Abstracts - Session 6 & 7

Day 2 Tuesday October 18th, 10.20-11.50

Session 6 - Purification of Protein Complexes

6.01 A Simple Complex	Radu Aricescu	Wellcome Trust Centre for Human Genetics, Division of Structural Biology,University of Oxford, Oxford, UK.

6.02 Expression from Polycistronic Vectors as a Novel Approach to Study Multimeric Protein Complexes – Insights from the Nicotinic Acetylcholine Receptor	Viktoria Kukhtina	Institute of Chemistry and Biochemistry, Free University Berlin, Takustraße 6, DE-14195 Berlin, Germany.
Overexpression of multidomain proteins is a challenging task. Our research topic is a multidomain transmembrane receptor protein, which makes the task even more exciting. The nicotinic acetylcholine receptor (nAChR) remains underinvestigated in terms of structure. No crystal structure of a ligand-gated ion channel was obtained until now. The intracellular domain of the nAChR is the least studied part of the whole receptor. This intracellular domain is comprised of five big loops, in the case of the Torpedo nAChR of loops of the five receptor subunits, 2 alpha, beta, gamma, delta. In this study the loop proteins were expressed in a heterologous system with the purpose of structural characterization. The creation of a polycistronic vector allowed us to produce all domains together in one E. coli cell under the control of a single promoter. Protein expression using a polycistronic vector has the main advantage over the individually expressed intracellular domains that all proteins of the receptor complex are present at the same time. This may help correct folding and assembly of the protein domain complex. The expression of all intracellular domains (a, b, g, d) in the polycistronic system was confirmed by MALDI MS as well as by Western blotting using protein-specific antibodies. Refolding of the proteins, extracted with urea from the purified inclusion bodies, was achieved successfully by size exclusion chromatography. Purification was performed by affinity chromatography on Ni-NTA. As only one of the domains, the d-loop had been carrying a (His)6-tag the occurrence of the intracellular domains of all subunits in the elution fraction after affinity chromatography indicated the formation of a complex of the intracellular domains of all receptor subunits.

6.03 Two-step Purification of Outer Membrane Proteins	Konstantinos Beis1#, Chris Whitfield2, Ian Booth3, and James H. Naismith1	1 Centre for Biomolecular Sciences, University of St. Andrews, KY16 9ST, UK. # Current address: The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 2 Department of Microbiology, University of Guelph, Guelph, Ontario, N1G 2W1 Canada. 3 Department of Molecular & Cell Biology, University of Aberdeen, AB25 2ZD, UK.
Here, we describe a simple and efficient method for the purification of E. coli outer membrane proteins. We have tested this protocol for the purification of Wza and OmpC proteins. Both proteins were purified to homogeneity, in two-steps, by anion exchange and size exclusion chromatography with a final yield of 2 mg/l for the Wza protein and 100 mg/l for the OmpC protein. The purity of the samples was judged by electrophoretic analysis, mass spectrometry, and single particle analysis, 3D crystallisation and X-ray diffraction.

6.04 Pyrococcus furiosus RNA Polymerase: In vitro Reconstitution and Subunits Interactions Analysis	Souad Naji1, Bernd Goede2, Karin Ilg1, and Michael Thomm1	1 Lehrstuhl für Mikrobiologie und Archaeenzentrum, Universität Regensburg, DE-93053 Regensburg, Germany. 2 Institut für Allgemeine Mikrobiologie, Universität Kiel, Am Botanischen Garten 1-9, DE-24118 Kiel, Germany.
Archaeal RNA Polymerases (RNAPs) resemble the eukaryotic nuclear RNAPs in architecture and complexity. Many of their subunits show high primary sequence similarity to their eukaryotic counterparts. Pyrococcus furiosus RNAP consists of 11 different subunits ranging in size from 5.7 to 127 kDa. In order to gain insights into P. furiosus RNAP assembly and function at the molecular level, all 11 subunits were over-expressed in E. coli and purified to homogeneity. We present the successful in vitro assembly of a fully active P. furiosus RNAP from its recombinant subunits. This reconstitution system allowed us also to study the structural and functional contributions of the various subunits and specific amino acids to RNAP activity.

