Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (μ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Genetic and environmental influences on blood pressure and physical activity: a study of nuclear families from Muzambinho, Brazil

Blood pressure (BP) and physical activity (PA) levels are inversely associated. Since genetic factors account for the observed variation in each of these traits, it is possible that part of their association may be related to common genetic and/or environmental influences. Thus, this study was designed to estimate the genetic and environmental correlations of BP and PA phenotypes in nuclear families from Muzambinho, Brazil. Families including 236 offspring (6 to 24 years) and their 82 fathers and 122 mothers (24 to 65 years) were evaluated. BP was measured, and total PA (TPA) was assessed by an interview (commuting, occupational, leisure time, and school time PA). Quantitative genetic modeling was used to estimate maximal heritability (h²), and genetic and environmental correlations. Heritability was significant for all phenotypes (systolic BP: h² = 0.37 ± 0.10, P < 0.05; diastolic BP: h² = 0.39 ± 0.09, P < 0.05; TPA: h² = 0.24 ± 0.09, P < 0.05). Significant genetic (r g) and environmental (r e) correlations were detected between systolic and diastolic BP (r g = 0.67 ± 0.12 and r e = 0.48 ± 0.08, P < 0.05). Genetic correlations between BP and TPA were not significant, while a tendency to an environmental cross-trait correlation was found between diastolic BP and TPA (r e = -0.18 ± 0.09, P = 0.057). In conclusion, BP and PA are under genetic influences. Systolic and diastolic BP share common genes and environmental influences. Diastolic BP and TPA are probably under similar environmental influences.

Defining Sales and Operations Planning Process in an Environmental Management Company

Sales and operations research publications have increased significantly in the last decades. The concept of sales and operations planning (S&OP) has gained increased recognition and has been put forward as the area within Supply Chain Management (SCM). Development of S&OP is based on the need for determining future actions, both for sales and operations, since off-shoring, outsourcing, complex supply chains and extended lead times make challenges for responding to changes in the marketplace when they occur. Order intake of the case company has grown rapidly during the last years. Along with the growth, new challenges considering data management and information flow have arisen due to increasing customer orders. To manage these challenges, case company has implemented S&OP process, though initial process is in early stage and due to this, the process is not managing the increased customer orders adequately. Thesis objective is to explore extensively the S&OP process content of the case company and give further recommendations. Objectives are categorized into six different groups, to clarify the purpose of this thesis. Qualitative research methods used are active participant observation, qualitative interviews, enquiry, education, and a workshop. It is notable that demand planning was felt as cumbersome, so it is typically the biggest challenge in S&OP process. More proactive the sales forecasting can be, more expanded the time horizon of operational planning will turn out. S&OP process is 60 percent change management, 30 percent process development and 10 percent technology. The change management and continuous improvement can sometimes be arduous and set as secondary. It is important that different people are required to improve the process and the process is constantly evaluated. As well as, process governance is substantially in a central role and it has to be managed consciously. Generally, S&OP process was seen important and all the stakeholders were committed to the process. Particular sections were experienced more important than others, depending on the stakeholders’ point of views. Recommendations to objective groups are evaluated by the achievable benefit and resource requirement. The urgent and easily implemented improvement recommendations should be executed firstly. Next steps are to develop more coherent process structure and refine cost awareness. Afterwards demand planning, supply planning, and reporting should be developed more profoundly. For last, information technology system should be implemented to support the process phases.

A Web-Based Environmental Toolkit to Support Small and Medium-Sized Enterprises in the Implementation of Their Own Environmental Management System

With small and medium sized-enterprises (SMEs) taking up the majority of the global businesses, it is important they act in an environmentally responsible manner. Environmental management systems (EMS) help companies evaluate and improve their environmental impact but they often require human, financial, and temporary resources that not all SMEs can provide. This research encompasses interviews with representatives of two small enterprises in Germany to provide insights into their understanding, and knowledge of an EMS and how they perceive their responsibility towards the environment. Furthermore, it presents a toolkit created especially for small and medium-sized enterprises that serves as a simplified version of an EMS based on the ISO 14001 standard and is evaluated by the representatives of the SMEs. Some of the findings are: while being open to the idea of improving their environmental impact, SMEs do not always feel it is their responsibility to do so; they seem to lack the means to fully implement an EMS. The developed toolkit is considered useful and usable and recommendations are drawn for its future enhancement.

