Hydrophobic Fusion Tags: Implications For Bioseparation and Cellular Expression

The studies in this thesis have shown that the partitioning of endoglucanase I (EGI, Cel7B) from T. reesei could be significantly improved by relatively minor genetic engineering. By adding short peptides composed of tryptophan and proline residues to EGI, extreme partitioning could be obtained. The site of the tag fusion was shown to be crucial for the efficiency of the tag. Methods suitable for large-scale purification of genetically modified EGI by a single-step extraction in aqueous two-phase systems have been established. The most optimal fusion protein, with respect to partitioning enhancement resulted, however, in impaired production in T. reesei. This was further elucidated and the low production was suggested to be caused by several factors such as proteolysis, impaired secretion and possible down-regulation of the promoter due to intracellular accumulation of the hydrophobic fusion protein. At certain stages during growth of the transformant expressing EGIcore-P5(WP)4 slight induction of the gene encoding the ER residual chaperone BIPI was detected. Furthermore, the amphiphilic protein hydrophobin I was utilized as a fusion tag to direct partitioning in aqueous two-phase systems. A system with improved separation features was evaluated, which is a clear progression from previously used systems both with respect to both robustness and purification properties. Applications towards large-scale purification with this system might be possible in the foreseeable future. Additionally, a novel approach for detergent removal after two-phase extraction in detergent based systems was developed. By addition of thermoseparating polymers, HM-EOPO or EOPO, phase separation could be induced by temperature increase, and thus the fusion protein could be recovered in a water phase. This method is both environmentally benign and displays compatibility with subsequent purification steps and handling of the target protein

Hypertension and cardiovascular risk factors in women

The aims of the thesis were to investigate the impact of pregnancy blood pressure, a current diagnosis of hypertension and blood pressure levels on neurohumoral, cardiovascular and metabolic status in postmenopausal women and thus to explore possible contributing mechanisms to the increased cardiovascular risk following hypertensive pregnancies. In this follow-up study after hypertensive- and normotensive pregnancies, 105 women were evaluated with the following methods: microneurography; office-, ambulatory- and central blood pressure measurements; anthropometric measurements; pulse wave velocity and augmentation index; carotid intima-media thickness; cardiovascular response to mental stress test and evaluation of perceived stress; echocardiography and laboratory analyses regarding metabolic and neurohumoral values. Another 160 women responded to a questionnaire regarding previous and present health. Women with previous hypertensive pregnancies had an increased prevalence of a diagnosis of hypertension, increased pulse wave velocity and affected metabolic parameters compared to women with previous normotensive pregnancies. These findings may partly explain the increased cardiovascular risk following hypertensive pregnancies. The sympathetic activity was only increased in women with previous hypertensive pregnancies and present hypertension. High self-reported perceived stress was associated with increased waist circumference which, in turn is related to an increased cardiovascular risk. Higher blood pressure levels were related to early signs of left ventricular diastolic dysfunction, emphasizing the importance of rigorous blood pressure control. Our study contributes with unique knowledge regarding women’s health many years after hypertensive and normotensive pregnancies. A diagnosis of present hypertension seems to be of major importance for the increased cardiovascular risk after hypertensive pregnancies, why maintenance of normotension is essential for women with previous hypertension pregnancies in order to retain cardiovascular health after menopause

Genetically engineered peptide fusions for improved protein partitioning in aqueous two-phase systems - Effect of fusion localization on endoglucanase I of Trichoderma reesei

Genetic engineering has been used for fusion of the peptide tag, Trp-Pro-Trp-Pro, on a protein to study the effect on partitioning in aqueous two-phase systems. As target protein for the fusions the cellulase, endoglucanase I (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, EGI, Cel7B) of Trichoderma reesei was used. For the first time a glycosylated two-domain enzyme has been utilized for addition of peptide tags to change partitioning in aqueous two-phase systems. The aim was to find an optimal fusion localization for EGI. The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker. The different constructs were expressed in the filamentous fungus T. reesei under the gpdA promoter from Aspergillus nidulans. The expression levels were between 60 and 100 mg/l. The partitioning behavior of the fusion proteins was studied in an aqueous two-phase model system composed of the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymer EO-PO (50:50) (EO50PO50) and dextran. The Trp-Pro-Trp-Pro tag was found to direct the fusion protein to the top EO50PO50 phase. The partition coefficient of a fusion protein can be predicted with an empirical correlation based on independent contributions from partitioning of unmodified protein and peptide tag in this model system. The fusion position at the end of the CBD, with the spacer Pro-Gly, was shown to be optimal with respect to partitioning and tag efficiency factor (TEF) was 0.87, where a fully exposed tag would have a TEF of 1.0. Hence, this position can further be utilized for fusion with longer tags. For the other constructs the TEF was only 0.43 and 0.10, for the tag fused to the truncated EGI and in the linker region of the full length EGI, respectively

Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells

The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B-max values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged MC4r corresponded to 0.25 mg active receptor/litre culture volume. Addition of a viral signal peptide at the N-terminus of the His-tagged MC4r did not improve the expression level. Confocal laser microscopy studies revealed that both the N- and C-terminally tagged MC4r did not accumulate intracellularly and were mainly located in the plasma membrane. The recombinant receptors showed similar affinity for the agonist NDP-MSH (K-d = 11 nM) as to MC4r expressed in mammalian cells. Functional coupling of the highest expressed C-terminal tagged receptor to endogenous Galpha protein was demonstrated through 6TPgammaS binding upon agonist stimulation of the receptor. K-i values for the ligands MTII, HS014, alpha-, beta-, and gamma-MSH are comparable to the values obtained for MC4r expressed in mammalian cells. (C) 2004 Elsevier Inc. All rights reserved

Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers

Endoglucanases (endo-1,4---glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide–propylene oxide random copolymer (EO50PO50) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)4 tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)2 tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)4 improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)

Extraction of endoglucanase I (Cel7B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system

Endoglucanases (EGI) (endo-1,4-β-d-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)2, (WP)4 or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)4 extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)4 fused to the catalytic module and a short sequence of the linker [EGIcore-P5(WP)4] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)4 tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran system. Hence, this construct will be suitable for further optimization of the extraction of the enzyme in a PEG 4000-sodium/potassium phosphate system from culture filtrate

Protein production and induction of the unfolded protein response in Trichoderma reesei strain Rut-C30 and its transformant expressing endoglucanase I with a hydrophobic tag

The effect of induction of protein production was studied in bioreactor cultures of T. reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag. The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production. Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations. Induction of the cellulase genes followed a similar temporal pattern in both strains, The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed. Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter. The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions. However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain

Parameters influencing protein extraction for whole broths in detergent based aqueous two-phase systems

The parameters important for an optimisation of cloud point extraction in technical scale were investigated using a genetically engineered fusion protein derived from endoglucanase I expressed in Trichoderma reesei and the nonionic polyoxyethylene Agrimul NRE 1205. The key parameters are temperature, detergent concentration, and additional salts. These parameters are interdependent, thus there is an optimum in the partition coefficient with respect to detergent concentration and a maximum for the partition coefficient and the yield with respect to temperature. These results were confirmed for the detergent C12E5 to demonstrate that these optima are due to the nature of polyoxyethylenes. Cloud point extraction was found to be only slightly affected by pH. In the case studied extraction of whole broth is favourable for a high yield and partition coefficient, since fusion protein adhering to the cells can be solubilized. However some loss of detergent which remains in the fungal biomass was observed

Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.

Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials