Membrane Pharmacy Structure Dynamics 

Research group : Priv.Doz. Dr. Thomas Nawroth 

Protein Purification   

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As shown in fig.1, the first step in protein purification is the prowth of cells from a genetically defined organism, of coarse after genetic modification. While the manipulation of small amounts of cells may be easy in a small scale, the isolation and purification of proteins in amounts sufficient for structural biology and molecular biochemistry requires larger cell mass, e.g. several 100 g to some kg. This is most easy with bacteria, but large amounts of cells are also suitable from yeast, plants and some animal tissues, e.g. beef heart or liver. The MPSD group has isolated the majority of purified proteins from bacteria and a few from yeast and beef heart mitochondria. The bacteria were strains of Micrococcus luteus, Micrococcus species, Halobacterium salinamrium (formerly called Halobaterium halobium), Rhodospirillum rubrum and Escherichia coli. Most proteins were integral or peripheral membrane proteins of the cellular energy metabolism (bioenergetics). Thus the second step in the isolation procedure was the preparation of purified membranes after degradation of cell walls.
Fig.1: The strategy of structure and membrane research leads from heterogenous cells over isolated homogenous systems (proteins, lipids, membranes, nucleic acids) to reconstituted model membranes and protein-detergent complexes  (after T20). Molecular genetics supplies cells with specific proteins, chemical synthesis gives us lipids, polymers and compounds for optical system manipulation, e.g. caged-compounds. The molecular homogenous systems are suitable for structure investigation (SAXS, SANS, ASAXS, X-ray diffraction, EXAFS / XANES, especially with time resolution). 

The isolation and purification of the membrane proteins requires the solubilization of the protein by replacement of the lipids by detergents (both are amphiphiles). As shown in figure2, the native structure and thus the biological function is only retained if weak, non-denaturating detergents of highest chemical quality are used (containing no peroxides or long chain impurities). The non-denaturating detergents do not penetrate into the protein and have at least one of the following properties: i) large area of the hydrophobic domain, and ii) low charge density in the hydrophilic head. This is the case for example in the detergents CHAPS, CHAPSO, TDOC, DOC, Digitonin, Deoxy-BigChap, Laurylmaltoside, Octylglucoside, Triton-X100 (has to be peroxide-free !).
Fig.2:The purification and investigation of membrane proteins requires the retainment of the function competent structure (native), which is obtained by solubilzation with weak, non-denaturating detergents [T20].

The choice of the detergent and conditions for the protein solubilization is the key for the isolation: i) The replacement of the membrane lipid by the detergent must not demage the protein structure and function; ii) the solubilization should be selective, i.e. in the ideal case the protein of interest should be solubilized only together with a few other proteins, which can then be removed by chromatography; and iii) the protein must not be demaged during isolation, e.g. by protease action, partial unfolding or oxidation, which might occur by detergent impurities. As shown in table1 and T20, the solubilization can be done following three methods: direct solubilization, fractionated detergent solubilization, and fractionated detergent exchange solubilization.

Table1: Methods of membrane protein solubilization by detergents.
Solubilization method procedure
direct solubilization membrane extraction with one detergent at a given concentration, the resdue after cenrifugation is removed
fractionated detergent solubilization membrane extraction with one detergent in two steps, the first extract and the residue is removed
fractionated detergent exchange solubilization membrane pre-extraction with one detergent - extract is removed;
then fractionated extract with another detergent - resiue removed

In living cells the constituents are subject of continous degradation and novel biosynthesis. The degradation is maintained by cellular proteases, which are down regulated in vivo. During disrupture of the cells, membarne preparation, deteregent solubilization and protein purification that regulation is lost. Thus several proteases may degrade the proteins of interest, which may lead to artefacts. Those protease artefacts can be avoided by i) cooling (ice); ii) rapid passage through the process steps from cells to the first chromatographic purification; and iii) inhibition of proteases by inhibitors during all steps of protein purification. A variety of intracellular proteases can be inhibited by the acid halogenides shown in Fig.3, which act on active serine residues. Those protease inhibitors are useful also during structure investigation, e.g. protein crystallization: They inhibit proteases and avoid microbiol problems as well. Note that the lifetime of those acid halides is  temperature and pH dependent (at 20C, pH7 about 1-2 days). Unfortunately the most powerful inhibitors are extremely toxic (xenobiotics), namely DFP (toxic dosis for man: 1 l). Thus they should handeled with care by experienced persons only (chemistry security training required !).
DFP (DiisopropyFluoroPhosphate) is a volatile toxin. The working concentraion is 0.1 mM. PMSF (PhenylMethylSulfonylFluoride) is a solid. The working concentraion is 0.1 mM. PefaBloc (AEBSF) is a solid well soluble in water. The working concentraion is 0.5 - 5 mM.
Fig.3: The protease inhibitors DFP, PMSF and PefaBloc are xenobiotics, which inhibit active serine residues.

