A loss-of-function screen reveals SNX5 and SNX6 as potential components of the mammalian retromer

The mammalian retromer is a multimeric protein complex involved in mediating endosome-to-trans-Golgi-network retrograde transport of the cation-independent mannose-6-phosphate receptor. The retromer is composed of two subcomplexes, one containing SNX1 and forming a membrane-bound coat, the other comprising VPS26, VPS29 and VPS35 and being cargo-selective. In yeast, an additional sorting nexin--Vps17p--is a component of the membrane bound coat. It remains unclear whether the mammalian retromer requires a functional equivalent of Vps17p. Here, we have used an RNAi loss-of-function screen to examine whether any of the other 30 mammalian sorting nexins are required for retromer-mediated endosome-to-trans-Golgi-network retrieval of the cation-independent mannose-6-phosphate receptor. Using this screen, we identified two proteins, SNX5 and SNX6, that, when suppressed, induced a phenotype similar to that observed upon suppression of known retromer components. Whereas SNX5 and SNX6 colocalised with SNX1 on early endosomes, in immunoprecipitation experiments only SNX6 appeared to exist in a complex with SNX1. Interestingly, suppression of SNX5 and/or SNX6 resulted in a significant loss of SNX1, an effect that seemed to result from post-translational regulation of the SNX1 level. Such data suggest that SNX1 and SNX6 exist in a stable, endosomally associated complex that is required for retromer-mediated retrieval of the cation-independent mannose-6-phosphate receptor. SNX5 and SNX6 may therefore constitute functional equivalents of Vps17p in mammals.

Mass spectrometry-based methods for identifying oxidized proteins in disease:advances and challenges

Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Occurrence and Function of Hoogsteen Base Pairs in Nucleic Acids

Nucleic acids (DNA and RNA) play essential roles in the central dogma of biology for the storage and transfer of genetic information. The unique chemical and conformational structures of nucleic acids – the double helix composed of complementary Watson-Crick base pairs, provide the structural basis to carry out their biological functions. DNA double helix can dynamically accommodate Watson-Crick and Hoogsteen base-pairing, in which the purine base is flipped by ~180° degrees to adopt syn rather than anti conformation as in Watson-Crick base pairs. There is growing evidence that Hoogsteen base pairs play important roles in DNA replication, recognition, damage or mispair accommodation and repair. Here, we constructed a database for existing Hoogsteen base pairs in DNA duplexes by a structure-based survey from the Protein Data Bank, and structural analyses based on the resulted Hoogsteen structures revealed that Hoogsteen base pairs occur in a wide variety of biological contexts and can induce DNA kinking towards the major groove. As there were documented difficulties in modeling Hoogsteen or Watson-Crick by crystallography, we collaborated with the Richardsons’ lab and identified potential Hoogsteen base pairs that were mis-modeled as Watson-Crick base pairs which suggested that Hoogsteen can be more prevalent than it was thought to be. We developed solution NMR method combined with the site-specific isotope labeling to characterize the formation of, or conformational exchange with Hoogsteen base pairs in large DNA-protein complexes under solution conditions, in the absence of the crystal packing force. We showed that there are enhanced chemical exchange, potentially between Watson-Crick and Hoogsteen, at a sharp kink site in the complex formed by DNA and the Integration Host Factor protein. In stark contrast to B-form DNA, we found that Hoogsteen base pairs are strongly disfavored in A-form RNA duplex. Chemical modifications N1-methyl adenosine and N1-methyl guanosine that block Watson-Crick base-pairing, can be absorbed as Hoogsteen base pairs in DNA, but rather potently destabilized A-form RNA and caused helix melting. The intrinsic instability of Hoogsteen base pairs in A-form RNA endows the N1-methylation as a functioning post-transcriptional modification that was known to facilitate RNA folding, translation and potentially play roles in the epitranscriptome. On the other hand, the dynamic property of DNA that can accommodate Hoogsteen base pairs could be critical to maintaining the genome stability.

