Optimized production strategy of the major capsid protein HPV 16L1 non-assembly variant in E. coli.

The capsid of human papillomavirus (HPV) consists of two capsid proteins - the major capsid protein L1 and the minor capsid protein L2. Assembled virus-like particles, which only consist of L1 proteins, are successfully applied as prophylactic vaccines against HPV infections. The capsid subunits are L1-pentamers, which are also reported to protect efficiently against HPV infections in animals. The recombinant production of L1 has been previously shown in E. coli, yeast, insect cells, plants and mammalian cell culture. Principally, in E. coli-based expression system L1 shows high expression yields but the protein is largely insoluble. In order to overcome this problem reported strategies address fusion proteins and overexpression of bacterial chaperones. However, an insufficient cleavage of the fusion proteins and removal of co-purified chaperones can hamper subsequent down streaming. We report a significant improvement in the production of soluble L1-pentamers by combining (I) a fusion of a N-terminal SUMO-tag to L1, (II) the heterologous co-expression of the chaperon system GroEL/ES and (III) low expression temperature. The fusion construct was purified in a 2-step protein purification including efficient removal of GroEL/ES and complete removal of the N-terminal SUMO-tag. The expression strategy was transferred to process-controlled high-cell-density fermentation with defined media according to the guidelines of good manufacturing practice. The produced L1 protein is highly pure (>95%), free of DNA (260:280 = 0.5) and pentameric. The production strategy yielded 5.73 mg of purified L1-pentamers per gram dry biomass. The optimized strategy is a suitable alternative for high yield L1-pentamer production and purification as a cheaper process for vaccine production.

Influence of microorganism and plant oils on the structure of mannosylerythritol lipid (MEL) biosurfactants revealed by a novel thin layer chromatography mass spectrometry method.

Mannosylerythritol lipids (MEL) are microbial glycolipid biosurfactants with great potential for application in cosmetics and household detergents. In current biotechnological processes, they are produced by basidiomycetous fungi, the Ustilaginaceae, as a complex mixture of different chemical structures. It was the aim of this paper to study the influence of producer organisms and substrates on the resulting MEL structures with a novel high-resolution HPTLC-MALDI-TOF method. Given the seven different microbes and four plant oils, our analysis revealed that the product concentrations varied strongly between organisms, while they were similar for the different substrates. Coconut oil presented an exception, since only one organism was able to synthesize MEL from this substrate in considerable yields. Analysis by GC-FID further showed that the chain length pattern of hydrophobic fatty acid side-chains was very specific for individual organisms, while substrates had only a minor influence on the chain length. Our novel HPTLC-MALDI-TOF combination method finally demonstrated the presence of multiple MEL sub-variants with differing acetylation and fatty acid chain lengths. It also revealed the production of a more hydrophilic biosurfactant mannosylmannitol lipid (MML) as a side-product in certain fungi. Overall, it was concluded that the pattern of produced biosurfactant structures are mainly governed by producer organisms rather than substrates.

Refolding and in vitro characterization of human papillomavirus 16 minor capsid protein L2.

The minor capsid protein L2 of papillomaviruses exhibits multiple functions during viral entry including membrane interaction. Information on the protein is scarce, because of its high tendency of aggregation. We determined suitable conditions to produce a functional human papillomavirus (HPV) 16 L2 protein and thereby provide the opportunity for extensive in vitro analysis with respect to structural and biochemical information on L2 proteins and mechanistic details in viral entry. We produced the L2 protein of high-risk HPV 16 in Escherichia coli as inclusion bodies and purified the protein under denaturing conditions. A successive buffer screen resulted in suitable conditions for the biophysical characterization of 16L2. Analytical ultracentrifugation of the refolded protein showed a homogenous monomeric species. Furthermore, refolded 16L2 shows secondary structure elements. The N-terminal region including the proposed transmembrane region of 16L2 shows alpha-helical characteristics. However, overall 16L2 appears largely unstructured. Refolded 16L2 is capable of binding to DNA indicating that the putative DNA-binding regions are accessible in refolded 16L2. Further the refolded protein interacts with liposomal membranes presumably via the proposed transmembrane region at neutral pH without structural changes. This indicates that 16L2 can initially interact with membranes via pre-existing structural features.