WO2005003784A1 - A method of screening for cell colonies that express a soluble variant of a target protein - Google Patents
A method of screening for cell colonies that express a soluble variant of a target protein Download PDFInfo
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- WO2005003784A1 WO2005003784A1 PCT/GB2004/002271 GB2004002271W WO2005003784A1 WO 2005003784 A1 WO2005003784 A1 WO 2005003784A1 GB 2004002271 W GB2004002271 W GB 2004002271W WO 2005003784 A1 WO2005003784 A1 WO 2005003784A1
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- Prior art keywords
- proteins
- filter
- protein
- soluble
- colonies
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
Definitions
- the invention relates to a method of screening protein molecules, in particular to methods for identifying soluble proteins and for detecting colonies expressing a soluble target protein.
- the production of pure or semi-pure proteins is important in many commercial and academic research and development programs . Often such proteins are produced recombinantly.
- Recombinant proteins can constitute products (e.g. enzymes for use in biomedical assays or in industrial processes) and are also used in the process of developing pharmaceutical drugs. In pharmaceutical drug development processes, these proteins are often used for structural studies (where methods such as NMR and x-ray crystallography are employed) , and in biochemical or biophysical studies of the target protein. In academic research, recombinantly expressed proteins are used for biochemical, biophysical and structural characterisation.
- recombinant proteins are produced by the overexpression of a gene of interest.
- proteins aggregate (e.g. in inclusion bodies) when overexpressed and fail to fold into their native conformation.
- Such protein aggregates must be dissolved and correctly refolded before they can be used in many of the above methods .
- refolding proteins f om inclusion bodies usually results in very low yields of proteins and often it is not possible to determine if the protein isolated is in fact correctly folded. To improve the yields of proteins obtained, it is therefore important for proteins to have a high solubility.
- Mutant libraries of proteins must further be screened to detect the recombinant clone (s) containing soluble variants.
- s recombinant clone
- Several screening methods have been described, which select proteins with increased solubility. Maxwell et al . (Protein Science, 1999, 8, 1908-1911) , described a simple assay for assessing solubility using chloramphenicol acetyltransferase (CAT) fusion proteins, this was based on the principle that cells expressing fusions of an insoluble protein to CAT exhibit decreased resistance to chloroamphenicol compared to fusions with soluble proteins.
- CAT chloramphenicol acetyltransferase
- Knaust and Nordlund (Analytical Biochemistry, 2001, 297, 79-85) , a screen for soluble proteins was developed using filtration of cells lysed in culture in 96 well plates. Recombinant bacteria were grown on agar plates and then used to inoculate media in 96 well plates. The cultures were grown, lysed and filtered.
- the present inventors have now developed a screening method which can process large numbers of recombinant bacteria in a short period of time. Surprisingly, it has been found that lysis of cells and filtration of lysates can be carried out directly on colonies of cells, hence eliminating the requirement to grow colonies in culture and the multiple pipetting steps involved with this technique.
- the present invention is hence capable of operating on large numbers of variants, is inexpensive and has a high reliability of predicting soluble variants.
- the present invention provides a method of treating one or more colonies of cells which method comprises : (a) subjecting the cell colonies to conditions which are capable of causing lysis thereof; and (b) filtering the lysate of step (a) through a filter having pores which allow only soluble proteins to pass through the filter.
- the invention provides a method of detecting one or more colonies expressing a soluble target protein, where the selection occurs on the basis of the ability of the protein of interest to pass through the filter.
- the invention particularly covers methods wherein the target protein is a membrane protein.
- a membrane protein is one which is associated with one of the membranes of a cell, and can either be found within the membrane or bound to it .
- the membrane may be an intracellular membrane or the outer cell membrane.
- the membrane is an intracellular membrane, and most preferably is an intracellular membrane of E. coli .
- the methods of the invention detect soluble proteins and colonies expressing soluble variants of a given protein.
- Membrane proteins are generally considered insoluble and therefore the target membrane proteins will be those which are capable of being made soluble, conveniently this solubilising may be achieved by subjecting the protein to mutagenesis, e.g. as described in Example 4.
- the invention also covers a method of selecting one or more colonies expressing a soluble non-enzymatic target protein.
- Non-enzymatic ' proteins are proteins which do not themselves possess enzymatic activity and are not capable of catalysing a biochemical reaction.
- Non-enzymatic 1 target proteins can be fused to other proteins e.g. tags such as horseradish peroxidase which do have enzymatic activity.
- this method is used as a screening method to identify colonies expressing soluble proteins.
- the filter through which the proteins pass acts as the screen, i.e. the identification of colonies expressing soluble proteins occurs as a direct result of whether or not the target protein has passed through the filter.
- the present invention also provides a method of determining whether or not a cell colony expresses a soluble variant of a target protein which method comprises : (a) subjecting said cell colony to conditions which are capable of causing lysis thereof; (b) filtering the lysate of step (a) through a filter having pores which allow only soluble proteins to pass through the filter; and (c) detecting target protein which has passed through the filter.