Bioengineering strategies to improve functional qualities of nisin

The abuse of antibiotics and the emergence of multi-drug resistant bacterial strains have created the need to explore alternative methods of controlling microbial pathogens. The bacteriocin family of antimicrobial peptides has been proposed as one such alternative to classic antibiotics. Nisin A belongs to the subgroup of bacteriocins called the lantibiotics, which contain several unusual amino acids as a consequence of enzyme-mediated post-translational modifications. As nisin is produced by generally regarded as safe (GRAS) microorganisms, it could potentially be applied in a clinical setting. However, as lantibiotics are naturally produced in such small quantities, this can hinder their industrial potential. In order to overcome this, several approaches can be utilised. For example, given the gene encoded nature of lantibiotics, genetic engineering approaches can be implemented in order to yield variants with enhanced properties. Here, the use of mutagenesis-based strategies was employed to obtain a derivative of nisin with enhanced bioactivity in vitro. Investigations with purified peptide highlighted the enhanced specific activity of this variant, nisin M21V, against food-borne Listeria monocytogenes strains. Furthermore, this specific enhanced bioactivity was evident in a mouse model of listeriosis. Reductions in bioluminescence and microbial counts in organs from infected mice were observed following treatment with nisin M21V compared to that of wild-type nisin A. Peptide bioengineering approaches were also implemented to obtain additional novel derivatives of nisin. The generation of “S5X” and “S33X” banks (representing a change of natural serines at positions 5 and 33 to all possible alternative residues) by a combination of site-saturation and site-directed mutagenesis led to the identification of several derivatives exhibiting improved stability. This allowed the rational design of variants with enhanced stability compared to that of wild type nisin. Another means of tackling issues associated with lantibiotic yield is to combine lantibiotics with other antimicrobials. This could circumvent the need for enhanced production while also reducing concentrations of the peptide antimicrobials. We observed that combinations of nisin variants and low levels of plant essential oils (thymol, carvacrol, trans-cinnamaldehyde) significantly controlled Gram negative foodborne pathogens in in vitro assays compared to nisin A-essential oil combinations. This enhanced control was also evident in model food systems. Nisin variants used in conjunction with carvacrol significantly reduced numbers of E. coli O157:H7 in apple juice while a commercial nisin preparation used in combination with citric acid significantly controlled C. sakazakii in infant milk formula. It is noteworthy that while nisin is generally associated with Gram positive targets, upon combination with plant essential oils the spectrum of inhibition was broadened to Gram negative targets.

Epigenetics within the matrix:a neo-regulator of fibrotic disease

Fibrosis of any tissue is characterized by excessive extracellular matrix accumulation that ultimately destroys tissue architecture and eventually abolishes normal organ function. Although much research has focused on the mechanisms underlying disease pathogenesis, there are still no effective antifibrotic therapies that can reverse, stop or delay the formation of scar tissue in most fibrotic organs. As fibrosis can be described as an aberrant wound healing response, a recent hypothesis suggests that the cells involved in this process gain an altered heritable phenotype that promotes excessive fibrotic tissue accumulation. This article will review the most recent observations in a newly emerging field that links epigenetic modifications to the pathogenesis of fibrosis. Specifically, the roles of DNA methylation and histone modifications in fibrotic disease will be discussed.

The synthetic progestin norgestrel modulates Nrf2 signaling and acts as an antioxidant in a model of retinal degeneration

Retinitis pigmentosa (RP) is one of the most common retinal degenerative conditions affecting people worldwide, and is currently incurable. It is characterized by the progressive loss of photoreceptors, in which the death of rod cells leads to the secondary death of cone cells; the cause of eventual blindness. As rod cells die, retinal-oxygen metabolism becomes perturbed, leading to increased levels of reactive oxygen species (ROS) and thus oxidative stress; a key factor in the secondary death of cones. In this study, norgestrel, an FDA-approved synthetic analog of progesterone, was found to be a powerful neuroprotective antioxidant, preventing light-induced ROS in photoreceptor cells, and subsequent cell death. Norgestrel also prevented light-induced photoreceptor morphological changes that were associated with ROS production, and that are characteristic of RP. Further investigation showed that norgestrel acts via post-translational modulation of the major antioxidant transcription factor Nrf2; bringing about its phosphorylation, subsequent nuclear translocation, and increased levels of its effector protein superoxide dismutase 2 (SOD2). In summary, these results demonstrate significant protection of photoreceptor cells from oxidative stress, and underscore the potential of norgestrel as a therapeutic option for RP.