13.20-15.00: Session 7 - Overcomming Difficulties

7.01 Production of PolyHistidine-Tagged TAT-Fusion Proteins	Matteo Bolomini-Vittori and Carlo Laudanna	Department of Pathology, University of Verona, Verona, Italy.
Intracellular delivery of expression vectors, proteins and/or pharmacologically relevant compounds is normally problematic due to low solubility, reduced percentage of cells targeted, low gene expression efficiency, size constrains and toxic effects. Previous works have shown that HIV-1 TAT protein freely translocates into the cell cytosol when added to culture media. Subsequently the identification in TAT of a short basic peptide sequence (Tat [45-57], YGRKKRRQRRR), conferring cell protein uptake capability, led the usage of this carrier aminoacidic sequence to transduce recombinant proteins fused to TATpeptide in a variety of primary human cells under several physio-pathological situations. The extensive application of this extremely valuable technology raises the need of developing efficient procedures for massive production of recombinant TAT-fusion proteins, which are both stable and able to cross the plasma membrane. Here we describe an efficient procedure for massive production of polyHistidinetagged TAT-fusion proteins. We constructed prokaryotic expression vectors coding for a (His)6 tag fused to a TAT-derived carrier peptide and including a Multiple Cloning Sites (MCS) allowing in frame cloning of the protein of interest. To express the protein we utilized E. coli strain which, under standard conditions, tends to accumulate high quantities of recombinant, un-folded, proteins in inclusion bodies. We then compared protein isolation under denaturing conditions, which facilitates protein purification and was previously suggested to allow a more efficient cellular internalization, to isolation under non-denaturing conditions. The procedure was developed in the prototype contest of the human small GTPase Rac1N17. The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography (IMAC), anion-exchange chromatography and desalting. Although isolation under denaturing conditions allowed very high yield of purified recombinant protein, we have been unable to remove the denaturing agent without preventing protein aggregation and precipitation, thus making the denaturing protocol unsuitable for subsequent cell biology experiments. In contrast, purification of the protein under native (folded) condition, allowed purification of soluble protein that was highly efficiently internalized by the cells.

7.02 An Optimized Method for the Purification to Homogeneity and Characterization of a Latent Polyphenol Oxidase from Loquat Fruit (Eriobotrya japonica Lindl.)	S. Sellés, J. Casado-Vela, M. J. Martínez, and R. Bru	Dpt. Agroquímica y Bioquímica. Fac. Ciencias. Universidad de Alicante, Alicante, Spain.
The purification of plant proteins to homogeneity has always been a challenging task for biochemist due to the low amount of proteins in plant tissues in general and in fruit tissues, in particular. Many reports have been published dealing with partial purification of the conspicuous and ubiquitous enzyme in plant kingdom polyphenol oxidase (PPO), which is responsible for the so-called enzymatic browning. Therefore, its control is highly desired in the plant food industry. To approach such a goal, the preparation of homogeneous PPO and its further characterization is an unavoidable task. Difficulties in continuing the chromatographic purification process of this enzyme after some extraction steps have hurdled the progress in the achievement of effective methods for controlling PPO activity, especially in minimally treated plant commodities. The characteristic latency of plant PPOs has been in part responsible for such technical difficulties in its purification, since the enzyme may become activated during the purification process. In fact, there is no report on a homogeneous latent PPO from fruit tissue. Here we report on a complete protocol for the purification to homogeneity of a latent loquat fruit PPO. In our study the enzyme has been extracted from loquat fruit in both, soluble and particulate fractions. The soluble PPO was partially purified and was recovered after ammonium sulphate precipitation in its active state. Two different PPO isoforms were detected in the particulate fraction of a loquat fruit (Eriobotrya japonica Lindl.) homogenate. The pellet obtained after 20000 × g was solubilized in a buffer containing 4% (v/v) Triton X-114 and submitted to phase partitioning. After DEAE anionic exchange chromatography of the detergent-depleted upper phase containing PPO activity, a 40.0 kDa active and a 59.2 kDa latent PPO isoforms could be separated and further identified as PPOs by using HPLC-MS/MS of the excised and trypsin-digested bands on SDS-PAGE gels. Further purification for the latent PPO using, first, HiTrap™ Phenyl HP hydrophobic interaction and, second, HiLoad™ 16/60 Superdex 75 pg gel filtration led to a unique band in silver stained SDS-PAGE. Such a preparation displayed an infinite degree of activation, indicating that no active contaminant PPOs were present. The molecular weight of the latent PPO was estimated to be about 59.2 and 61.2 by SDS-PAGE and gel filtration chromatrography, respectively indicating that this latent PPO is a monomer. Kinetic characterization of both, the pure latent PPO and the partly purified soluble PPO was carried out, including optimum pH and temperature, thermal stability, inhibitors, substrate specificity and determination of Vmax, Km and kcat.

