Investigation of cloning strategies for A. thaliana G Protein [alpha]-subunit gene in Pichia Pastoris

In this thesis a strategy was developed to clone and express the gene of the A. thaliana heterotrimeric G-protein [alpha] subunit (GPA1). For this purpose an appropriate eukaryotic expression system was chosen to produce large quantities of high purity recombinant protein. GPA1 was amplified by PCR and cloned using a Pichia pastoris expression system. Two different plasmids pPICZC+GPA1 and pPICZ[alpha]B+GPA1' were constructed. pPICZC+GPA1 was designed for intracellular expression whereas pPICZ[alpha]B+GPA1' contained a signal peptide facilitating secretion of the recombinant protein into the extracellular medium. The possibility of using different yeast strains that may improve expression was explored. Recombinant synthesis of GPA1 was achieved with the pPICZC+GPA1 construct using the strain GS115, which shows Mut[+] phenotype. Expression was followed by monitoring growth of yeast as well as western blots of cellular extracts at different time points during induction. This study describes the first report of expression of A. thaliana GPA1 gene in a eukaryotic system and constitutes a critical step forward in studies of G-proteins in plants. It follows to reason that the availability of purified recombinant GPA1 will enable biochemical characterization, comparison with its mammalian counterparts and facilitate structural studies

Structural investigation of g-protein signaling in plants

Heterotrimeric G proteins, composed of alpha, beta and gamma subunits, are a major group of signaling molecules in eukaryotic organisms. There is lack of direct biophysical and structural data for the plant heterotrimer unlike its mammalian counterparts. Heterotrimeric G protein subunits from Arabidopsis were cloned and purified. The alpha subunit, GPA1 was purified from Pichia, with a GTP binding ratio of 0.3 mole GTP/ mole protein. The recombinant beta (AGB1) and gamma (AGG1 and AGG2) subunits were isolated from E.coli and preliminary purification strategies were optimized. This is to our knowledge, the first study to report recombinant production of a plant beta subunit and in vitro dimerization of purified AGB1-AGG2 subunits. GPA1 was purified in two different biophysical states, as characterized by UVspectroscopy, dynamic light scattering, circular dichroism spectropolarimetry and mass spectrometry. The stable oligomeric form had higher GTP hydrolysis activity and a GDP binding ratio of 1.4 mole GDP/mole protein. Indirect biophysical evidence points to interaction of GPA1 with receptor mimetic compounds, membrane fractions of yeast cells and the recombinant AGB1-AGG2 dimer. This is to our knowledge, the first study showing the expression and purification of the plant alpha subunit from a eukaryotic expression system and its detailed biophysical characterization. Small angle solution X-ray scattering (SAXS) measurements verified the oligomeric nature of the protein, which was stabilized via detergent micelles. The detergent content was verified by proton nuclear magnetic resonance spectroscopy. SAXS patterns were consistent with dimeric protein complexing with micelles. Rigid body modeling was used for further modeling

Structural Modelling and Structure-Function Analysis of <i>Zymomonas mobilis</i> Levansucrase

Levansucrases are bacterial enzymes which produce fructan polymers from sucrose via hydrolysis and transfructosylation activities. These polymers; levan and fructooligosaccharides are valuable for food and pharmaceutical industries. Levansucrases from Gram-positive bacteria such as Bacillus subtilis tend to produce levan, while those from Gram-negative bacteria preferentially produce fructooligosaccharides. Zymomonas mobilis is an efficient levansucrase producer and its extracellular levansucrase can produce both fructooligosaccharides and levan depending on the reaction parameters. In this study, the structure of Z. mobilis levansucrase was modeled in order to help to understand the structure-function relationship of the enzyme. Furthermore, amino acids previously reported to be important for levansucrase activity were mapped on the model. The structural model presents a five-bladed propeller with a deep, negatively charged central pocket, similar to other bacterial levansucrases. Mapping showed that amino acids which previously reported to affect fructan length are located on the periphery of the structure covering the active site central pocket. Thus it is showed that, for the first time, that hydrolysis and transfructosylation reactions are catalyzed on different parts of Z. mobilis levansucrase structure. The structural location of the critical amino acids will pave the way to identify other residues which control fructan length by site directed mutagenesis without altering the overall fold of the enzyme

Zymomonas mobilis LEVANSUKRAZ ENZİMİNİN LEVAN ÜRETİMİNDE KULLANILMASI

Levan gıda, kozmetik ve ilaç sanayi gibi birbirinden farklı endüstrilerde kullanım alanına sahip bir fruktoz polimeridir. Levansukrazlar sakkarozu substrat olarak kullanarak fruktoz polimerleri oluşumunu katalizleyen enzimlerdir. Bakteriler tarafından hücre dışına salgılanan levansukrazlar bakterinin yüksek sakkaroz konsantrasyonunda levan ve/veya fruktooligosakkarit polimerlerini sentezlemesini sağlar. Bu çalışmada Zymomonas mobilis NRRL B-14023 kullanılarak levansukraz enziminin üretilmesi ve levan polimeri üretme koşulları incelenmiştir. Yapılan çalışmada Z. mobilis levansukrazı üretilmiş ve ham enzimin levan aktivitesi gösterilmiştir. Levansukraz, Z. mobilis B-14023 hücreleri tarafından 36 saat boyunca 30.3 °C’de 159 g/L sakkaroz, pH 4.91 ortamında statik kültürde üretilmiştir. Levansukrazın levan üretimi için optimum inkübasyon süresi ve sıcaklığı 24 saat ve 25 °C olarak bulunduktan sonra aktiviteye etki eden faktörler araştırılmıştır. Artan NaCl konsantrasyonunda levan üretiminde azalma olduğu görülmüştür. Ayrıca 10 mM EDTA ve MgCl2 varlığında enzimin levan üretim aktivitesinde azalma olduğu bulunmuştur. Sonuç olarak Z. mobilis NRRL B-14023 levansukrazı ile ortalama 62.42 g/L levan üretilmiştir.Levan has a wide range of uses in different industries such as food, cosmetics and pharmaceutical. Levansucrases are enzymes, which catalyze the formation of fructose polymers using sucrose as substrate. Levansucrases are produced and secreted by bacteria and enable them to synthesize levan/fructooligosaccharides in medium containing high sucrose concentrations. In this study, Zymomonas mobilis levansucrase was produced and its levan activity was investigated. Levansucrase was produced by Z. mobilis NRRL B-14023 cells in static culture in medium with 159 g/L sucrose, pH 4.91 at 30.3 °C for 36 h. The optimum incubation time, temperature of crude enzyme for levan production were found as 24 h and 25 °C. The levan production of levansucrase decreased with increasing NaCl concentrations. Furthermore the presence of 10 mM EDTA and MgCl 2 showed an inhibitory effect on levan production. As a result, on average 62.42 g/L levan was produced using Z. mobilis NRRL B-1423 levansucrase