Prediction of Phosphorylation Sites from Protein Sequences

Phosphorylation is amongst the most crucial and well-studied post-translational modifications. It is involved in multiple cellular processes which makes phosphorylation prediction vital for understanding protein functions. However, wet-lab techniques are labour and time intensive. Thus, computational tools are required for efficiency. This project aims to provide a novel way to predict phosphorylation sites from protein sequences by adding flexibility and Sezerman Grouping amino acid similarity measure to previous methods, as discovering new protein sequences happens at a greater rate than determining protein structures. The predictor – NOPAY - relies on Support Vector Machines (SVMs) for classification. The features include amino acid encoding, amino acid grouping, predicted secondary structure, predicted protein disorder, predicted protein flexibility, solvent accessibility, hydrophobicity and volume. As a result, we have managed to improve phosphorylation prediction accuracy for Homo sapiens by 3% and 6.1% for Mus musculus. Sensitivity at 99% specificity was also increased by 6% for Homo sapiens and for Mus musculus by 5% on independent test sets. In this study, we have managed to increase phosphorylation prediction accuracy for Homo sapiens and Mus musculus. When there is enough data, future versions of the software may also be able to predict other organisms.

Statistical modeling of protein lysate array data

The protein lysate array is an emerging technology for quantifying the protein concentration ratios in multiple biological samples. It is gaining popularity, and has the potential to answer questions about post-translational modifications and protein pathway relationships. Statistical inference for a parametric quantification procedure has been inadequately addressed in the literature, mainly due to two challenges: the increasing dimension of the parameter space and the need to account for dependence in the data. Each chapter of this thesis addresses one of these issues. In Chapter 1, an introduction to the protein lysate array quantification is presented, followed by the motivations and goals for this thesis work. In Chapter 2, we develop a multi-step procedure for the Sigmoidal models, ensuring consistent estimation of the concentration level with full asymptotic efficiency. The results obtained in this chapter justify inferential procedures based on large-sample approximations. Simulation studies and real data analysis are used to illustrate the performance of the proposed method in finite-samples. The multi-step procedure is simpler in both theory and computation than the single-step least squares method that has been used in current practice. In Chapter 3, we introduce a new model to account for the dependence structure of the errors by a nonlinear mixed effects model. We consider a method to approximate the maximum likelihood estimator of all the parameters. Using the simulation studies on various error structures, we show that for data with non-i.i.d. errors the proposed method leads to more accurate estimates and better confidence intervals than the existing single-step least squares method.

Phospholipases and the network of auxin signal transduction with ABP1 and TIR1 as two receptors: a comprehensive and provocative model

Three types of phospholipases, phospholipase D, secreted phospholipase A2, and patatin-related phospholipase A (pPLA) have functions in auxin signal transduction. Potential linkage to auxin receptors ABP1 or TIR1, their rapid activation or post-translational activation mechanisms, and downstream functions regulated by these phospholipases is reviewed and discussed. Only for pPLA all aspects are known at least to some detail. Evidence is gathered that all these signal reactions are located in the cytosol and seem to merge on regulation of PIN-catalyzed auxin efflux transport proteins. As a consequence, auxin concentration in the nucleus is also affected and this regulates the E3 activity of this auxin receptor. We showed that ABP1, PIN2, and pPLA, all outside the nucleus, have an impact on regulation of auxin-induced genes within 30 min. We propose that regulation of PIN protein activities and of auxin efflux transport are the means to coordinate ABP1 and TIR1 activity and that no physical contact between components of the ABP1-triggered cytosolic pathways and TIR1-triggered nuclear pathways of signaling is necessary to perform this.

Influência do estímulo por PAF no perfil de abundância proteica em neutrófilos : uma abordagem proteômica

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2015.

Differential effects of low-temperature inhibition on the propylene induced autocatalysis of ethylene production, respiration and ripening of 'Hayward' kiwifruit

