BiomedCentral Biotech

BMC Biotechnology - Latest Articles

The latest research articles published by BMC Biotechnology

Bioconversion of D-galacturonate to keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) using filamentous fungi

Background: The D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-threo-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives. Keto-deoxy-L-galactonate is a natural intermediate in the fungal D-galacturonate metabolic pathway, and thus keto-deoxy-L-galactonate can be produced in a simple biological conversion. Results: Keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) accumulated in the culture supernatant when Trichoderma reesei delta lga1 and Aspergillus niger delta gaaC were grown in the presence of D-galacturonate. Keto-deoxy-L-galactonate accumulated even if no metabolisable carbon source was present in the culture supernatant, but was enhanced when D-xylose was provided as a carbon and energy source. Up to 10.5 g keto-deoxy-L-galactonate /l was produced from 20 g D-galacturonate /l and A. niger delta gaaC produced 15.0 g keto-deoxy-L-galactonate /l from 20 g polygalacturonate /l, at yields of 0.4 to 1.0 g keto-deoxy-L-galactonate per g D-galacturonate consumed. Keto-deoxy-L-galactonate accumulated to concentrations of 12 to 16 g/l intracellularly in both producing organisms. This intracellular concentration was sustained throughout production in A. niger delta gaaC, but decreased in T. reesei. Conclusions: Bioconversion of D-galacturonate to keto-deoxy-L-galactonate was achieved with both A. niger delta gaaC and T. reesei delta lga1, although production (titre, volumetric and specific rates) was better with A. niger than T. reesei. A. niger was also able to produce keto-deoxy-L-galactonate directly from pectin or polygalacturonate demonstrating the feasibility of simultaneous hydrolysis and bioconversion. Although keto-deoxy-L-galactonate accumulated intracellularly, concentrations above ~12 g/l were exported to the culture supernatant. Lysis may have contributed to the release of keto-deoxy-L-galactonate from T. reesei mycelia.
 

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

Background: Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins. Results: The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin beta-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, the capacity for glycosylation of recombinant glycoproteins expressed at very high levels, such as the magnICON-expressed E. coli enterotoxin B subunit:H.sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform. Conclusion: Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.
 

Stabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins.

Background: Whereas T cell receptors (TCRs) detect peptide/major histocompatibility complexes (pMHCs) with exquisite specificity, there are challenges regarding their expression and use as soluble detection molecules due to molecular instability. We have investigated strategies for the production of TCR-immunoglobulin (Ig) fusion proteins. Two different TCRs that are characteristic of a mouse model for idiotype (Id) dependent immune regulation were engineered. They are structurally unrelated with different variable (V), diversity (D) and joining (J) segments, but each share one V gene segment, either Valpha or Vbeta, with the well characterized murine TCR, 2C. Results: Several TCR-Ig formats were assessed. In one, the TCR V domains were fused to Ig constant (C) regions. In others, the complete extracellular part of the TCR was fused either to a complete Ig or an Ig Fc region. All molecules were initially poorly secreted from eukaryotic cells, but replacement of unfavourable amino acids in the V regions improved secretion, as did the introduction of a disulfide bridge between the TCR C domains and the removal of an unpaired cysteine. A screening strategy for selection of mutations that stabilize the actual fusion molecules was developed and used successfully. Molecules that included the complete heterodimeric TCR, with a stabilizing disulfide bridge, were correctly folded as they bound TCR-specific antibodies (Abs) and detected pMHC on cells after specific peptide loading. Conclusions: We show that fully functional TCR-Ig fusion proteins can be made in good yields following stabilizing engineering of TCR V and C region genes. This is important since TCR-Ig fusions will be important probes for the presence of specific pMHCs in vitro and in vivo. In the absence of further affinity maturation, the reagents will be very useful for the detection of kinetic stability of complexes of peptide and MHC.
 

Oligonucleotide indexing of DNA barcodes: identification of tuna and other scombrid species in food products

Background: DNA barcodes are a global standard for species identification and have countless applications in the medical, forensic and alimentary fields, but few barcoding methods work efficiently in samples in which DNA is degraded, e.g. foods and archival specimens. This limits the choice of target regions harbouring a sufficient number of diagnostic polymorphisms. The method described here uses existing PCR and sequencing methodologies to detect mitochondrial DNA polymorphisms in complex matrices such as foods. The reported application allowed the discrimination among 17 fish species of the Scombridae family with high commercial interest such as mackerels, bonitos and tunas which are often present in processed seafood. The approach can be easily upgraded with the release of new genetic diversity information to increase the range of detected species. Results: Cocktail of primers are designed for PCR using publicly available sequences of the target sequence. They are composed of a fixed 5' region and of variable 3' cocktail portions that allow amplification of any member of a group of species of interest. The population of short amplicons is directly sequenced and indexed using primers containing a longer 5' region and the non polymorphic portion of the cocktail portion. A 226 bp region of CytB was selected as target after collection and screening of 148 online sequences; 85 SNPs were found, of which 75 were present in at least two sequences. Primers were also designed for two shorter sub-fragments that could be amplified from highly degraded samples. The test was used on 203 samples of seafood (canned tuna and scomber, tuna salad, tuna sauce) and could successfully detect the presence of different or additional species that were not identified on the labelling of canned tuna, tuna salad and sauce samples. Conclusions: The described method is largely independent of the degree of degradation of the DNA source and can thus be applied to processed seafood. Moreover, the method is highly flexible: publicly available sequence information on mitochondrial genomes are rapidly increasing for most species, facilitating the choice of target sequences and the improvement of resolution of the test. This is particularly important for the discrimination of marine and aquaculture species for which genome information is still limited.
 

