Spatially Resolved Effects of Protein Freeze-Thawing in a Small-Scale Model Using Monoclonal Antibodies.

Protein freeze-thawing is frequently used to stabilize and store recombinantly produced proteins after different unit operations in upstream and downstream processing. However, freeze-thawing is often accompanied by product damage and, hence, loss of product. Different effects are responsible, including cold denaturation, aggregation effects, which are caused by inhomogeneities in protein concentration, as well as pH and buffer ingredients, especially during the freeze cycle. In this study, we tested a commercially available small-scale protein freezing unit using immunoglobin G (IgG) as monoclonal antibody in a typical formulation buffer containing sodium phosphate, sodium chloride, and Tween 80. Different freezing rates were used respectively, and the product quality was tested in the frozen sample. Spatially resolved tests for protein concentration, pH, conductivity, and aggregation revealed high spatial differences in the frozen sample. Usage of slow freezing rates revealed high inhomogeneities in terms of buffer salt and protein distribution, while fast rates led to far lower spatial differences. These protein and buffer salt inhomogeneities can be reliably monitored using straight forward analytics, like conductivity and photometric total protein concentration measurements, reducing the need for HPLC analytics in screening experiments. Summarizing, fast freezing using steep rates shows promising results concerning homogeneity of the final frozen product and inhibits increased product aggregation.

pH conditioning is a crucial step in primary recovery - A case study for a recombinant Fab from E. coli.

Primary recovery of recombinant proteins from E. coli often describes a major challenge in downstream processing. After product release, the target protein usually accounts for only a small amount of total protein and has to be separated from a complex mixture of host cell proteins (HCPs) and non-proteinogenic impurities, such as DNA and lipids. Non-optimized procedures as well as unfavorable conditions at the extraction step and conditioning cause significant product loss already prior capture. In this study, we investigated pH conditioning during primary recovery for a subsequent cation exchange chromatography (CEX)-based capture of a recombinant Fab produced in E. coli. We showed that pH ≤ 5.0, which is necessary for CEX, led to high product loss due to protein precipitation during cell disruption and pH conditioning. Thus, we developed a procedure that resulted in a 25% increased Fab recovery prior capture based on simple re-arrangement of process steps and the use of a low-cost stabilizing agent. Summarizing, we show the huge potential for simple and cheap improvement of overall downstream process recovery by optimization of pH conditioning during primary product recovery.

A quantitative indicator diagram for lytic polysaccharide monooxygenases reveals the role of aromatic surface residues in HjLPMO9A regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) have changed our understanding of lignocellulosic degradation dramatically over the last years. These metalloproteins catalyze oxidative cleavage of recalcitrant polysaccharides and can act on the C1 and/or C4 position of glycosidic bonds. Structural data have led to several hypotheses, but we are still a long way from reaching complete understanding of the factors that determine their divergent regioselectivity. Site-directed mutagenesis enables the investigation of structure-function relationship in enzymes and will be of major importance in unraveling this intriguing matter. In this context, it is crucial to have an enzyme assay or screening approach with a direct correlation with the desired functionality. LPMOs render this search extra challenging due to their insoluble substrates, complex pattern of reaction products and lack of synthetic standards of most oxidized products. Here, we describe a regioselectivity indicator diagram based on the time-course of only 2 HPAEC-PAD signals. The diagram was successfully used to confirm the hypothesis that aromatic surface residues influence the C1/C4 oxidation ratio in Hypocrea jecorina LPMO9A. Consequently, the diagram should become a valuable tool in the search towards better understanding and engineering of regioselectivity in LPMOs.

A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli.

Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E. coli and investigated and compared both strategies, (a) the production of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the production of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titre of 100mgL-1 cultivation broth, and were able to produce 48mg active HRP per litre cultivation broth in the periplasm. In terms of biochemical properties, soluble HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and production. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amounts, which are then refolded.

A novel cytosolic NADH:quinone oxidoreductase from Methanothermobacter marburgensis.

Methanothermobacter marburgensis is a strictly anaerobic, thermophilic methanogenic archaeon that uses methanogenesis to convert H2 and CO2 to energy. M. marburgensis is one of the best-studied methanogens, and all genes required for methanogenic metabolism have been identified. Nonetheless, the present study describes a gene (Gene ID 9704440) coding for a putative NAD(P)H: quinone oxidoreductase that has not yet been identified as part of the metabolic machinery. The gene product, MmNQO, was successfully expressed, purified and characterized biochemically, as well as structurally. MmNQO was identified as a flavin-dependent NADH:quinone oxidoreductase with the capacity to oxidize NADH in the presence of a wide range of electron acceptors, whereas NADPH was oxidized with only three acceptors. The 1.50 Å crystal structure of MmNQO features a homodimeric enzyme where each monomer comprises 196 residues folding into flavodoxin-like α/ß domains with non-covalently bound FMN (flavin mononucleotide). The closest structural homologue is the modulator of drug activity B from Streptococcus mutans with 1.6 Å root-mean-square deviation on 161 Cα atoms and 28% amino-acid sequence identity. The low similarity at sequence and structural level suggests that MmNQO is unique among NADH:quinone oxidoreductases characterized to date. Based on preliminary bioreactor experiments, MmNQO could provide a useful tool to prevent overflow metabolism in applications that require cells with high energy demand.