Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2001-01-16
2004-11-23
Weber, Jon P. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S412000, C530S417000, C530S350000, C435S004000, C427S132000
Reexamination Certificate
active
06822081
ABSTRACT:
The invention relates to a process for the isolation and/or purification of a proteinaceous material, wherein a solid phase comprising a mixture of hydrophobic and hydrophilic groups is used.
For many applications proteinaceous material is required to be in an isolated or purified form, in particular, to be free of interfering contaminations. Therefore, proteins obtained by conventional processes must be purified prior to the desired application in most cases. For example, sequencing of proteins requires products being free of contaminations which might interfere with the sequencing reaction. For a qualitative or quantitative determination of proteins the sample must also be free of contaminations which might interfere with the detection method. Using UV-spectroscopy the sample must be purified from constituents having an absorption in the same wavelength range as the analyte. Mass-spectrometric analysis of proteins, e.g. with MALDI-MS (Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry) also requires a sample preparation which provides the analyte essentially free of any other sample components which might interfere with analysis.
If only a few samples per day are to be processed, purification can be done manually, e.g. using conventional columns. However, if numerous samples have to be purified, a purification process is highly recommendable which is technically easy to perform in order to minimize personnel, money and time expenses. The process therefore desirably is easy to automate, fast and cost-efficient.
So far, several processes for the purification of proteins are known. Commonly used reversed phase chromatography is based on unspecific reversible binding of proteins and peptides to hydrophobic groups, being located and fixed to a support material within a column. For packing a column suitably for column-chromatographic purification of proteins organic solvents are usually employed to prevent the column fill material (stationary phase) from sticking together, since in an aqueous solution agglutination of the stationary phase material occurs because of its hydrophobic surfaces. However proteins are often present in aqueous solutions or samples. Binding of the proteins to the fixed stationary phase usually takes place in an aqueous solution, whereas for elution from the solid phase an organic solvent is added. Due to the hydrophobic character of the stationary phase, individually mobile solid phase particles cannot be used in an aqueous solution, since the solid phase particles would agglutinate. Thus a direct contact of a sample, such as urine or blood, with a conventional binding phase material in loose form is not feasible. Further, since conventional binding phase particles can be used only packed in a column, a combination of reversed phase purification with other separation technologies such as magnetic particle technology cannot be successfully performed. A further drawback of column-chromatographic purification processes is that they are rather complex and expensive and are difficult to automate.
Therefore, it was an object of the invention to provide a process for the isolation or purification of proteins, which overcomes the above-mentioned drawbacks of the known processes at least partly and which, in particular, provides easy and cost-efficient purification of a large number of samples.
The invention provides a process for the isolation and/or purification of a proteinaceous material comprising the steps:
(a) providing an aqueous sample comprising a proteinaceous material,
(b) contacting the aqueous sample with a solid phase comprising a mixture of hydrophobic groups and hydrophilic groups on at least one surface thereof, wherein said proteinaceous material binds to said at least one surface and
(c) separating off other sample components.
The process according to the invention allows for isolation, purification and/or concentration of proteinaceous material from aqueous samples. The proteinaceous material may be dissolved or dispersed in the aqeous sample, solutions being preferred. The proteinaceous material preferably is selected from peptides, proteins and any material which comprises at least partly peptidic bonds. Also comprised by the term proteinaceous material, as used herein, are adducts of peptides and/or proteins with other molecules, for example, sugars, lipids, nucleic acids and the like.
The solid phase used in the process according to the invention comprises a mixture of hydrophobic groups and hydrophilic groups. The hydrophobic groups serve to bind the proteinaceous material. The proteinaceous material is preferably bound reversibly and unspecifically to the hydrophobic groups located on at least one surface of the solid phase, Binding is, thus, not limited by specific binding pairs having high affinity such as streptavidin/avidin. In addition to the hydrophobic groups which are capable of binding proteinaceous material the solid phase used according to the invention comprises hydrophilic groups on at least one surface thereof, which hydrophilic groups prevent the solid phase from sticking together in an aqeous environment. By adding hydrophilic groups in adequate proportions the characteristics of the solid phase coated with hydrophobic groups can be varied. The configuration and numerical proportion of hydrophilic and hydrophobic groups are calculated in such a way that reversed phase binding of proteinaceous material, in particular, of peptides and proteins, will still take place, but no agglutination of the particles in an aqeous environment is observed. The choice of the functional hydrophilic and hydrophobic groups as well as the amount thereof can be individually adjusted for the respective analyte and the type and size of solid phase used.
Surprisingly, it was found that proteinaceous material can be bound to the solid phase used according to the invention with high yield, thus reducing the amount of solid phase required for purification.
Preferably, the solid phase comprises solid particles. Due to the addition of hydrophilic groups agglutination of small particles having hydrophobic groups located on their surface can be prevented according to the invention. Therefore, small individually mobile particles can be used in an aqueous solution without the necessity to pack them or to fix them on a support material. The solid phase preferably comprises solid particles having a diameter from at least 1 nm up to 10 nm. More preferred nanoparticles are used having a diameter of preferably at least 1 nm, in particular, at least 5 nm and, most preferred, at least 10 nm and up to 1.000 nm, preferably up to 500 nm, most preferably up to 100 nm and/or microparticles having a diameter of at least 1 &mgr;m, preferably at least 5 &mgr;m and up to 1.000 &mgr;m, preferably up to 500 &mgr;m.
Basically, however, any solid phase known to the skilled person may be used as solid phase such as microtiter plates, vessels such as Eppendorf vessels, Greiner tubes, Nunc tubes and the like. Preferably, solid particles having a diameter ≧1 nm to ≦1 mm are used as solid phase, thus providing a favorable specific surface per g of particle.
The method of the invention allows the use of most different solid phase materials. Silica, a plastic material such as polystyrene or a magnetic or magnetizable material is used preferably as solid phase material and, in particular, gamma-iron oxide. If a magnetic solid phase is used, further advantages can be achieved. In particular, it is possible this way to combine reversed phase purification of proteinaceous material and magnetic particle technology. Magnetic separation can be performed relatively easily and is easy to automate. Moreover, if paramagnetic or para- and ferromagnetic particles are used as solid phase, sticking together of the solid particles can be further reduced. Usually small magnetic particles having a diameter within nm or &mgr;m range are used in magnetic particle technology (e.g. by the company Dynal, Oslo, Norway), in the case of which the problem of sticking together is especially se
Kalkum Markus
Nordhoff Eckhard
Rauth Holger
Reinhardt Richard
Kam Chih-Min
Max-Planck Gesellschaft zur Förderung der Wissenschaften e.V.
Rothwell Figg Ernst & Manbeck P.C.
Weber Jon P.
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