermodynamic study of the fermentation process in optimum conditions determined in previous steps and also determined the volumetric mass transfer coefficient (kLa) comparing the fermentation systems in simple culture medium (SF) and in a extractive fermentation using aqueous two-phase system (ATPS) PEG/phosphate salts (SEF) medium with and without microbial growth. A strain of Streptomyces spp. selected as the best producer of AC was DAUFPE 3060, which showed the highest production of this inhibitor, 494 mg/L at 48h, in flasks under the conditions of pH 6.0, 32 °C, 150 rpm, 5 g/L of glycerol and 20 g/L of soybean flour. After the optimization step, the most significant variables in the study selection, temperature and concentration of soybean flour, were studied. The optimal values were 32 °C and 40 g/L of temperature and soybean flour concentration, respectively, with production of 629 mg/L of CA after 48h of cultivation. The thermodynamic study confirmed that 32 °C is the maximum temperature production of CA, after this value, starts gradually, the degradation of CA. In the study of volumetric mass transfer coefficient, kLa, without microbial growth, showed higher values of kLa for the SF, because the high viscosity of the PEG used in the SFE. The PEG molar mas and agitation velocity were the variables that most influenced the extraction of CA in flasks using a SFE, with similar behavior in a bioreactor. Finally, the study of oxygen transfer rate in SFE using ATPS with microbial growth was evaluated to optimize the production and extraction of CA. The results showed that there is an ideal range of agitation and aeration to prevent cell disruption and increase the CA recovery.

The bacterium Escherichia coli (E. coli) is in many situations an ideal host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve sufficiently high product quantities. However, there are several factors that may limit this host’s ability to produce large amounts of heterologous proteins in a soluble and native form. For many applications a high purity of the recombinant protein is demanded, which implies a purification strategy where the product efficiently can be isolated from the complex milieu of host cell contaminants. In this thesis, different strategies based on both rational and combinatorial genetic engineering principles have been investigated, aiming at improving and facilitating recombinant E. coli protein production and purification. One objective was to improve the PEG/salt aqueous two-phase system (ATPS) purification process of the lipase cutinase, by increasing the selectivity of the protein for the system top-phase. Peptide tags, with varying properties, were designed and genetically fused to the C-terminal end of ZZ-cutinase. Greatly increased partitioning values were observed for purified protein variants fused to tryptophan containing peptide tags, particularly a (WP)4 peptide. The partitioning properties of the ZZ-cutinase-(WP)4 protein were also retained when added to the ATPS directly from an E. coli total cell disintegrate, emphasizing the applicability of this genetic engineering strategy for primary protein purification in ATPSs. Further on, a combinatorial library approach using phage display technology was investigated as a tool for identification of peptide tags capable of improving partitioning properties of ZZ-cutinase in an ATPS. Repeated ATPS-based partitioning-selection cycles of a large phagemid (pVIII) peptide library, resulted in isolation of phage particles preferentially decorated with peptides rich in tyrosine and proline residues. Both a peptide corresponding to a phage library derived peptide sequence as well as peptides designed based on information of amino acid appearance frequencies in later selection rounds, were shown to improve partitioning several-fold when genetically fused to the C-terminal end of ZZ-cutinase. From the two- to four–fold increased production yields observed for these fusion proteins compared to ZZ-cutinase-(WP)4, it was concluded that the selection system used allowed for selection of desired peptide properties related to both partitioning and E. coli protein production parameters. Bacterial protein production is affected by several different mRNA and protein sequence-related features. Attempts to address single parameters in this respect are difficult due to the inter-dependence of many features, for example between codon optimization and mRNA secondary structure effects. Two combinatorial expression vector libraries (ExLib1 and ExLib2) were constructed using a randomization strategy that potentially could lead to variations in many of these sequence-related features and which would allow a pragmatic search of vector variants showing positive net effects on the level of soluble protein production. ExLib1 was constructed to encode all possible synonymous codons of an eight amino acid N-terminal extension of protein Z, fused to the N-terminal of an enhanced green fluorescent reporter protein (EGFP). In ExLib2, the same eight positions were randomized using an (NNG/T) degeneracy code, which could lead to various effects on both the nucleotide and protein level, through the introduction of nucleotide sequences functional as e.g. alternative ribosome binding or translation initiation sites or as translated codons for an Nterminal extension of the target protein by a peptide sequence. Flow cytometric analyses and sorting of library cell cultures resulted in isolation of clones displaying several-fold increases in whole cell fluorescence compared to a reference clone. SDS-PAGE and western blot analyses verified that this was a result of increases (up to 24-fold) in soluble intracellular ZEGFP product protein content. Both position specific codon bias effects and the appearance of new ribosomal binding sites in the library sequences were concluded to have influenced the protein production. To explore the possibility of applying the same combinatorial library strategy for improving soluble intracellular production of heterologous proteins proven difficult to express in E. coli, three proteins with either bacterial (a transcriptional regulator (DntR)) or human (progesterone receptor ligand binding domain (PRLBD) and 11-β Hydroxysteroid dehydrogenase type I (11-β)) origin, were cloned into the ExLib2 library. Flow cytometric sorting of libraries resulted in isolation of DntR library clones showing increased soluble protein production levels and PR-LBD library clones with up to ten-fold increases in whole cell fluorescence, although the product under these conditions co-separated with the insoluble cell material. / QC 20100623