High-throughput cloning and expression in recalcitrant bacteria

We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an organism-specific plasmid ready for high-efficiency transformation. We demonstrated VBEx proof of principle for Lactococcus lactis, but the method can be adapted to all organisms for which plasmids are available

Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors.

The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.MEK was supported by a Commonwealth (University of Cambridge) Scholarship awarded in conjunction with the Cambridge Commonwealth Trust and Cambridge Overseas Trust. AGC and KJM were supported by grants from the BBSRC (Grant No: BB/I019669/1 and BB/I019227/1). GGJ received funding from the Ogden Trust and the Isaac Newton Trust administered through the University of Cambridge Bursary Scheme. DSCH was supported by a Croucher Cambridge International Scholarship awarded in conjunction between the Croucher Foundation and the Cambridge Overseas Trust. SAH was supported by an Oliphant Cambridge Australia Scholarship (App No: 10132070) awarded by the Cambridge Commonwealth Trust. The contributions of LBM and LPSC were supported by funds from the Francis Crick Institute, which receives its core funding principally from Wellcome Trust, Cancer Research UK, and the UK Medical Research Council (to LPSC - MC_UP_A253_1111) and funds from FAPESP, CNPq and CAPES-PDSE (to LBM - 2011/21232-1, 140079/2013-0, 99999.003125/2014-09).This is the final version of the article. It first appeared from the American Chemical Society via http://dx.doi.org/10.1021/acs.jmedchem.6b0000

Fragment-Sized EthR Inhibitors Exhibit Exceptionally Strong Ethionamide Boosting Effect in Whole-Cell Mycobacterium tuberculosis\textit{Mycobacterium tuberculosis} Assays

Small-molecule inhibitors of the mycobacterial transcriptional repressor EthR have previously been shown to act as boosters of the second-line antituberculosis drug ethionamide. Fragment-based drug discovery approaches have been used in the past to make highly potent EthR inhibitors with ethionamide boosting activity both $\textit{in vitro}$ and $\textit{ex vivo}$. Herein, we report the development of fragment-sized EthR ligands with nanomolar minimum effective concentration values for boosting the ethionamide activity in $\textit{Mycobacterium tuberculosis}$ whole-cell assays.We would like to thank A. Coyne for help in the preparation of this manuscript. P.O.N. would like to thank the EPSRC for providing Ph.D. funding. We also thank the Bill and Melinda Gates Foundation and the EU FP7MM4TB Grant No. 260872, the ERC-STG INTRACELLTB Grant No. 260901, the Agence Nationale de la Recherche (ANR-10-EQPX-04-01), the Feder (12001407 (D-AL) Equipex Imaginex BioMed), the Intramural Research Program of the National Institutes of Health/NIAID, and the Région Nord Pas de Calais, France, for providing funding to support this work

The Response of Lactococcus lactis to Membrane Protein Production

Background: The biogenesis of membrane proteins is more complex than that of water-soluble proteins, and recombinant expression of membrane proteins in functional form and in amounts high enough for structural and functional studies is often problematic. To better engineer cells towards efficient protein production, we set out to understand and compare the cellular consequences of the overproduction of both classes of proteins in Lactococcus lactis, employing a combined proteomics and transcriptomics approach. Methodology and Findings: Highly overproduced and poorly expressed membrane proteins both resulted in severe growth defects, whereas amplified levels of a soluble substrate receptor had no effect. In addition, membrane protein overproduction evoked a general stress response (upregulation of various chaperones and proteases), which is probably due to accumulation of misfolded protein. Notably, upon the expression of membrane proteins a cell envelope stress response, controlled by the two-component regulatory CesSR system, was observed. Conclusions: The physiological response of L. lactis to the overproduction of several membrane proteins was determined and compared to that of a soluble protein, thus offering better understanding of the bottlenecks related to membrane protein production and valuable knowledge for subsequent strain engineering.

