Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant membrane proteins. By fusing green fluorescent protein and an erythromycin resistance marker (ErmC) to the C-terminus of a target protein, one simultaneously selects for variants with enhanced expression (increased erythromycin resistance) and correct folding (green fluorescent protein fluorescence). Three evolved hosts, displaying 2- to 8-fold increased expression of a plethora of proteins, were fully sequenced and shown to carry single-site mutations in the nisK gene. NisK is the sensor protein of a two-component regulatory system that directs nisin-A-mediated expression. The levels of recombinant membrane proteins were increased in the evolved strains, and in some cases their folding states were improved. The generality and simplicity of our approach allow rapid improvements of protein production yields by directed evolution in a high-throughput way.     

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD₆₀₀ compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.     

Protein interaction network for Alzheimer's disease using computational approach

Alzheimer''s disease (AD) is the most common form of dementia. It is the sixth leading cause of death in old age people. Despite recent advances in the field of drug design, the medical treatment for the disease is purely symptomatic and hardly effective. Thus there is a need to understand the molecular mechanism behind the disease in order to improve the drug aspects of the disease. We provided two contributions in the field of proteomics in drug design. First, we have constructed a protein-protein interaction network for Alzheimer''s disease reviewed proteins with 1412 interactions predicted among 969 proteins. Second, the disease proteins were given confidence scores to prioritize and then analyzed for their homology nature with respect to paralogs and homologs. The homology persisted with the mouse giving a basis for drug design phase. The method will create a new drug design technique in the field of bioinformatics by linking drug design process with protein-protein interactions via signal pathways. This method can be improvised for other diseases in future.     

Foreign protein production using plant cell and organ cultures: Advantages and limitations

Plants and plant tissue cultures are used as host systems for expression of foreign proteins including antibodies, vaccines and other therapeutic agents. Recombinant or stably transformed plants and plant cell cultures have been applied for foreign protein production for about 20 years. Because the product concentration achieved exerts a major influence on process economics, considerable efforts have been made by commercial and academic research groups to improve foreign protein expression levels. However, post-synthesis product losses due to protease activity within plant tissues and/or extracellular protein adsorption in plant cell cultures can negate the benefits of molecular or genetic enhancement of protein expression. Transient expression of foreign proteins using plant viral vectors is also a practical approach for producing foreign proteins in plants. Adaptation of this technology is required to allow infection and propagation of engineered viruses in plant tissue cultures for transient protein expression in vitro.     

Modulating secretory pathway pH by proton channel co‐expression can increase recombinant protein stability in plants

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host‐cell engineering approaches are being developed to ameliorate host‐cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid‐susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co‐expression. The co‐expression of a heterologous ion channel to stabilize acid‐labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.     

Plants as bioreactors for protein production: avoiding the problem of transgene silencing

Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures. The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations. Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories. Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level. Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.These authors contributed equally to this work     

Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs

The transient expression of recombinant biopharmaceutical proteins in plants can suffer inter‐batch variation, which is considered a major drawback under the strict regulatory demands imposed by current good manufacturing practice (cGMP). However, we have achieved transient expression of the monoclonal antibody 2G12 and the fluorescent marker protein DsRed in tobacco leaves with ～15% intra‐batch coefficients of variation, which is within the range reported for transgenic plants. We developed models for the transient expression of both proteins that predicted quantitative expression levels based on five parameters: The OD_600nm of Agrobacterium tumefaciens (from 0.13 to 2.00), post‐inoculation incubation temperature (15–30°C), plant age (harvest at 40 or 47 days after seeding), leaf age, and position within the leaf. The expression models were combined with a model of plant biomass distribution and extraction, generating a yield model for each target protein that could predict the amount of protein in specific leaf parts, individual leaves, groups of leaves, and whole plants. When the yield model was combined with a cost function for the production process, we were able to perform calculations to optimize process time, yield, or downstream costs. We illustrate this procedure by transferring the cost function from a production process using transgenic plants to a hypothetical process for the transient expression of 2G12. Our models allow the economic evaluation of new plant‐based production processes and provide greater insight into the parameters that affect transient protein expression in plants. Biotechnol. Bioeng. 2012; 109: 2575–2588. © 2012 Wiley Periodicals, Inc.     

Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5′UTR combination

The promoter and 5′‐untranslated region (5′UTR) play a key role in determining the efficiency of recombinant protein expression in plants. Comparative experiments are used to identify suitable elements but these are usually tested in transgenic plants or in transformed protoplasts/suspension cells, so their relevance in whole‐plant transient expression systems is unclear given the greater heterogeneity in expression levels among different leaves. Furthermore, little is known about the impact of promoter/5′UTR interactions on protein accumulation. We therefore established a predictive model using a design of experiments (DoE) approach to compare the strong double‐enhanced Cauliflower mosaic virus 35S promoter (CaMV 35SS) and the weaker Agrobacterium tumefaciens Ti‐plasmid nos promoter in whole tobacco plants transiently expressing the fluorescent marker protein DsRed. The promoters were combined with one of three 5′UTRs (one of which was tested with and without an additional protein targeting motif) and the accumulation of DsRed was measured following different post‐agroinfiltration incubation periods in all leaves and at different leaf positions. The model predictions were quantitative, allowing the rapid identification of promoter/5′UTR combinations stimulating the highest and quickest accumulation of the marker protein in all leaves. The model also suggested that increasing the incubation time from 5 to 8 days would reduce batch‐to‐batch variability in protein yields. We used the model to identify promoter/5′UTR pairs that resulted in the least spatiotemporal variation in expression levels. These ideal pairs are suitable for the simultaneous, balanced production of several proteins in whole plants by transient expression. Biotechnol. Bioeng. 2013; 110: 471–482. © 2012 Wiley Periodicals, Inc.     

Computational Assessment of Protein–protein Binding Affinity by Reversely Engineering the Energetics in Protein Complexes

The cellular functions of proteins are maintained by forming diverse complexes. The stability of these complexes is quantified by the measurement of binding affinity, and mutations that alter the binding affinity can cause various diseases such as cancer and diabetes. As a result, accurate estimation of the binding stability and the effects of mutations on changes of binding affinity is a crucial step to understanding the biological functions of proteins and their dysfunctional consequences. It has been hypothesized that the stability of a protein complex is dependent not only on the residues at its binding interface by pairwise interactions but also on all other remaining residues that do not appear at the binding interface. Here, we computationally reconstruct the binding affinity by decomposing it into the contributions of interfacial residues and other non-interfacial residues in a protein complex. We further assume that the contributions of both interfacial and non-interfacial residues to the binding affinity depend on their local structural environments such as solvent-accessible surfaces and secondary structural types. The weights of all corresponding parameters are optimized by Monte-Carlo simulations. After cross-validation against a large-scale dataset, we show that the model not only shows a strong correlation between the absolute values of the experimental and calculated binding affinities, but can also be an effective approach to predict the relative changes of binding affinity from mutations. Moreover, we have found that the optimized weights of many parameters can capture the first-principle chemical and physical features of molecular recognition, therefore reversely engineering the energetics of protein complexes. These results suggest that our method can serve as a useful addition to current computational approaches for predicting binding affinity and understanding the molecular mechanism of protein–protein interactions.