After the selective solubilization, the protein content of a preparation has reduced to a few %, if a proper procedure has been applied. The protein can then be subjected to chromatographic purification, which may enrich the protein of interest by a further factor of 10 - 100. For structural biology and molecular biochemistry a product of >95 purity (99% for crystallography and time resolved studies) with fully retained function is required.

Table2: Protein content during steps of a typical preparation for structural biology: ATP-synthase from Micrococcus luteus.
step / procuct 250g cells (wet weight) purified membranes (PMV) fractionated detergent exchange solubilisate 1st ion exchange chromatography IEC 2nd ion exchange chromatography IEC GPC: gel permeation with detergent exchage
protein content 40g 8 g 3 g 200 mg 100 mg 60 mg
purity 0,15% 3% 10% 80% 98% >99%

The first column of the chromatographic purification has to be a material capable of "heavy duty": i) it must be capableof handling large protein amounts and volume (several grams); ii) it has to be insensitive to impurities, e.g. lipids and easy to regenerate many times; and iii) is has to be stable to microbiol attack. For large scale preparation for structural biology studies we use in several preparations, e.g. ATP-synthase and F1ATPase a large tentaculus ion exchanger (Fractogel EMD650m-DEAE, Merch, Darmstadt) HPLC column of 60 g protein binding capacity (biotechnology scale). Then a column of higher separation power follows: for ATP-synthase Fractogel EMD650s-DMAE (HPLC); for Cytochrome-c oxidase or Quinol-oxidase: affinity chromatography colums (Lysine-Agarose and cytochrome-c-Agarose). In most preparations of membrane proteins the last purification step has to combinded with a detergent exchange to a detergent suitable for structure research. This can be done for large memrane proteins by gel permeation chromatography (GPC) using Superose6 (Pharmacia). The final product is concentrated by ultrafiltration and stored in liquid nitrogen. An addition of 10% glycerol during purification and freezing is useful (protein stabilizing). Furthermore this prevents the protein from radiation demage during the investigation at high-flux synchrotrons, e.g. by time resolved methods. Some protein purifications developed by the MPSD group are presented in table3. Preparation protocols are available on demand (email).

Table3: Some protein preparations of the MPSD group (overview in T20).
Protein organism / strain preparation type yield from cells references, comments
ATP-synthase Micrococcus luteus small scale 10 mg from 60 g
ATP-synthase Micrococcus luteus biotechnological scale 70 mg from 250 g for TR-studies and neutron scattering / liposomes
F1ATPase Micrococcus luteus small scale 25 mg from 60 g
F1ATPase Micrococcus luteus biotechnological scale 200 mg from 250 g for TR-studies and crystallization
F1ATPase Micrococcus species small scale 20 mg from 60 g
F1ATPase Escherichia coli small scale 10 mg from 200 g
F1ATPase Rhodospirillum rubrum small scale 5 mg from 60 g chloroform method
ATP-synthase Rhodospirillum rubrum small scale 10 mg from 60 g
ATP-synthase beef heart mitochondria small scale 20 mg from 1 beef heart
Cytochrome-c oxidase COX  Micrococcus luteus small scale 5 mg from 60 g
Quinol oxidase QOX Micrococcus luteus small scale 10 mg from 60 g
Cytochrome-bc1 complex Rhodospirillum rubrum small scale 5 mg from 60 g
Quinol oxidase QOX Rhodospirillum rubrum small scale 5 mg from 60 g
Bacteriorhodopsin purple membrane PM Halobacterium salinarium large scale 200 mg from 100g
monomeric Bacteriorhodopsin mBR Halobacterium salinarium small scale 30 mg from 50 mg PM

The methods and technology of membrane protein purification is subject of the lectures "Methods of membrane biochemistry". The methods for structure research of membranes and membrane proteins in solution are subjects of the lectures "Biophysical chemistry of membranes and membrane proteins". You can learn the technology of membrane protein purification and some techniques for biophysical chararacterization by participation in our practical course "Advanced practicum in membrane biochemistry". The analytical techniques are subject of the Bioanalytics lecture.

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email to:   update : 15.10.2013