Characterization of single chain antibody targets through yeast two hybrid

Background: Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. Results: We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions: Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.
 

Novel strong tissue specific promoter for gene expression in human germ cells

Background: Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results: Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS) was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102), where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter). To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD) suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1), whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293). In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X). The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions: We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12), and an important role - in the rest two cell lines.
 

Moisture-induced solid state instabilities in alpha-chymotrypsin and their reduction through chemical glycosylation

Background: Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state. Results: First, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme ?-chymotrypsin (?-CT) under controlled humidity conditions. Results showed that ?-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, ?-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that ?-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size. Conclusion: Water sorption leads to aggregation, inactivation, and structural changes of ?-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.
 

Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

Background: Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products. Results: Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed ?-lactamase (?W290) selection cassette contains a segment inside the ?-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests. Conclusions: Our results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway® or ligase-independent cloning (LIC) .
 

Testing the interaction between analytical modules: an example with Roundup Ready(R) soybean line GTS 40-3-2

Background: The modular approach to analysis of genetically modified organisms (GMOs) relies on the independence of the modules combined (i.e. DNA extraction and GM quantification). The validity of this assumption has to be proved on the basis of specific performance criteria. Results: An experiment was conducted using, as a reference, the validated quantitative real-time polymerase chain reaction (PCR) module for detection of glyphosate-tolerant Roundup Ready® GM soybean (RRS). Different DNA extraction modules (CTAB, Wizard and Dellaporta), were used to extract DNA from different food/feed matrices (feed, biscuit and certified reference material [CRM 1%]) containing the target of the real-time PCR module used for validation. Purity and structural integrity (absence of inhibition) were used as basic criteria that a DNA extraction module must satisfy in order to provide suitable template DNA for quantitative real-time (RT) PCR-based GMO analysis. When performance criteria were applied (removal of non-compliant DNA extracts), the independence of GMO quantification from the extraction method and matrix was statistically proved, except in the case of Wizard applied to biscuit. A fuzzy logic-based procedure also confirmed the relatively poor performance of the Wizard/biscuit combination. Conclusions: For RRS, this study recognises that modularity can be generally accepted, with the limitation of avoiding combining highly processed material (i.e. biscuit) with a magnetic-beads system (i.e. Wizard).
 

SIVsm Tat, Rev, and Nef1: functional characteristics of r-GV internalization on isotypes, cytokines, and intracellular degradation

Background: Recombinant gas vesicles (r-GV) from Halobacterium sp. strain SD109 expressing cassettes with different SIVsm inserts, have potential utility as an effective antigen display system for immunogen testing in vivo and for initial epitope assessments in vitro. Previous mouse model studies demonstrated immunization with r-GV expressing selected exogenous sequences elicited a prolonged immune response. Here we tested segments from three SIVsm genes (tat, rev, and nef) each surface displayed by r-GV. As with HIV, for SIVsm the proteins encoded by tat, rev and nef respectively serve critical and diverse functions: effects on efficient viral RNA polymerase II transcription, regulation of viral gene expression and effects on specific signaling functions through the assembly of multiprotein complexes. Humoral responses to r-GVTat, Rev or Nef1 elicited in vivo, associated changes in selected cell cytokine production following r-GV internalization, and the capacity of J774A.1 macrophage cells to degrade these internalized display/delivery particles in vitro were examined. Results: The in vivo studies involving r-GV immunizations and in vitro studies of r-GV uptake by J774A.1 macrophages demonstrated: (i) tests for antibody isotypes in immunized mice sera showed activation and re-stimulation of memory B cells, (ii) during long term immune response to the epitopes, primarily the IgG1 isotype was produced, (iii) in vitro, macrophage degradation of r-GV containing different SIVsm inserts occurred over a period of days resulting in an inherent slow breakdown and degradation of the SIVsm peptide inserts, (iv) vesicle specific GvpC, a larger protein, degraded more slowly than the recombinant peptide inserts and (v) in vitro uptake and degradation of the r-GV populations tested was associated with SIVsm insert specific patterns for cytokines IL-10, IL-12 and IL-18. Conclusions: Together these findings provide new information underscoring r-GV potential. They can clearly: display various exogenous peptides, be intracellularly degraded in vitro over a period of days, affect cell cytokine levels, and retain their self-adjuvanting capacity irrespective of the specific peptide expressed within the GvpC protein. These features support the cost effective generation of vaccine components, and provide a simple, self-adjuvanting system for assessing immune visibility of and specific responses to individual pathogen peptides.