LaBaer Lab | Research
Research in the LaBaer Lab
The study of functional proteomics requires a multidisciplinary approach – merging biology, biochemistry, cell biology, engineering, molecular biology, bioinformatics, software development, database management, and biostatistics – to aid in the evaluation of the entire collection of human proteins (proteome) and their specific role(s) in health and disease.
Nucleic Acid-Programmable Protein Array (NAPPA)
The foundation of our proteomics research is a novel protein microarray technology, called Nucleic Acid-Programmable Protein Array (NAPPA), which replaces the complex process of spotting purified proteins with the simple process of spotting plasmid DNA. This core technology is a key innovation in the quest for identifying and characterizing proteins that regulate crucial events in the progression of diseases such as type I diabetes, breast and lung cancer, or infectious pathogens that are currently the focus of our research.
Protein microarrays display proteins in high spatial density on a microscopic surface. They can be used to test a variety of functions of many proteins simultaneously including interactions with other macromolecules, functional activity, and their suitability as substrates of enzymes. Historically, these arrays have been produced by expressing and purifying proteins, which are then printed on the array surface. However, many challenges accompany this approach including the difficulty in purifying many proteins, a dynamic range in yields that spans several orders of magnitude (which is reflected in the widely varying amount of protein displayed on the array surface) and the danger or proteins becoming unfolded during the many manipulations, such as purification, storage and printing.
To avoid these challenges and to produce the most functional arrays possible, we have developed a novel protein microarray technology, called Nucleic Acid-Programmable Protein Array (NAPPA), which replaces the complex process of spotting purified proteins with the simple process of spotting plasmid DNA. Our technology exploits the ability to transfer protein encoding regions (open reading frames; ORFs) into specialized tagged expression vectors. These new expression clones are then spotted on the array and the proteins are then produced in situ in a cell-free system and immobilized in place upon their synthesis. This minimizes direct manipulation of the proteins and produces them just-in-time for the experiment, avoiding problems with protein purification and stability (Science. 2004 305:86). A next generation method for these arrays has been developed that allows thousands of proteins to be produced simultaneously in situ, and with remarkably consistent protein levels displayed (Nat Methods. 2008 5:535).
The power of this approach is that by expressing many proteins on a single array, it is possible to test the function of many proteins simultaneously. In our laboratory we use NAPPA technology to explore several biological questions including: 1. Identifying autoantibody biomarkers in sera that can be readily used for the detection of cancer, such as breast cancer, ovarian cancer, prostate cancer, and lung cancer. 2. Identifying novel autoantibodies for Type I diabetes by serological screening of diabetic sera against a 6000+ library of human antigens. 3. Developing a better vaccine against B. anthracis. 4. Screening human serum in order to find antibodies against specific pathogens such as Pseudomonas aeruginosa and V. chlorea to identify immunogenic proteins as a first step towards developing an effective vaccine.
Using protein microarrays to examine small molecule inhibitory specificity
There is a strong interest in our group in developing next generation applications for protein microarrays. One such project uses NAPPA to look at kinase activity. The human kinome contains more than 500 proteins well known for their importance in normal cell physiology and for their role in many diseases. Many kinases printed on NAPPA retain their kinase activity and can be used to test the IC50 of drugs used to inhibit them. This method may provide a high throughput rapid approach for evaluating drug selectivity as well as structure activity relationships.
A second project involves coupling two technologies, NAPPA and surface plasmon resonance imaging (SPRi), to create a high-throughput platform to detect and characterize a variety of protein interactions using a label-free detection system that is sensitive, quantitative and provides information on binding kinetics. We have developed new chemistries for NAPPA compatible with a SPRi device that has been adapted to be compatible with protein microarrays and detecting multiplex binding events. This method allows for real time and label-free detection of binding events for multiple proteins simultaneously, making this the first approach that will allow the simple determination of strength and selectivity of binding at scale. This technology has potential to revolutionize the study of protein interaction networks by enabling quantitative comparisons of binding affinities across many molecular species, as well as determining the kinetic data of interactions pathways.
Cell based research
We also have a strong interest in the high throughput study of protein function in vivo using cell based assays. Our large collection of full length gene clones can be employed in the ectopic expression of proteins in cells. We also have a complete collection of shRNAs that target human genes, allowing us to knock down expression of virtually any gene of interest. Both ectopic expression and RNAi are introduced into cells using retro and lenti-viral vectors, allowing us to target virtually any cell type of interest. By coupling these tools with state of the art robotics, high throughput screening studies are executed to look for proteins that alter cellular phenotypes.
One example of this approach focused on identifying and characterizing genes that regulate critical events involved in the progression of breast cancer and in understanding the development of resistance to anti-estrogen drug treatment. We have produced several matching drug sensitive and resistant breast cancer cultured cell lines that we used to find proteins that can make a drug sensitive cell become resistant. We used our clone collection of more than 500 human kinases in high throughput unbiased screens to identify 29 kinases that confer drug resistance on sensitive MCF7 subclones in repeated screens. This research has the potential to find genes that are involved in breast development and resistance to hormonal treatment in breast cancer. We hope that defining the gene pathways that are responsible for drug resistance will lead to combined treatments that will more effectively treat resistant cancers.
Our technology exploits the ability to transfer protein encoding regions of the human genome (ORFs) into specialized tagged expression vectors. These new expression clones are then spotted on the array, and the proteins are then produced in situ in a cell-free system and immobilized in place upon their synthesis. This minimizes direct manipulation of the proteins and produces them just-in-time for the experiment, avoiding problems with protein purification and stability (Science. 2004 305:86). A next generation method for these arrays has been developed that allows thousands of proteins to be produced simultaneously in situ, and with remarkably consistent protein levels displayed (Nat Methods. 2008 5:535).
In concert with NAPPA technology, we have assembled a key scientific resource: the DNASU Plasmid Repository (Nucleic Acids Res. 2007 35:D680-4., Nucleic Acids Res. 2010 38:D743-9., J Struct Funct Genomics. 2011 Mar 1. [Epub ahead of print]) a massive library of more than 131,000 expression-ready plasmid clones for genes found in human and other commonly studied organisms. This repository is maintained in a robotically controlled, rapid-access storage facility, with clone variants openly available to laboratories all over the world.
In addition to the plasmid repository, DNASU also has a high throughput sequencing facility and Next Gen Sequencing core.