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Applicability of Hooke’s and Jeeves Direct Search S olution Method to Metal cutting [Turning] Operation.

Role of optimization in engineering design is prominent one with the advent of computers. Optimization has become a part of computer aided design activities. It is prima rily being used in those design activities in which the goal is not only to achieve just a feasible design, but also a des ign objective. In most engineering design activities, the design objective could be simply to minimize the cost of production or to maximize the efficiency of the production. An optimization algorithm is a procedure which is executed it eratively by comparing various solutions till the optimum or a satisfactory solution is found. In many industri al design activities, optimization is achieved indirectly by comparing a few chosen design solutions and accept ing the best solution. This simplistic approach never guarantees and optimization algorithms being with one or more d esign solutions supplied by the user and then iteratively check new design the true optimum solution. There ar e two distinct types of optimization algorithms which are in use today

Heterologous production and functional and thermodynamic characterization of cation diffusion facilitator (CDF) transporters of mesophilic and hyperthermophilic origin

The members of the cation diffusion facilitator (CDF) family transport heavy metal ions and play an important function in zinc ion homeostasis of the cell. A recent structure of an Escherichia coli CDF transporter protein YiiP has revealed its dimeric nature and autoregulatory zinc transport mechanism. Here, we report the cloning and heterologous production of four different CDF transporters, two each from the pathogenic mesophilic bacterium Salmonella typhimurium and from the hyperthermophilic bacterium Aquifex aeolicus, in E. coli host cells. STM0758 of S. typhimurium was able to restore resistance to zinc ions when tested by complementation assays in the zinc-sensitive GG48 strain. Furthermore, copurification of bicistronically produced STM0758 and cross-linking experiments with the purified protein have revealed its possible oligomeric nature. The interaction between heavy metal ions and Aq_2073 of A. aeolicus was investigated by titration calorimetry. The entropy-driven, high-affinity binding of two Cd2+ and two Zn2+ per protein monomer with Kd values of around 100 nm and 1 μm, respectively, was observed. In addition, at least one more Zn2+ can be bound per monomer with low affinity. This low-affinity site is likely to possess a functional role contributing to Zn2+transport across membranes

Research data supporting "Fragment-based approaches to the development of Mycobacterium tuberculosis CYP121 inhibitors"

Supporting data for the article "Fragment-based approaches to the development of Mycobacterium tuberculosis CYP121 inhibitors", published in the Journal of Medicinal Chemistry, 2016. LCMS, HRMS, IR and NMR spectra from the characterisation of compounds reported in the Experimental Methods section of the article are provided as PDFs. Isothermal titration calorimetry enthalpy plots and data fitting for compounds reported in Table 2 of the article are provided as PDFs. Data for human cytochrome P450 inhibition studies with compounds 19a, 25a and 26a provided as a PDF.This research data supports “Fragment-based approaches to the development of Mycobacterium tuberculosis CYP121 inhibitors” which will be published in “Journal of Medicinal Chemistry”.This work was supported by the BBSRC [grant number BB/I019669/1 and BB/I019227/1], Wellcome Trust, Cancer Research UK and the UK Medical Research Council (to LPSC - MC_UP_A253_1111) FAPESP, CNPq and CAPES-PDSE (to LBM - 2011/21232-1, 140079/2013-0, 99999.003125/2014-09), SAH was supported by an Oliphant Cambridge Australia Scholarship (App No: 10132070), MEK was supported by a Commonwealth (University of Cambridge) Scholarship awarded in conjunction with the Cambridge Commonwealth Trust and Cambridge Overseas Trust. GGJ received funding from the Ogden Trust and the Isaac Newton Trust administered through the University of Cambridge Bursary Scheme. DSCH was supported by a Croucher Cambridge International Scholarship awarded in conjunction between the Croucher Foundation and the Cambridge Overseas Trust., São Paulo Research Foundation (FAPESP), National Council for Scientific and Technological Development (CNPq), Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-PDSE) and Various personal funding administered by the University of Cambridge