Immobilization of bacterial recombinant proteins

Recombinant α-L Rhamnosidase has several potential applications in citrus fruit juice processing industries. Immobilized recombinant α-L Rhamnosidase further provides an added advantage to this industrially important enzyme. Various techniques have been used to immobilize native rhamnosidase from fungal origin and applications were explored in great details by several workers. (Puri et al., 1996, 2000, 2001)

A recombinant rhamnosidase from a bacterial source was expressed in E.coli has been immobilized in calcium alginate beads (entrapment method). A batch bioreactor was created for the hydrolysis of naringin using immobilized recombinant α-L Rhamnosidase under shaking and stationary conditions and it was found to hydrolyze naringin effectively. The system was efficient to hydrolyze narigin under shaking conditions and was operationally stable up to 9 days. A high percent hydrolysis of naringin was achieved at pH 7.5 and 60˚C by immobilized rhamnosidase. Entrapped rhamnosidase was able to hydrolyze naringin content in kinnow juice repeatedly and this feature makes this technique economically suitable for debittering of fruit juices.

Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli

The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.

Production and immunological analysis of IgE reactive recombinant egg white allergens expressed in Escherichia coli.

IgE-mediated allergy to chicken egg affects a large number of children and adults worldwide. The current management strategy for egg allergy is strict avoidance, however this is impractical due to the presence of eggs in a range of foods and pharmaceutical products including vaccines. Strict avoidance also poses nutritional disadvantages due to high nutritional value of eggs. Allergen specific immunotherapy is being pursued as a curative treatment, in which an allergic individual is gradually exposed to the allergen to induce tolerance. Use of recombinant proteins for immunotherapy has been beneficial due to the purity of the recombinant proteins compared to natural proteins. In this study, we produced IgE reactive recombinant egg white proteins that can be used for future immunotherapy. Using E. coli as an expression system, we successfully produced recombinant versions of Gal d 1, 2 and 3, that were IgE reactive when tested against a pool of egg allergic patients' sera. The IgE reactivity indicates that these recombinant proteins are capable of eliciting an immune response, thus being potential candidates for immunotherapy. We have, for the first time, attempted to produce recombinant versions of all 4 major egg white allergens in E. coli, and successfully produced 3, with only Gal d 4 showing loss of IgE reactivity in the recombinant version. The results suggest that egg allergy in Australian populations may mainly be due to IgE reactivity to Gal d 3 and 4, while Gal d 1 shows higher IgE reactivity. This is the first report of a collective and comparative immunological analysis of all 4 egg white allergens. The significance of this study is the potential use of the IgE reactive recombinant egg white proteins in immunotherapy to treat egg allergic patients.

Studies to prevent degradation of recombinant fc-fusion protein expressed in mammalian cell line and protein characterization

Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1-D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

Beacon interacts with cdc2/cdc28-like kinases

Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.

Molecular and immunological analysis of hen's egg yolk allergens with a focus on YGP42 (Gal d 6)

Allergy to hen's (Gallus domesticus) egg white is one of the most common forms of food allergy. Allergy to hen's yolk also exists however, to a lesser extent when compared to egg white allergy. Two minor allergens from the hen's egg yolk known as α-livetin (Gal d 5) and YGP42 (Gal d 6) were discovered recently. In this study, we investigated whether sensitization to egg white is associated with reactivity to egg yolk as well. Sera obtained from 25 patients with allergy to egg white were tested for specific IgE binding for egg yolk proteins through western immunoblotting. 36% of patients were found with true IgE-sensitization against egg yolk proteins. It was found that most of the IgE reactive yolk proteins were fragments of major precursor proteins of hen; vitellogenin-1 (VTG-1), vitellogenin-2 (VTG-2) and apolipoprotein B (Apo B). The egg yolk allergen Gal d 6 is the C-terminal part of VTG-1 and was found to be allergenic in significant percentage of egg white allergy patients. These results highlight the significance of Gal d 6 as an important allergen of egg yolk. Therefore, the secondary aim of this study involved developing a recombinant version of YGP42 in an Escherichia coli expression system. Recombinant Gal d 6 (rGal d6) was expressed as a fusion peptide with a 6 × His tag and purified using metal chelating resin. The inhibition ELISA results showed that rYGP42 was IgE reactive and was able to inhibit IgE binding to crude egg yolk (CEY) by up to 30%. Traditionally, it was thought that allergy to egg yolk occurred independently from sensitization to egg white. This study underlies the importance of concomitant sensitization to egg yolk proteins in patients allergic to egg white. Evidence reported in this study strongly suggests that egg yolk has potentially undiscovered allergens and therefore warrants further investigation. Furthermore, IgE reactive Gal d 6 presented in this study has the potential to be used in diagnosis and immunotherapy to treat egg allergy.

Biological properties of the Hendra virus envelope proteins

Outbreaks of Hendra virus in Queensland, during 1994-1999, resulted in the deaths of 17 horses and 2 humans from respiratory or neurological disease. Immunological, ultrastructural and expressional studies of the viral envelope proteins using recombinant poxviruses and DNA vectors contributed to improving our understanding of the biology of Hendra virus.

Industrial biotechnology for the production of proteins/enzymes for a sustainable future

Worldwide emergence of Industrial biotechnology (IB) is providing opportunities to produce enzymes/proteins with variety of industrial/therapeutic applications. In transitioning the Australian economy towards a sustainable future, Federal government identified the development of IB pathway which would ensure increased productivity, enhanced sustainability, health, safety and reduced environmental footprint. The presentation will revolve around specific stories that drives Deakin University newest technology platform which applies biology and fermentation in an integrated way to play a crucial role in developing cost-effective technologies for the development of molecules that can benefit pharmaceutical and food industry in regional Victoria and Australia in general. The talk will also highlight specific examples where new products like recombinant rhamnosidase (an enzyme used for the production of flavonoids with health benefits) and ribosome inactivating proteins (detected in medicinal plants which possess RNA N- glycosidase activity that depurinates the major rRNA, thus damaging ribosome in an irreversible manner and arresting protein synthesis) would be made available through bioprocessing.

Addition of magnesium chloride to enhance mono-dispersity of a coiled-coil recombinant mouse macrophage protein

X-ray crystallography for the determination of three-dimensional structures of protein macromolecules represents an important tool in function assignment of uncharacterized proteins. However, crystallisation is often difficult to achieve. A protein sample fully characterized in terms of dispersity may increase the likelihood of successful crystallisation by improving the predictability of the crystallisation process. To maximize the probability of crystallisation of a novel mouse macrophage protein (rMMP), target molecule was characterized and refined to improve monodispersity. Addition of MgCl2 at low concentrations resolves the rMMP into a monodisperse solution, and finally successful crystallization of rMMP was achieved. The effect of MgCl2 was studied using gel filtration chromatography and dynamic light scattering.

Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax, and show no preference for Bak versus Bax

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.