- step (c) the target protein is detected and the presence of target protein indicates that the corresponding cell colony produces a soluble variant of that target protein.
- the target protein is not detected on the basis of its own enzymatic activity.
- detection can occur on the basis of the enzymatic activity of a tag fused to the target protein of interest, e.g. horseradish peroxidase, G-S-T or luciferase.
- the tag may participate as a substrate in an enzymatic detection method, His tags being particularly suitable in this regard.
- INDIA His Probe-HRP Pierre, Rockford IL, USA
- the target protein of protein of interest is either poly-histidine tagged or is histidine rich and where the target protein is detected by a Nickel activated derivative of horse radish peroxidase which binds to His tags .
- a non-enzymatic i.e.
- non-catalytic detection method i.e. no method based on substrate conversion
- detection is based on affinity binding between the target protein and a detection moiety, for example an antibody or antibody fragment or affibody (non Ab based protein binding partner) .
- detection methods allow for rapid and reliable analysis of a wide variety of target molecules, including those which themselves possess no catalytic activity.
- the present invention provides a method of identifying a cell colony which expresses a soluble variant of a target protein, which method comprises : (a) subjecting said cell colony to conditions which are capable of causing lysis thereof; (b) filtering the lysate of step (a) through a filter having pores which allow only soluble proteins to pass through the filter; and (c) detecting target protein which has passed through the filter, wherein the target is not detected on the basis of its own enzymatic activity.
- the proteins in the filtrate are localised on a solid support, e.g.
- colony or “colonies” describes a circumscribed group of cells, normally derived from a single cell or small cluster of cells, growing on a solid or semi-solid medium. Colonies can be formed from any cell type which can be made to express recombinant proteins and which can grow on solid or semi solid media. For example, colonies can be formed of prokaryotic e.g. bacteria or eukaryotic cells e.g.
- yeast unicellular eukaryotes such as Leishmania, insect cells or mammalian cells or cell lines.
- colonies are formed of E. coli, Bacillus subtilis , Streptococcus lactis, Streptomyces lividens, Lactococcus lactis, Staphylococcus aureas, Aspergillus niger, Picia pastoris, Saccharomyces cerevisiae or Schizosaccaromyces pombe.
- Semi-solid or solid media used to grow colonies typically consists of culture media with the addition of 0.1% or greater agar. More preferably, solid or semi- solid media contains at least 0.2%, e.g. at least 1.0% or at least 1.5% agar.
- the lysis step of the present invention can be carried out chemically or otherwise using reagents which are well known in the art e.g. urea, lysozyme containing buffers or detergents.
- the degree of lysis must be sufficient to allow the proteins of the cell to pass freely out of the cell.
- lysis is performed in the presence of detergents or amphiphiles, for example Triton X-100 or dodecylmaltoside, to release the protein from the membrane.
- the lysis step is non- denaturing, allowing proteins to retain a native, i.e.
- the lysis step can alternatively be carried out by freeze thawing the colonies. More preferably, lysis is carried out using both native lysis buffer and freeze thawing the colonies.
- the native lysis buffer contains lysozyme, for example at 50-750 ⁇ g/ml, more preferably at 100-200 ⁇ g/ml. DNAse can also be found in native lysis buffer preferably at 250-750 ⁇ g/ml.
- Native lysis buffer may contain, for example 20 mM Tris, pH 8, 100 mM NaCI, lysozyme (200 ⁇ g/ml) and DNAse I (750 ⁇ g/ml) .
- the colonies will be exposed to the lysis mixture (buffer) for 15-60 minutes, preferably around 30 minutes.
- the step of freeze thawing is preferably repeated, i.e. two or more cycles, preferably 3 or more cycles of freeze thawing are performed.
- lysis is achieved by a 30 minute incubation at room temperature with lysis buffer and three x 10 minutes freeze thawing.
- the percentage of cells lysed within a colony during the lysis step is 5-50%.
- Colonies to be lysed preferably contain at least 10 4 cells e.g. at least 10 5 , at least 10 6 , at least 10 7 or at least 10 8 cells.
- the size of such colonies is typically 0.1-3 mm 2 , preferably 0.2-2 mm 2 and more preferably 0.25-1 mm 2 .
- a "native-like" protein or “native” protein refers to a soluble intracellular, extracellular or membrane protein wherein the protein exhibits a native-like conformation and functions similarly or identically to the naturally occurring protein. "Native" or “nativelike” proteins are expressed in soluble form and/or are correctly folded.
- Native-like membrane proteins do not have to be present free in solution, but may be present in membrane vesicles rather than inclusion bodies .
- “native-like” proteins are generally not insoluble, present in inclusion bodies, aggregated or misfolded. According to the methods of the present invention, it is the soluble proteins which are able to pass through the filter and thus be separated from insoluble proteins. As described herein, this allows the identification of colonies expressing soluble proteins, e.g. through the use of blotting techniques.
- solubility and correct, i.e. native or native-like folding means that the method is able to separate proteins with their native conformation from misfolded/aggregated proteins.
- a "soluble" protein can thus be defined with reference to possession of a native or native-like conformation. Further, a soluble protein can be described as a protein which remains in the supernatant after cell lysis and centrifugation thereof. Centrifugation can be carried out at at least lOOOg, preferably at at least 3000g, preferably at at least lOOOOg and more preferably at around 20000g. Centrifugation can be carried out at lOOOOOg. The duration of centrifugation can be from 1 minute (typically at least 10 mins) to at least 1 hour, where the duration required generally decreases as the centrifugal force increases.
- Particularly suitable conditions for providing only soluble proteins in the resultant supernatant include 10 minutes at 10000Og, 30 minutes at 3000g or 15 minutes at 20000g. 15 minutes at 20000g as described in the Examples being especially suitable.
- Some proteins which pass through the filter will not correspond exactly to any naturally occurring protein.
- a library of proteins may be generated based on a target protein in order to identify related mutants with improved solubility in a given expression system compared to a problematic target protein. In these circumstances it is appropriate to consider the soluble mutants as having a "native-like" conformation.
- the filtration step of the invention can be carried out using standard filter membranes for the filtering of biological samples.
- the filters will typically have a pore size from 0.015 ⁇ m to 12 ⁇ m, preferably from 0.35 to 1.2 ⁇ m, preferably from 0.45 to 1.2 ⁇ m, more preferably from 0.45 to 0.8 ⁇ m.
- the filters Preferably, have pore sizes below 4.0 ⁇ m, typically below 2.0 ⁇ m, more preferably below 1.0 ⁇ m.
- an optimal pore size may be 0.1-1.5 ⁇ m.
- preferred pore sizes may be larger.
- filters are manufactured and marketed as having a particular pore size but the manufacturing process may occasionally result in a few smaller or larger pores; the sizes listed, which refer to the diameter, are thus the most common pore size of a given filter. Although reference is made to a range of potential pore sizes, any single filter will usually have one designated pore size, e.g. 0.45 ⁇ m.
- Suitable filters are Super and GH polypro (from Pall) and Nucleopore (from Whatman) . It will be appreciated that different cell types may require the use of filters with different pore sizes, due for example to their different tendencies to harbour aggregated proteins, which aggregates may also have varying properties in different cell types. Selection of a suitable filter is well within the competency of someone skilled in this field.
- the filter is overlayed on the colonies to lift the colonies/protein from the semi-solid or solid growth media ( Figure 1) .
- filters could be placed on the growth media and cells seeded directly onto the filter, the filter could then simply be lifted off with the colonies already on it.
- the lifting of colonies from their growth media can be carried out prior to the lysis step. The lysis can hence be carried out directly on colonies on a filter.
- the filter with colonies attached can be treated with lysis buffer ( Figure 2) or overlayed on other membranes/ filters treated with lysis buffer Filtration is carried out after lysis, i.e. it is the lysate which is filtered. It will be appreciated however that filtration and lysis may occur simultaneously when considering a whole colony since some cells may undergo lysis before others and hence may be filtered before or at the same time as others are lysed.
- proteins which pass through the filter are held on a solid support, e.g. a capture membrane, to allow screening/detection of any proteins of interest and then to allow the identification of colonies expressing such proteins.
- capture membranes may typically comprise nitrocellulose.
- the detection of the target protein is not on the basis of its own enzymatic activity. Detection can however occur on the basis of the enzymatic activity of a tag fused to the target protein or where the tag acts as a substrate in an enzymatic detection method.
- the first filter which separates soluble from insoluble proteins acts as the essential screen for this invention.
- proteins can simply be allowed to pass through the filter, possibly as a result of an active capillary action.
- force may be applied to aid filtration. The force can be applied vertically on the filter paper, wherein such forces can include the application of pressure or a vacuum.
- the capture membrane can fix the soluble proteins from the individual colonies and their positions on the capture membrane can then be compared to the filter carrying the original colonies.
- the colony filtration blot it is possible to track back and identify the original colonies expressing the soluble proteins of interest on the growth media.
- positive controls can be used. These are clearly seen on the final colony filtration blots and can enable the membrane/blot to be correctly orientated can enable the membrane/blot to be correctly orientated with the original colonies (Figs. 7 and 8) .
- a solid support such as a capture membrane allows the ready identification of the colonies expressing the soluble proteins of interest.
- the filter with colonies attached can be placed colony side down onto a material soaked in lysis buffer.
- a (nitrocellulose) capture membrane can then be placed on top of the filter with colonies and several layers of filter paper (and paper towels) can be placed on top of this ( Figure 3a) .
- Force can then be applied to the top of this "sandwich” and ideally transfer buffer poured around the bottom to facilitate filtration and transfer of proteins onto the capture membrane .
- the filter is placed colony side up onto a capture membrane and a vacuum is applied to "pull" protein through the filter paper and onto the capture membrane (Figure 3b) .
- lysis and filtration may conveniently take place in one overall step, e.g. during the application of conditions capable of causing lysis (e.g.
- the cell lysate is filtered and captured on a capture membrane.
- the present invention provides a method of separating soluble from insoluble proteins, which method comprises: (a) subjecting one or more colonies of cells to conditions which are capable of causing lysis thereof; (b) filtering the lysate of step (a) through a filter having pores which allow only soluble proteins to pass through the filter, thereby generating a filtrate containing soluble proteins. According to this method, most or all insoluble proteins will fail to pass through the filter and hence separation of soluble from insoluble proteins occurs . It will therefore be appreciated that the filter through which the soluble proteins and the colonies from which they are produced.
- the methods of the present invention can also include the detection of a protein of interest after filtration.
- Proteins of interest can be detected using various tags which are well known in the art, e.g. histidine tag, VS tag, T7 tag, FLAG tag or any short protein sequence to which a specific antibody is available, glutathione-S-transferase, thioredoxin, green fluorescent protein and maltose-binding protein.
- Tags are preferably between 1-100 amino acids in length, preferably between 1-70, 2-50, 1-30, or 1-20 amino acids in length. More preferably tags can be 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, e.g.
- His tags are generally 6 amino acids in length. Tags can be attached to a protein of interest generally by expressing such proteins as fusion proteins. As such, short tags are preferred, to allow proteins of interest to maintain a native-like conformation. Further, C-terminal tags are preferred, although N-terminal His tags are also used. Proteins of interest can also be detected using antibodies, monoclonal or polyclonal, either directed to a tag or directly to the protein of interest (expressed on its own or as a fusion) . Proteins can also be detected if an enzymatic activity is exhibited, for example fusion tags that possess enzymatic activity include green fluorescent protein, horseradish peroxidase and glutathione-S- transferase.
- Proteins can further be detected via fusion tags which act as the substrate in enzymatic detection methods. Preferably target proteins are not detected on the basis of their own enzymatic activity. If the different colonies are each representative of a particular variant from a generated library, proteins of interest will typically be soluble members of the library. Conveniently therefore the members of the library will be expressed as fusions with a small tag to aid detection or antibodies to the library members may be used.
- the filtrates of soluble proteins can further be used in assays to test for the biochemical activity of the protein of interest. In one embodiment, the filtrate can be simultaneously screened for the presence/amount of a soluble protein of interest (e.g.
- a tag and an antibody directed thereto for the activity of that protein using a suitable assay.
- an assay it will (preferably) be in addition to a method detecting soluble variants, e.g. a method based on affinity binding.
- a positive colony i.e. one expressing a target protein in soluble form
- Such colonies can be cultured and soluble or total protein isolated for Western blotting if confirmation of the screening results are required.
- Such clones can be used for the overexpression of the protein of interest for many different purposes, e.g. for structural studies to elucidate the protein sequence .
- the methods of the invention are of utility in the separation of soluble and insoluble proteins and for the detection of colonies producing soluble proteins.
- Soluble proteins can often be expressed in greater amounts and can be used in techniques such as NMR/X-ray crystallography for structural genomics .
- the methods of the present invention can also be used to screen cDNA libraries for particular clones expressing soluble proteins.
- the invention can also be used to screen for soluble variants of a particular protein for example, a membrane protein.
- the methods of the invention can also be used to screen vector libraries and libraries composed of different expression bacteria or strains. Many standard expression vectors provide poor protein expression due to sequence variation in the promoter regions, translation initiation sites and reporter proteins. By randomising these regions and varying reporter proteins the most efficient sequence for soluble protein expression can be selected.
- mutants in growing cultures arose independent of the environmental agents, rather than being generated by any agent.
- target proteins a variation in expression levels and solubility among bacteria can be seen and it is thought that some of the differences may be a result of differences in genotype of the bacteria.
- mutagenic agents and high expressing soluble proteins selected using the CoFi-blot technique Filtration is performed so that the majority of proteins passing through the filter, in particular the majority of a target protein or proteins passing through the filter are in soluble form.
- the filter hence acts as the detection screen for this invention, allowing colonies expressing soluble target proteins to be identified.
- Genes/cDNAs/coding regions encoding a protein of interest can be mutated to produce variants of that protein with varying degrees of solubility. These mutants can be produced in an expression system, wherein the most soluble variants can be selected using the lysis and filtration steps of the present methods performed on transformed colonies.
- Genes/cDNAs/coding regions can be transformed or transfected into expression systems in vectors/constructs, such as plasmids, viral vectors, cosmids and YACs .
- vectors may contain regulatory sequences and other elements well known in the art.
- the gene/cDNA/coding region may be placed under the control of a promoter in a vector. Promoters used are generally capable of expressing the protein of interest within a particular host.
- the promoter used is inducible i.e. the expression of the protein of interest can be controlled.
- inducible promoters/systems include lac wherein induction of expression is controlled by the addition of IPTG and tet on/off, wherein induction of expression is controlled by the presence/absence of tetracycline and others are known in the field.
- Many different methods of mutagenesis are known in the art which could be employed to create a library of variants of a protein of interest. Possible procedures include truncation of the sequence, use of an exonuclease enzyme, introduction of a randomised cassette or site-directed mutagenesis.
- the number of nucleotides removed may be less than 2000, preferably less than 1000, and more preferably less than 800.
- Introduction of a randomised cassette for mutagenesis preferably uses a cassette containing less than 100 nucleotides. Mutagenesis is preferably carried out on several copies of a nucleic acid sequence encoding a protein of interest so that a set of different mutated sequences can be screened, hence increasing the probability of identifying a native-like protein with improved solubility. The use of random mutagenesis is especially preferred where there is no prior knowledge of which particular mutations may yield a soluble variant.
- Libraries of proteins can be created where the coding region has been randomly mutagenised and where different length constructs have been generated by erase-a-base or random priming reactions.
- vectors can be randomly mutagenised (or vector libraries) , preferably in the promoter region.
- N or C terminal tags or in the origin of replication and hosts can be randomly mutagenised, for example by random knockouts or from preselected libraries of strains.
- the methods of the invention can hence be used to screen directly for the expression of soluble protein from a limited number of genes (transferring cells with just cloned genes) or to screen large numbers of genes e.g. from cDNA expression libraries.
- the present invention provides a kit for use in the methods described above which comprises : (a) a filter having pore sizes which only allow soluble proteins to pass through the filter; (b) a capture membrane; and optionally (c) reagents for use in native lysis of the cell colonies.
- (b) is preferably a nitrocellulose membrane and (c) preferably includes one or more components of a native lysis buffer as described herein. Suitable filters and capture membranes are also discussed herein.
- the kit also contains (d) detection means, e.g. affinity binding partners, for the target protein of interest . Such kits can be used to detect colonies expressing a soluble variant of a membrane protein.
- Figure 1 shows a method of peeling/lifting colonies from the semi-solid/solid growth media using a filter
- Figure ⁇ 2 depicts one procedure to obtain colony lysis, wherein the filter with colonies is placed colony side up onto nitrocellulose and the filter soaked in native lysis buffer; following lysis of the colonies, soluble proteins can pass through the filter onto the membrane
- Figure 3a shows one embodiment of the invention where the "sandwich" method is employed.
- a filter with colonies is placed colony side down on filters soaked in lysis buffer. Nitrocellulose is placed on top of this and filter paper and paper towels on top of this.
- Figure 3b shows another embodiment of the invention where a filter with colonies is placed colony side up on a nitrocellulose membrane in a column with buffer. A vacuum is then applied to aid filtration;
- Figure 4a shows a colony filtration blot for total protein for all 48 different constructs used to screen for soluble proteins of 24 E. coli proteins;
- Figure 4b (i) shows a colony filtration blot of the 48 constructs (24 different E.
- Figure 5 shows results of a dot blot comparing the number of positive GST tagged clones obtained when denaturing or native lysis buffer is used
- Figure 6 shows a schematic presentation of the Erase-A-Base process
- the plasmid is linearized by endonuclease digestion in two unique restriction sites introduced in the cloning primer (2)
- the enzymes are chosen in such a way that one leaves a 3 ' overhang protected from ExoIII digestion and one leaves 5' which is susceptible to the digestion
- samples from the ExoIII digestion are removed at timed intervals (3) and added to tubes containing SI Nuclease, which removes the remaining single-stranded tails (4)
- the plasmid is religated (5) and transformed into a E.
- FIG. 7a shows examples of four different colony filtration blots; two blots that are from the RORa library and two from the SOCS-2 library; the time points where the aliquots were removed are noted under the pictures; the later time points do not contain any or very few colonies judged as positives; this is due to the fact that constructs at these time points correspond to proteins which have their start inside a domain;
- Figure 7b shows RORA 5-8 minutes in more detail, where the positive controls are located and some examples of colonies judged as positive/soluble;
- Figure 8 shows positive controls being added to a plated library; positive controls will form positive colonies and can serve as reference points to help identify other positive colonies expressing soluble protein;
- Figure 9 shows Western blot of soluble protein fractions of 24 different clones picked from a filtration colony screen of RORa library; the cells were grown in liquid culture and lysed using freeze th
- Figure 12 shows colony filtration blot for an E. coli integral membrane protein predicted to contain 13 transmembrane segments (amino acid permease) in a) native lysis buffer (as in Example 1) b) denaturing lysis buffer (+8M Urea) c) + Triton X-100 (1%) d) dodecylmaltoside (5 mM) .