Improving Cost-Efficiency and Reducing Environmental Impacts of Intermodal Transportation with Dry Port Concept – Major Rail Transport Corridor in Baltic Sea Region

Transportation plays a major role in the gross domestic product of various nations. There are, however, many obstacles hindering the transportation sector. Cost-efficiency along with proper delivery times, high frequency and reliability are not a straightforward task. Furthermore, environmental friendliness has increased the importance of the whole transportation sector. This development will change roles inside the transportation sector. Even now, but especially in the future, decisions regarding the transportation sector will be partly based on emission levels and other externalities originating from transportation in addition to pure transportation costs. There are different factors, which could have an impact on the transportation sector. IMO’s sulphur regulation is estimated to increase the costs of short sea shipping in the Baltic Sea. Price development of energy could change the roles of different transport modes. Higher awareness of the environmental impacts originating from transportation could also have an impact on the price level of more polluting transport modes. According to earlier research, increased inland transportation, modal shift and slowsteaming can be possible results of these changes in the transportation sector. Possible changes in the transportation sector and ways to settle potential obstacles are studied in this dissertation. Furthermore, means to improve cost-efficiency and to decrease environmental impacts originating from transportation are researched. Hypothetical Finnish dry port network and Rail Baltica transport corridor are studied in this dissertation. Benefits and disadvantages are studied with different methodologies. These include gravitational models, which were optimized with linear integer programming, discrete-event and system dynamics simulation, an interview study and a case study. Geographical focus is on the Baltic Sea Region, but the results can be adapted to other geographical locations with discretion. Results indicate that the dry port concept has benefits, but optimization regarding the location and the amount of dry ports plays an important role. In addition, the utilization of dry ports for freight transportation should be carefully operated, since only a certain amount of total freight volume can be cost-efficiently transported through dry ports. If dry ports are created and located without proper planning, they could actually increase transportation costs and delivery times of the whole transportation system. With an optimized dry port network, transportation costs can be lowered in Finland with three to five dry ports. Environmental impacts can be lowered with up to nine dry ports. If more dry ports are added to the system, the benefits become very minor, i.e. payback time of investments becomes extremely long. Furthermore, dry port network could support major transport corridors such as Rail Baltica. Based on an analysis of statistics and interview study, there could be enough freight volume available for Rail Baltica, especially, if North-West Russia is part of the Northern end of the corridor. Transit traffic to and from Russia (especially through the Baltic States) plays a large role. It could be possible to increase transit traffic through Finland by connecting the potential Finnish dry port network and the studied transport corridor. Additionally, sulphur emission regulation is assumed to increase the attractiveness of Rail Baltica in the year 2015. Part of the transit traffic could be rerouted along Rail Baltica instead of the Baltic Sea, since the price level of sea transport could increase due to the sulphur regulation. Both, the hypothetical Finnish dry port network and Rail Baltica transport corridor could benefit each other. The dry port network could gain more market share from Russia, but also from Central Europe, which is the other end of Rail Baltica. In addition, further Eastern countries could also be connected to achieve higher potential freight volume by rail.

Enzymatic Synthesis, Recovery, and Purification of Ethyl β-ᴅ-glucopyranoside

A method to synthesize ethyl β-ᴅ-glucopyranoside (BEG) was searched. Feasibility of different ion exchange resins was examined to purify the product from the synthetic binary solution of BEG and glucose. The target was to produce at least 50 grams of 99 % pure BEG with a scaled up process. Another target was to transfer the batch process into steady-state recycle chromatography process (SSR). BEG was synthesized enzymatically with reverse hydrolysis utilizing β-glucosidase as a catalyst. 65 % of glucose reacted with ethanol into BEG during the synthesis. Different ion exchanger based resins were examined to separate BEG from glucose. Based on batch chromatography experiments the best adsorbent was chosen between styrene based strong acid cation exchange resins (SAC) and acryl based weak acid cation exchange resins (WAC). CA10GC WAC resin in Na+ form was chosen for the further separation studies. To produce greater amounts of the product the batch process was scaled up. The adsorption isotherms for the components were linear. The target purity was possible to reach already in batch without recycle with flowrate and injection size small enough. 99 % pure product was produced with scaled-up batch process. Batch process was transferred to SSR process utilizing the data from design pulse chromatograms and Matlab simulations. The optimal operating conditions for the system were determined. Batch and SSR separation results were compared and by using SSR 98 % pure products were gained with 40 % higher productivity and 40 % lower eluent consumption compared to batch process producing as pure products.

Impact of increased environmental awareness on product marketing

This Bachelor thesis studies how companies use a variety of different sustainable marketing approaches to sell their green products. Empirical part is done by researching and interviewing two B2B furniture companies.