Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors.

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

Unexpected tobacco etch virus (TEV) protease cleavage of recombinant human proteins.

The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.

Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases.

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment.

Producing recombinant proteins in Vibrio natriegens.

The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli.

Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA.

The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

Improved production of class I phosphatidylinositol 4,5-bisphosphate 3-kinase.

Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the ß and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.

Combinatorial assembly of clone libraries using site-specific recombination.

Generation of DNA clones for use in proteomic and genomic research often requires a significant level of parallel production, as the number of downstream options for these experiments increases. Where a single fluorescently tagged construct may have sufficed before, there is now the need for multiple types of labels for different readouts and different assays. Protein expression, which once utilized a very small set of vectors because of low throughput expression and purification, has now rapidly matured into a high throughput system in which dozens of conditions can be tested in parallel to identify the best candidate clones. This has returned the bottleneck in many of these technologies to the generation of DNA clones, and standard cloning techniques often dramatically limit the throughput and success of such processes. In order to overcome this bottleneck, higher-throughput and more parallel cloning processes need to be developed which would allow rapid, inexpensive production of final clones. In addition, there is a strong need to utilize standardized elements to avoid unnecessarily remaking fragments of clones that could be used in multiple constructs. The advent of recombinational cloning helped to increase the parallel processing of DNA clones, but was still limited by the need to generate different vector backbones for each specific need. The solution to this problem emerged with the introduction of combinatorial approaches to clone construction, based on either homologous or site-specific recombination processes. In particular, the Gateway Multisite system provides all of the necessary components for a highly parallel, inexpensive, rapid, and diverse platform for clone construction in many areas of proteomic and genomic research. Here we describe our optimized system for combinatorial cloning, including improvements in cloning protocols and construct design that permit users to easily generate libraries of clones which can be combined in parallel to create an unlimited number of final constructs. The system is capable of utilizing the tens of thousands of commercially available Gateway clones already in existence, and allows easy adaptation of most DNA vectors to the system.

Optimizing transient recombinant protein expression in mammalian cells.

Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression.

The apoptosis modulator and tumour suppressor protein RBM5 is a phosphoprotein.

RBM5/LUCA-15/H37 is a nuclear SR-related RNA binding protein with the ability to modulate both apoptosis and the cell cycle, and retard tumour formation. How RBM5 functions to carry out these, potentially interrelated, biological activities is unknown. Since reversible phosphorylation has been shown to play an important role in the regulation of SR protein function, apoptosis and cell cycle control, in an attempt to elucidate the underlying mechanisms regulating RBM5 function, the phosphorylation status of RBM5 was investigated. Whole cell lysate from growing cell cultures was treated with the broad phosphatase spectrum of CIP, resulting in a decrease in the molecular mass of RBM5. A similar decrease in molecular mass, of a subset of RBM5 proteins, was observed during growth factor deprivation, in a manner consistent with partial dephosphorylation of RBM5. Molecular mass increased upon growth factor addition, demonstrating that this apoptosis-associated alteration in molecular mass was a reversible process. Immunoprecipitation and mutagenesis experiments strongly suggested that phosphotyrosines are not present in RBM5 under normal growth conditions, and that serine 69 is phosphorylated, but not by Akt kinase. Taken together, these results suggest that reversible phosphorylation of RBM5 is a mechanism capable of regulating RBM5 participation in modulating apoptosis, and perhaps tumour suppression.

Expression of RBM5-related factors in primary breast tissue.

The aim of this study was to examine the expression of the RBM5 tumor suppressor, in relation to RBM6 and RBM10, to obtain a better understanding of the potential role played by these RBM5-related factors in the regulation of RBM5 tumor-suppressor activity. Paired non-tumor and tumor samples were obtained from 73 breast cancer patients. RNA and protein expression were examined by semi-quantitative reverse transcription-polymerase chain reaction and immunoblot, respectively. Data were analyzed using various statistical methods to test for correlations amongst the RBM5-related factors, and between the factors and various pathological parameters. Most notably, RBM5, RBM10v1, and HER2 protein expression levels were elevated in tumor tissue (P < 0.0001). RBM5 and RBM10v1 protein expression were significantly positively correlated (P < 0.001), as were RBM5 and HER2 protein expression (P < 0.01), in both non-tumor and tumor tissue, whereas RBM10v1 and HER2 protein expression were only marginally correlated, in non-tumor tissue (P < 0.05). Interestingly, RBM5 and RBM10v1 protein expression were both deregulated in relation to RNA expression in tumor tissue. RBM10v2 and RBM6 RNA were highly significantly positively correlated in relation to various factors relating to poor prognosis (P < 0.0001). To our knowledge, this study is the first to examine RBM5 expression at both the RNA and protein level in primary breast tumor tissue, and the first to examine expression of all RBM5-related factors in a comprehensive manner. The results provide a graphic illustration that RBM5-related factors are significantly differentially expressed in breast cancer, and suggest complex inter-related regulatory networks involving alternative splicing, oncogenic expression, and tissue-specific function.