- Figures 13a and 13b show colony filtration blots of mutation libraries of two different E. coli membrane proteins (a and b) . Positive and surrounding colonies were picked for further analysis.
- Figures 14a and 14b show dot blots made of the soluble fractions from clones picked from the colony filtration blots of Figures 13a and 13b.
- the non- mutated wild type (WT) protein was included on the blots .
- An increase in membrane protein expression as compared to the WT can be seen among several of the clones from both proteins (a and b) .
- E. coli proteins in two different expressions vectors N-terminal His- or Flag-tag, with known solubility characteristics were used to test the Colony Filtration blot procedure (CoFi blot) .
- the method uses an antibody-reaction for detection of soluble protein, and is therefore universally applicable to any protein containing a suitable tag, or other fusion polypeptide moiety, against which antibodies can be generated.
- test clones Small-scale expression of test clones: For control expression tests, the plasmids were freshly transformed into E. coli strain B121 (DE3) , single colonies were picked and grown overnight in LB medium (Difco, Detroit, MI, USA) at 37°C while shaking at 250 rpm. For test expression, 1 ml LB was inoculated 1:10 with overnight culture and grown to an OD 600 of about 0.6. Cultures were induced by addition of IPTG to a final concentration of 1 mM and grown for another 4 h in a shaker running at 250 rpm at 37°C. The cells were harvested by centrifugation at 2 000 g.
- LB medium Difco, Detroit, MI, USA
- Culturing on LB media plates Freshly transformed cells from the 48 different constructs (24 different proteins in two expression vectors) were arrayed on two LB plates containing appropriate antibiotics at 37 degrees. The colonies were transferred to a Durapore filter membrane with 0.45 ⁇ m pore size (Millipore, Bedford, MA, USA) by gently applying the filter membrane on top of the LB plate, thereby putting the filter membrane in contact with the colonies . Through this procedure most of the colonies are transferred to the surface of the filter membrane. The filter membranes were then transferred with the colony side up to a new LB plates containing IPTG resulting in induced expression. After 4 hours the filter membrane containing the colonies was subjected to lysis.
- the procedure described was done for two membranes in parallel, one to be used for detection of soluble proteins and one to be used to detect total protein, ie the sum of the aggregated protein and the soluble protein.
- Lysis of colonies and transfer to detection membrane The lysis for total protein was done by placing one of the filter membranes on top of a "lysis and detection sandwich" constituting of one Protran BA 45 nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) and a 3MM paper (Whatman) drenched in denaturing lysis buffer. (8M Urea, 20 mM Tris, pH 8, 100 mM NaCI) . The colonies are subjected to this treatment for 1 hour at room temperature.
- the "lysis and detection sandwich” is then frozen at -80°C for 10 minutes and thawed for 10 minutes at 37 degrees. This freeze/thaw procedure is repeated 3 times.
- Example 2 Construction and screening of deletion libraries : To obtain deletion clones, a procedure was employed which has been described before in standard molecular Biology Protocol manuals such as Ausubel et al (Short
- the targets were amplified using Touch down PCR with a plasmid containing the cDNA as template.
- the PCR primers were designed in such a way that suitable restriction sites were located in front of the open reading frame.
- the PCR products were cloned into a pET based expression vector using conventional restriction/ ligation methods. Creation of deletion libraries
- the pET based vectors containing the coding sequence for the two proteins were linearized by endonucelase digestion in the two unique restriction sites introduced in the cloning primer. RORa was digested with Sphl and Hindlll and SOCS-2 with Sphl and Xhol both digestions were made in NEB buffer 2 and at * 37 degrees for 3 hours .
- the digests were verified by agarose gel electrophoresis and purified (QIAquick PCR Purification Kit, QIAGEN) .
- the Erase-a-Base Kit from Promega was used according to the manufacturers instructions with the following alterations.
- the Exonuclease III digestions were made at 27 degrees to achieve a rate at of approximately 70 bp/min.
- 24 aliquots were removed from the ExoIII digestion mix, every minute for RORa and for SOCS-2 every 30 seconds.
- the 24 samples were pooled into six and treated with SI nuclease.
- the DNA was precipitated using Ethanol and Ammoniumacetate.
- the precipitated DNA was resuspended in lOmM Tris pH 8 and treated with Klenow polymerase to flush the ends .
- the plasmids were religated and transformed into a cloning strain, DH5a or ToplO plated on LB plates containing the appropriate antibiotics.
- the library was recovered from the cloning strain by making a plasmid preparation from the colonies on the plate. Screening for soluble protein expression
- the deletion libraries were transformed into an expression strain BL21(DE3) and plated onto plates containing the appropriate antibiotics. Positive controls, constructs expressing soluble proteins, were also added to the plate to facili tate orientation . The plates were incubated at 37 degrees overnight.