7.03 Challenging Protein Purification from Anammox Bacteria	I. Cirpus2, W. Geerts1, J. Hermans1, H. J. Op den Camp1, M. Strous1, G. J. Kuenen2, and M. S. M. Jetten1,2	1 Department of Microbiology, Radboud University Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen,The Netherlands. 2 Department of Biotechnology, University of Technology, Julianalaan 67, NL-2628 BC Delft,The Netherlands.
The anaerobic ammonium oxidation (anammox) is a fascinating microbial pathway contributing to the global biogeochemical nitrogen cycle. To investigate the anammox pathway of energy generation the responsible proteins have to be identified and purified for characterisation. The anammox bacteria themselves have a complex cell envelope consisting of protein and lipopolysaccharide and grow in a biofilm. Preparing cell extract and purifying proteins from the cell aggregates is hampered by the extracellular polymeric material and by aggregation of proteins. Intramolecular disulfide formation was also a factor of gelation. Cell extract gelled at extended storage on ice, upon freezing/thawing and boiling. Additionally, proteins aggregated on various chromatography media upon concentration and during desalting. The polysaccharides clogged the matrix of chromatographic materials and the pores of ultrafiltration membranes. The precipitation of proteins and polysaccharides caused very low resolution and streaking on SDS- and two-dimensional polyacrylamide gels. High resolution gel electrophoresis of the cell extract was only achieved after clarification from polymeric substances with denaturating phenol extraction. The present work describes the causes for gelation of anammox cell extract. Methods for sample preparation for polyacrylamide gel electrophoresis and chromatography are given, which will enable isolation of anammox proteins and their characterization.

7.04 Challenging Expression and Purification of Two Novel Golgi Proteins	Maarit Takatalo and Ragna Rönnholm	Department of Biological and Environmental Sciences, Viikinkaari 5D, P.O.Box 56, SF-00014 University of Helsinki, Finland.
The proper function of the Golgi complex, a central organelle of the membrane traffic, is essential for the cell’s well-being. Despite its important role, many Golgi proteins are still unidentified or uncharacterized. Several proteomic approaches undertaken over the years have focused on identifying some of these novel participants. The aim of this study was to express and purify two novel Golgi proteins for NMR analysis. Nothing is known of their function, they share no sequence similarity to known proteins or domains. The typical approach of the protein expression in E. coli and purification as His6- or GST-tagged fusion was unsuited for these proteins. Despite the several different expression and purification conditions tested, protein yields remained low. The smaller of the two proteins had low solubility and was mainly expressed in inclusion bodies. The larger protein displayed repeated fragmentation pattern even though protease inhibitors were included. As E. coli is not natural host for these proteins, the improper folding or processing can not be excluded. To assess this, protein expression in insect cell culture will be evaluated. Purification of the smaller protein form inclusion bodies will also be optimized.