Membrane Pharmacy Structure Dynamics  Research group : Priv.Doz. Dr. Thomas Nawroth  Protein Purification

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 20°C, pH7 about 1-2 days). Unfortunately the most powerful inhibitors are extremely toxic (xenobiotics), namely DFP (toxic dosis for man: 1 µl). 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.