- Example 1 The screening for soluble protein was performed as described in Example 1 with the following exceptions. Expression was induced for 6 hours at 25 degrees instead of 37 and 4 hours. For RORa 12 positive clones and 12 negative clones and for SOCS-2 14 positive clones were taken for further analysis. Solubility assay, SDS PAGE and Western blot transfer The clones that were picked for further analysis were characterized as described in Example 1. With the exception that the cells were grown for 6 hours at 25 degrees after induction of expression. Antibody-incubation and development of colony blots and Western blots The colony blots and western blots were developed as described in Example 1.
- E. coli clones DNA fragments coding for 42 different E. coli membrane proteins were amplified from genomic E. coli DNA by PCR, using specific primers. PCR products were cloned into Gateway vectors by homologous recombination. The plasmids were transformed into E. coli strain C41.
- the colonies were transferred to filter membranes of different type and origin (Millipore, Bedford, MA, USA; or Pall Life Sciences, Ann Arbor, MI, USA), or of different pore size (0.45 to 3 ⁇ m) by gently placing the filter membrane on top of the LB plate, thereby putting the membrane in contact with the colonies.
- the filter membrane were then transferred with the colony side up to a new LB plate containing IPTG for induced expression at +37°C for 3 hours. Lysis of colonies and transfer to detection membrane : Lysis under native and denaturing conditions was performed as described in example 1, for 60-90 min..
- Lysis in presence of detergents was performed in the same way as the native lysis in Example 1, except Triton X-100 (1%) or dodecylmaltoside (5mM) was added to the native lysis buffer. Detection was done using an antibody specific for a C-terminal His-tag. Results The membrane proteins from the colonies which were lysed in a native lysis buffer with detergent present were clearly visible in the blots, while proteins from colonies lysed without detergent present were not visible (See Figure 12) . The addition of detergent solubilised membrane proteins allowing them to pass the filtration membrane and to be visible on the colony blot.
- PCR reactions were performed with a Gene-amp PCR system 9700 thermocycler. Mutagenesis was performed with Stratagen GeneMorph Random Mutagenesis kit (Stratagen, La Jolla, USA) according to manufacturer's protocol for medium range mutation frequency (3-7 mutations/kb) . The PCR program suggested in manufacturer's manual was used with a denaturing temperature of 94°C and an annealing temperature of 55°C *
- the PCR products were cloned into an expression plasmid, transformed into DH5 ultracompetent cells and plated on LA plates with tetracyclin (30 ⁇ g/ml) . After overnight growth, the colonies were pooled using 9 ml of LB, and plasmid DNA was isolated using a miniprep kit according to manufacturer's protocol. Plasmids were transformed into a C41 expression system and plated on LA plates with tetracyclin (30 ⁇ g/ml) . Expression screening:
- Clones of interest were grown in liquid culture as described in example 1 and the cells were lysed as described in example 1 with the exception that DDM was added to the lysis buffer. Dot blots were made of the solubilized fractions. The dot blots were developed as described above.
- a vector library can be designed consisting of a randomised stretch of five amino acids adjacent to the start codon. This creates a great vector variation and ideally the randomised region can be fitted individually to the different protein targets.
- the randomised nucleotides after the ATG in the expression vector can either be added to the vector directly or added together with the gene of interest by cloning of a PCR product generated from randomised primers.
- the principle of primer design for adding randomised nucleotides is ATG NNN NNN NNN NNN NNN NNN. The selection of highly expressing soluble clones will be performed as described with the CoFi-blot technique .
- the mutator frequencies were determined by the Rifr (Rifampicin revertants) method described by Miller (Miller et al . 1992 supra) where the mutation frequencies should optimally be in the range of 10_ 4 -10_ 3 .
- Rifampicins inhibit the RNA polymerase and thereby cell growth.
- the mutation frequencies were calculated as the amount of revertants growing on Rif containing plates compared to the number of viable cells.
- Mutant expression strain libraries were screened with the CoFi-blot technique as described above.
