Development of an E. coli strain for cell-free ADC manufacturing.

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody-drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.

Structural distributions from single-molecule measurements as a tool for molecular mechanics.

A mechanical view provides an attractive alternative for predicting the behavior of complex systems since it circumvents the resource-intensive requirements of atomistic models; however, it remains extremely challenging to characterize the mechanical responses of a system at the molecular level. Here, the structural distribution is proposed to be an effective means to extracting the molecular mechanical properties. End-to-end distance distributions for a series of short poly-L-proline peptides with the sequence P(n)CG(3)K-biotin (n = 8, 12, 15 and 24) were used to experimentally illustrate this new approach. High-resolution single-molecule Förster-type resonance energy transfer (FRET) experiments were carried out and the conformation-resolving power was characterized and discussed in the context of the conventional constant-time binning procedure for FRET data analysis. It was shown that the commonly adopted theoretical polymer models-including the worm-like chain, the freely jointed chain, and the self-avoiding chain-could not be distinguished by the averaged end-to-end distances, but could be ruled out using the molecular details gained by conformational distribution analysis because similar polymers of different sizes could respond to external forces differently. Specifically, by fitting the molecular conformational distribution to a semi-flexible polymer model, the effective persistence lengths for the series of short poly-L-proline peptides were found to be size-dependent with values of ~190 Å, ~67 Å, ~51 Å, and ~76 Å for n = 8, 12, 15, and 24, respectively. A comprehensive computational modeling was carried out to gain further insights for this surprising discovery. It was found that P(8) exists as the extended all-trans isomaer whereas P(12) and P(15) predominantly contained one proline residue in the cis conformation. P(24) exists as a mixture of one-cis (75%) and two-cis (25%) isomers where each isomer contributes to an experimentally resolvable conformational mode. This work demonstrates the resolving power of the distribution-based approach, and the capacity of integrating high-resolution single-molecule FRET experiments with molecular modeling to reveal detailed structural information about the conformation of molecules on the length scales relevant to the study of biological molecules.

Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain.

The Mycobacterium tuberculosis protein tyrosine phosphatase PtpB shows resistance to the oxidative conditions that prevail within an infected host macrophage, but the mechanism of this molecular adaptation is unknown. Crystal structures of PtpB revealed previously that a closed, two-helix lid covers the active site. By measuring single-molecule Forster-type resonance energy transfer to probe the dynamics of two helices that constitute the lid, we obtained direct evidence for large, spontaneous opening transitions of PtpB with the closed form of both helices favored approximately 3:1. Despite similar populations of conformers, the two helices move asynchronously as demonstrated by different opening and closing rates under our experimental conditions. Assuming that lid closure excludes oxidant, the rates of opening and closing quantitatively accounted for the slow observed rate of oxidative inactivation. Increasing solvent viscosity using glycerol but not PEG8000 resulted in higher rates of oxidative inactivation due to an increase in the population of open conformers. These results establish that the rapid conformational gating of the PtpB lid constitutes a reversible physical blockade that transiently masks the active site and retards oxidative inactivation.

Illuminating the mechanistic roles of enzyme conformational dynamics.

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.

Quantitation of steroid hormones in thin fresh frozen tissue sections.

As analytical technologies in proteomics and metabolomics continue to mature, there is an increasing need to apply these to clinically relevant biologic samples. In this study, a liquid chromatography-tandem mass spectrometry method that utilizes selected reaction monitoring was used to measure the absolute quantity of estrogens and estrogen metabolites and testosterone in 8-microm tissue sections obtained from a fresh frozen lymph node tumor infiltrated by metastatic breast carcinoma. Total (conjugated plus unconjugated) and unconjugated levels of these steroid hormones were measured using two cohorts, each containing five adjacent serial sections cut from this tumor. The results were highly reproducible across replicate samples, showing that typical histological tissue sections represent an important sample type for the measurement of these specific metabolites.

A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems.

Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell-free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re-examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell-free system with a 95% reduction in reagent costs (excluding cell-extract, plasmid, and T7 RNA polymerase made in-house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze-thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high-throughput screening, and biotherapeutics.

Confocal single-molecule FRET for protein conformational dynamics.

Single-molecule FÓ§rster-type resonance energy transfer (smFRET) is a unique technique capable of following conformational motions of individual protein molecules. The direct observation of individual proteins provides rich information that would be washed away in ensemble measurements, hence opening up new avenues for establishing the protein structure-function relationships through dynamics. Retrieving dynamics information of biomolecular motions via smFRET, though, requires careful experiment design and rigorous treatment of single-molecule statistics. Here, we describe the rudimentary steps for an smFRET experiment, including sample preparation for the microscope, building of critical parts for single-molecule FRET detection, and a robust methodology for photon-by-photon data analysis.

Engagement of arginine finger to ATP triggers large conformational changes in NtrC1 AAA+ ATPase for remodeling bacterial RNA polymerase.

The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to σ54-RNA polymerase to activate transcription from σ54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the γ-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods. First, the arginine finger induces rigid-body roll, extending surface loops above the plane of the ATPase ring to bind σ54. Second, ATP hydrolysis permits Pi release and retraction of the arginine with a reversed roll, remodeling σ54-RNAP. This model provides a fresh perspective on how ATPase subunits interact within the ring-ensemble to promote transcription, directing attention to structural changes on the arginine-finger side of an ATP-bound interface.

Direct Mg(2+) binding activates adenylate kinase from Escherichia coli.

We report evidence that adenylate kinase (AK) from Escherichia coli can be activated by the direct binding of a magnesium ion to the enzyme, in addition to ATP-complexed Mg(2+). By systematically varying the concentrations of AMP, ATP, and magnesium in kinetic experiments, we found that the apparent substrate inhibition of AK, formerly attributed to AMP, was suppressed at low magnesium concentrations and enhanced at high magnesium concentrations. This previously unreported magnesium dependence can be accounted for by a modified random bi-bi model in which Mg(2+) can bind to AK directly prior to AMP binding. A new kinetic model is proposed to replace the conventional random bi-bi mechanism with substrate inhibition and is able to describe the kinetic data over a physiologically relevant range of magnesium concentrations. According to this model, the magnesium-activated AK exhibits a 23- +/- 3-fold increase in its forward reaction rate compared with the unactivated form. The findings imply that Mg(2+) could be an important affecter in the energy signaling network in cells.

Gene promoter methylation in prostate tumor-associated stromal cells.

BACKGROUND: Gene expression can be silenced through the methylation of specific sites in the promoter region. This mechanism of gene silencing has an important role in the carcinogenesis of prostate and other cancers. Although tumor-associated stromal cells also exhibit changes in gene expression, promoter methylation has not been described in these cells. METHODS: Tumor epithelia, tumor-associated stroma and normal epithelia, and stroma adjacent to tumor tissues were isolated from whole-mount prostatectomy specimens (two per patient) of patients (n = 5) with localized prostate cancer and from normal epithelia and stroma from benign prostate hyperplasia specimens (two per patient) from men (n = 5) without prostate cancer by using laser capture microdissection or expression microdissection. The methylation status of three genes important in prostate carcinogenesis, GSTP1, RARbeta2, and CD44, were evaluated using quantitative methylation-sensitive polymerase chain reaction. RESULTS: GSTP1 and RARbeta2 were methylated in the tumor epithelium of all five prostate cancer patients and in the tumor-associated stroma in four of the five patients. CD44 was methylated in the tumor epithelium from four of the five patients but not in the tumor stroma. GSTP1 and RARbeta2 were methylated in normal epithelium of two and four patients, respectively, and in normal stroma of one and two patients, respectively, that were isolated from regions adjacent to the tumors and may have resulted from a tumor-field effect; CD44 methylation was not observed in normal epithelium or stroma. In contrast, normal epithelia and stroma from benign prostate hyperplasia specimens showed no promoter methylation in GSTP1, RARbeta2, or CD44. CONCLUSIONS: The observation of promoter methylation in the non-neoplastic cells of the prostate tumor microenvironment may advance our understanding of prostate cancer development and progression and lead to new diagnostic and prognostic markers and therapeutic targets.

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations--implications for the study of ribosome dynamics.

Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg(2+) is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg(2+) concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg(2+) that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg(2+) concentrations. In low Mg(2+) concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.