Previous studies (Stavroulakis and Sfakiotakis, 1993) have shown an inhibition of propylene-induced ethylene production in kiwifruit below a critical temperature range of 11-14.8 degrees C. The aim of this research was to identify the biochemical basis of this inhibition in kiwifruit below 11-14.8 degrees C. 'Hayward' kiwifruit were treated with increasing propylene concentrations at 10 and 20 degrees C. Ethylene biosynthesis pathways and fruit ripening were investigated. Kiwifruit at 20 degrees C in air started autocatalysis of ethylene production and ripened after 19 d with a concomitant increase in respiration. Ethylene production and the respiration rise appeared earlier with increased propylene concentrations. Ripening proceeded immediately after propylene treatment, while ethylene autocatalysis needed a lag period of 24-72 h. The latter event was attributed to the delay found in the induction of 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) activity and consequently to the delayed increase of l-aminocyclopropane l-carboxylic acid (ACC) content. In contrast propylene treatment induced 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) activity with no lag period. Moreover, transcription of ACC synthase and ACC oxidase genes was active only in ethylene-producing kiwifruit at 20 degrees C. In contrast, treatment at 10 degrees C with propylene strongly inhibited ethylene production, which was attributed to the low activities of both ACC synthase and ACC oxidase as well as the low initial ACC level. Interestingly, fruit treated with propylene at 10 degrees C appeared to be able to transcribe the ACC oxidase but not the ACC synthase gene. However, propylene induced ripening of that fruit almost as rapidly as in the propylene-treated fruit at 20 degrees C. Respiration rate was increased together with propylene concentration. It is concluded that kiwifruit stored at 20 degrees C behaves as a typical climacteric fruit, while at 10 degrees C behaves like a non-climacteric fruit. We propose that the main reasons for the inhibition of the propylene induced (autocatalytic) ethylene production in kiwifruit at low temperature (less than or equal to 10 degrees C), are primarily the suppression of the propylene-induced ACC synthase gene expression and the possible post-transcriptional modification of ACC oxidase.

Mechanistic Dissection of the Cop9 Signalosome’s Deneddylation Activity on Cullin-RING Ligases

We set out to understand the precise mechanisms that regulate the activation and deactivation of Cullin-RING Ligases (CRLs). While a great deal of work has already gone into identifying the players involved in these pathways and the cellular consequences associated with the loss of each, the biochemical mechanisms regulating these steps have remained elusive. In this work we sought to gain a better understanding of the mechanisms behind these steps by teasing apart specific their biochemical reactions. By measuring the individual microscopic rate constants of the reactions we have shed light on both the proper sequence of events in the regulation of CRLs as well as how they are in fact controlled.

Prior to this work, it was believed that CSN deactivated CRLs by binding them and enzymatically removing the activating post-translation modification Nedd8. It was believed that CSN could not bind to CRLs while they were active due to the steric hindrance by the CRL substrates, and that they would remain bound to deneddylated CRLs as a sequestering agent until a new substrate could displace it. We now have some insight that substrates themselves cannot inhibit CSN very well, but that the active ubiquitination by an E2 enzyme precludes CSN binding and activity. When the substrate for a CRL becomes depleted, CSN then binds to the CRL in a low affinity, low activity conformation. This triggers a conformational change that pulls the autoinhibitory Ins-1 loop away from the active site in the catalytic subunit Csn5, resulting in a large increase in affinity and cleavage of the isopeptide bond between CRLs and Nedd8. Upon dissociation of Nedd8, CSN rapidly returns to the low affinity state and dissociates from the CRL, allowing it reenter its activation cycle.

Differential effects of low-temperature inhibition on the propylene induced autocatalysis of ethylene production, respiration and ripening of 'Hayward' kiwifruit

Previous studies (Stavroulakis and Sfakiotakis, 1993) have shown an inhibition of propylene-induced ethylene production in kiwifruit below a critical temperature range of 11-14.8 degrees C. The aim of this research was to identify the biochemical basis of this inhibition in kiwifruit below 11-14.8 degrees C. 'Hayward' kiwifruit were treated with increasing propylene concentrations at 10 and 20 degrees C. Ethylene biosynthesis pathways and fruit ripening were investigated. Kiwifruit at 20 degrees C in air started autocatalysis of ethylene production and ripened after 19 d with a concomitant increase in respiration. Ethylene production and the respiration rise appeared earlier with increased propylene concentrations. Ripening proceeded immediately after propylene treatment, while ethylene autocatalysis needed a lag period of 24-72 h. The latter event was attributed to the delay found in the induction of 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) activity and consequently to the delayed increase of l-aminocyclopropane l-carboxylic acid (ACC) content. In contrast propylene treatment induced 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) activity with no lag period. Moreover, transcription of ACC synthase and ACC oxidase genes was active only in ethylene-producing kiwifruit at 20 degrees C. In contrast, treatment at 10 degrees C with propylene strongly inhibited ethylene production, which was attributed to the low activities of both ACC synthase and ACC oxidase as well as the low initial ACC level. Interestingly, fruit treated with propylene at 10 degrees C appeared to be able to transcribe the ACC oxidase but not the ACC synthase gene. However, propylene induced ripening of that fruit almost as rapidly as in the propylene-treated fruit at 20 degrees C. Respiration rate was increased together with propylene concentration. It is concluded that kiwifruit stored at 20 degrees C behaves as a typical climacteric fruit, while at 10 degrees C behaves like a non-climacteric fruit. We propose that the main reasons for the inhibition of the propylene induced (autocatalytic) ethylene production in kiwifruit at low temperature (less than or equal to 10 degrees C), are primarily the suppression of the propylene-induced ACC synthase gene expression and the possible post-transcriptional modification of ACC oxidase.

