Better Tools and Better Knowledge for Structural Genomics
Auto Induction Media – F.W. Studier, Brookhaven National Laboratory
Studier at BNL has formulated growth media in which expression strains can grow uninduced to relatively high cell densities and then be induced automatically without any intervention by the experimenter. Cell densities attained in these auto-inducing cultures have produced 10-fold more target protein per volume of culture than with the standard IPTG induction protocol. Auto-induction also allows many cultures to be inoculated in parallel and induced simply by growing to saturation, making auto-induction a powerful tool for screening clones for expression and solubility in an automated setting. Our first test of a medium formulated for labeling proteins with Se-Met by auto-induction produced 5mg of purified, fully labeled protein from 500mL of culture, which sufficed for structure determination. We continue to evaluate the effectiveness of these media and protocols as we begin to use them to produce proteins routinely. Once new vectors being developed in Studier’s laboratory (for expression of toxic protein in E. coli) are functional, we plan to explore systematically the capabilities and limits of the new combinations of expression vectors and media.
Auto-induction protocols and recipes for producing proteins were developed and distributed to more than 150 laboratories and described at several national and international scientific meetings. A patent has been applied for, products based on this work have been commercialized by Novagen as Overnight Express Auto induction Systems, and this work has been recognized by an R&D 100 Award from R&D Magazine as one of the 100 most significant innovations commercialized in 2004. Auto-induction provides substantial improvements in convenience and efficiency of high-throughput protein production and is used routinely by NYSGXRC and several other phase one Protein Structure Initiative (PSI-1) structural genomics centers for parallel screening, production of 10-100mg amounts in shake flasks, and labeling with Se-Met.
A Novel, Facile System for Protein Expression – C.D. Lima, Sloan-Kettering Institute
Lima’s novel SUMO-based protein expression system (developed while at Cornell; see US Patent Application 10/188,343) for high-throughput cloning and protein expression has been utilized within the NYSGXRC to rapidly clone, affinity purify, and proteolytically liberate NYSGXRC targets from the His6-SMT3 fusion partner for further purification. This proprietary vector has been Topo-adapted for rapid (5 min) topoisomerase mediated directional flap-ligation to facilitate high-throughput and highly efficient restriction enzyme independent cloning for DNA generated from the coding regions containing our protein targets. This measure alleviates the need for engineering longer than necessary primers to contain convenient restriction sites, the need to enzymatically digest and purify the DNA inserts, and the need for long, low-temperature ligations traditionally utilized to clone DNA fragments into expression vectors. A description of the commercially available system can be found by searching the Invitrogen Web site for the keyword “SUMO” or visiting:
A Mammalian Cell Expression System for Disulfide Rich Proteins – L. Shapiro, Columbia University
Production of natively folded mammalian proteins in heterologous systems is frequently challenging. Proteins can be mis-folded due to the lack of cognate chaperones or the absence of the proper cellular machinery for post-translational modification. Shapiro has developed a system for the selection of highly expressing stable mammalian cells based on detection of the fluorescence intensity of a co-expressed marker, the green fluorescent protein (GFP).
In the Shapiro system, the coding sequence for the gene of interest is placed under the control of a strong constitutive promoter (such as the promoter element derived from cytomegalovirus, CMV). Downstream, after the termination codon for the gene of interest, an internal ribosome entry site (IRES) is followed by the coding sequence for GFP. Transcription from this construct produces a single bicistronic messenger RNA encoding both genes. The IRES element enables binding of the ribosome at the initiation site for production of GFP. Thus, two separate proteins – the target and GFP – are translated from the same messenger RNA, and expression levels of both proteins are thereby coupled. This system enables efficient selection of highly expressed targets by monitoring the fluorescence intensity of mammalian cells expressing variable amounts of GFP. Use of fluorescence-activated cell sorting (FACS) technology allows for rapid selection of either clonal or non-clonal populations of highly expressing cells. We have used this method to identify efficiently producing HEK-293 cell lines for production of the highly-disulfide-bonded proteins of the resistin hormone family (Mancia et al., 2004). Protein expressed in mammalian cells using this system allowed us to determine crystal structures for resistin and RELM-b (Patel et al., 2004).
References
Patel, S.D., Rajala, M.W., Rossetti, L., Scherer, P.E., and Shapiro, L. (2004) Disulfide-dependent multimeric assembly of resistin family hormones. Science 304, 1154-1158.
Mancia, F., Patel, S.D., Rajala, M.W., Scherer, P.E., Nemes, A., Ira Schieren, I., Hendrickson, W.A., and Shapiro, L. (2004) Optimization of protein production in mammalian cells with a co-expressed fluorescent marker. Structure, 12, 1355-1360.
High-Throughput Metallo-Protein Detection – M.R. Chance, Albert Einstein College of Medicine
During PSI-1 Year Four, Chance and co-workers at AECOM put into routine operation a novel, automated system to screen purified proteins for metal content to provide functional information as well as facilitate SAD/MAD structure determinations.
Up to 1/3 of all proteins, contain metal ions. Among the transition metals found in proteins, iron and zinc are among the most common. This method relies on illuminating samples with high-energy synchrotron X-rays that are capable of ejecting a 1s electron from the first electron shell surrounding a metal nucleus. Passage of another electron from the second shell to fill the “hole” in the first shell yields an emitted X-ray (Ka emission) of energy that is characteristic for each element.
The instrument developed by Chance, now routinely used by the NYSGXRC, utilizes advanced X-ray absorption spectroscopy equipment on BNL NSLS Beamline X9B. It is one of only two places in the US (the other being Stanford Synchrotron Radiation Laboratory) at which such experiments can be performed. A multi-element, fast count rate, high-resolution Germanium detector has been combined with a multi-plate rail for high-throughput sample screening that can hold 5, 16-well plates. Rapid data collection and easy setup and takedown of the system were design and engineering requirements and have been achieved. The system is designed to detect the following metals (each with an accessible anomalous absorption edge): Mn, Fe, Co, Cu, Ni, Zn, and Se.