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Application Number | Priority Date | Filing Date | Title |
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DE602004022414T DE602004022414D1 (en) | 2003-07-02 | 2004-05-27 | METHOD FOR FINDING CELL COLONIES THAT EXPRESS A SOLUBLE VARIANT OF A TARGET PROTEIN |
EP04735027A EP1639373B1 (en) | 2003-07-02 | 2004-05-27 | A method of screening for cell colonies that express a soluble variant of a target protein |
US10/562,734 US7718381B2 (en) | 2003-07-02 | 2004-05-27 | Method of screening for cell colonies that express a soluble variant of a target protein |
AU2004254096A AU2004254096B2 (en) | 2003-07-02 | 2004-05-27 | A method of screening for cell colonies that express a soluble variant of a target protein |
CA2531026A CA2531026C (en) | 2003-07-02 | 2004-05-27 | A method of screening for cell colonies that express a soluble variant of a target protein |
AT04735027T ATE438858T1 (en) | 2003-07-02 | 2004-05-27 | METHOD FOR FINDING CELL COLONIES THAT EXPRESS A SOLUBLE VARIANT OF A TARGET PROTEIN |
DK04735027T DK1639373T3 (en) | 2003-07-02 | 2004-05-27 | Method for screening cell colonies expressing a soluble variant of a target protein |
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GBGB0315525.6A GB0315525D0 (en) | 2003-07-02 | 2003-07-02 | A method of screening |
GB0315525.6 | 2003-07-02 |
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WO2005003784A1 true WO2005003784A1 (en) | 2005-01-13 |
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PCT/GB2004/002271 WO2005003784A1 (en) | 2003-07-02 | 2004-05-27 | A method of screening for cell colonies that express a soluble variant of a target protein |
Country Status (10)
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US (1) | US7718381B2 (en) |
EP (1) | EP1639373B1 (en) |
AT (1) | ATE438858T1 (en) |
AU (1) | AU2004254096B2 (en) |
CA (1) | CA2531026C (en) |
DE (1) | DE602004022414D1 (en) |
DK (1) | DK1639373T3 (en) |
ES (1) | ES2331311T3 (en) |
GB (1) | GB0315525D0 (en) |
WO (1) | WO2005003784A1 (en) |
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GB0419628D0 (en) * | 2004-09-03 | 2004-10-06 | European Molecular Biology Lab Embl | Method for determining protein solubility |
US20140024555A1 (en) * | 2011-03-31 | 2014-01-23 | Los Alamos National Security, Llc | Method of identifying soluble proteins and soluble protein complexes |
GB201106548D0 (en) | 2011-04-18 | 2011-06-01 | Evitraproteoma Ab | A method for determining ligand binding to a target protein using a thermal shift assahy |
US9523693B2 (en) | 2011-04-18 | 2016-12-20 | Biotarget Engagement Interest Group Ab | Methods for determining ligand binding to a target protein using a thermal shift assay |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2152214A (en) * | 1983-11-08 | 1985-07-31 | Univ Surrey | Detection of enterotoxigenic E. coli |
US20020127587A1 (en) * | 2001-02-13 | 2002-09-12 | Domenica Simms | Methods and compositions for isolation of biological macromolecules |
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DE4237113B4 (en) * | 1992-11-03 | 2006-10-12 | "Iba Gmbh" | Peptides and their fusion proteins, expression vector and method of producing a fusion protein |
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2003
- 2003-07-02 GB GBGB0315525.6A patent/GB0315525D0/en not_active Ceased
-
2004
- 2004-05-27 ES ES04735027T patent/ES2331311T3/en not_active Expired - Lifetime
- 2004-05-27 DK DK04735027T patent/DK1639373T3/en active
- 2004-05-27 DE DE602004022414T patent/DE602004022414D1/en not_active Expired - Lifetime
- 2004-05-27 US US10/562,734 patent/US7718381B2/en active Active
- 2004-05-27 AU AU2004254096A patent/AU2004254096B2/en not_active Ceased
- 2004-05-27 CA CA2531026A patent/CA2531026C/en not_active Expired - Lifetime
- 2004-05-27 AT AT04735027T patent/ATE438858T1/en not_active IP Right Cessation
- 2004-05-27 WO PCT/GB2004/002271 patent/WO2005003784A1/en active Application Filing
- 2004-05-27 EP EP04735027A patent/EP1639373B1/en not_active Expired - Lifetime
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2152214A (en) * | 1983-11-08 | 1985-07-31 | Univ Surrey | Detection of enterotoxigenic E. coli |
US20020127587A1 (en) * | 2001-02-13 | 2002-09-12 | Domenica Simms | Methods and compositions for isolation of biological macromolecules |
Non-Patent Citations (2)
Title |
---|
KNAUST R K C ET AL: "Screening for soluble expression of recombinant proteins in a 96-well format", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, NEW YORK, NY, US, vol. 297, no. 1, 1 October 2001 (2001-10-01), pages 79 - 85, XP002240117, ISSN: 0003-2697 * |
WALDO G S ET AL: "Rapid protein-folding assay using green fluorescent protein.", July 1999, NATURE BIOTECHNOLOGY. JUL 1999, VOL. 17, NR. 7, PAGE(S) 691 - 695, ISSN: 1087-0156, XP002291069 * |
Also Published As
Publication number | Publication date |
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GB0315525D0 (en) | 2003-08-06 |
CA2531026C (en) | 2010-09-21 |
US7718381B2 (en) | 2010-05-18 |
EP1639373B1 (en) | 2009-08-05 |
DE602004022414D1 (en) | 2009-09-17 |
DK1639373T3 (en) | 2009-12-07 |
US20070087325A1 (en) | 2007-04-19 |
ATE438858T1 (en) | 2009-08-15 |
EP1639373A1 (en) | 2006-03-29 |
CA2531026A1 (en) | 2005-01-13 |
AU2004254096B2 (en) | 2009-06-11 |
AU2004254096A1 (en) | 2005-01-13 |
ES2331311T3 (en) | 2009-12-29 |
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