Production of Recombinant Trichoderma Reesei Endoglucanase Protein CEL7B by Using Kluyveromyces Lactis

This research is about producing recombinant Trichoderma reesei endoglucanase Cel7B by using Kluyveromyces lactis, transformed with chromosomally integrated Cel7B cDNA, as a host cell (K. lactis Cel7B). Cel7B is one of the glycoside hydrolyze family of proteins that are produced by T. reesei. Cel7B together with other endoglucanases, exoglucanases, and â-glucosidases hydrolyze cellulose to glucose, which can then be fermented to biofuels or other value-added products. The research objective of this MS project is to examine favorable fermentation conditions for recombinant Cel7B enzyme production and improved activity. Production of enzyme on different types of media was examined, and the activity of the enzyme was measured by using different tools or procedures. The first condition tested for was using different concentrations of galactose as a carbon and energy source; however galactose also acts as a potent promoter of recombinant Cel7B expression in K. lactis Cel7B. The purpose of this method is to determine the relationship between production of enzyme with increasing sugar concentration. The second culture condition test was using different types of media: a complex medium-yeast extract, peptone, galactose (YPGal); a minimal medium-yeast nitrogen base (YNB) with galactose; and a minimal medium with supplement-yeast nitrogen base with casamino acid (YBC), a nitrogen source, with galactose. The third condition was using different types of reactors or fermenters: a small reactor (shake flask) and a larger automated bioreactor (BioFlo 3000 fermenter). The purpose of this method is to determine the quantity of the protein produced by using different environments of production. Different tools to determine the presence and activity of Cel7B enzyme were used. For the presence of enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. Secondly, to detect enzyme activity, the carboxymethyl cellulose- 3,5-dinitrosalicylic acid (CMC- DNS) assay was employed. SDS-PAGE showed that the enzyme band was at 67 kDa, which is larger than native Cel7B (52 kDa.), likely due to over glycolylation during post-translational processing in K. lactis. For the different types of media used in our fermentation, recombinant Cel7B was produced from yeast extract peptone galactose (YPGal), and yeast nitrogen base with casamino acid (YBC), but was not produced and no activity was detected from yeast nitrogen base (YNB). This experiment concluded that the Cel7B production requires the amino acid resources as part of fermentation medium. In experiments where recombinant Cel7B net activity was measured at 1% galactose initial concentration in YPGal and YBC media, higher enzyme activity was detected for the complex medium YPGal. Higher activity of recombinant Cel7B was detected for flask culture in 2% galactose compared to 1% galactose for YBC medium. Two bioreactor experiments were conducted under these culture conditions at 30°C, pH 7.0, dissolved oxygen of 50% of saturation, and 250 rpm agitation (variable depending on DO control) K. lactis-Cel7B yeast growth curves were quite reproducible with maximum optical density (O.D) at 600 nm of between 7 and 8 (when factoring dilution of 10:1). Galactose was consumed rapidly during the first 15 hours of bioreactor culture and recombinant Cel7B started to appear in the culture at 10-15 hours and increased thereafter up to a maximum of between 0.9 and 1.6 mg/mL/hr in these experiments. These bioreactor enzyme activity results are much higher than comparable experiments conducted with flask-scale culture (0.5 mg/mL/hr). In order to achieve the highest recombinant Cel7B activity from batch culture of K. lactis-Cel7B, based on this research it is best to use a complex medium, 2% initial galactose concentration, and an automated bioreactor where good control of temperature, pH, and dissolved oxygen can be achieved.