CN101490548A

CN101490548A - Protein surface remodeling

Info

Publication number: CN101490548A
Application number: CNA2007800271393A
Authority: CN
Inventors: 大卫·R·刘; 凯文·约翰·菲利普斯; 迈克尔·S·劳伦斯
Original assignee: Harvard College
Current assignee: Harvard College
Priority date: 2006-06-02
Filing date: 2007-06-01
Publication date: 2009-07-22
Also published as: JP5396270B2; JP2013063093A; JP2015033392A; JP2009539351A

Abstract

Aggregation is a major cause of the misbehavior of proteins. A system for modifying a protein to create a more stable variant is provided. The method involves identifying non-conserved hydrophobic amino acid residues on the surface of a protein, suitable for mutating to more hydrophilic residues (e.g., charged amino acids). Any number of residues on the surface may be changed to create a variant that is more soluble, resistant to aggregation, has a greater ability to re-fold, and/or is more stable under a variety of conditions. The invention also provides GFP, streptavidin, and GST variants with an increased theoretical net charge created by the inventive technology. Kits are also provided for carrying out such modifications on any protein of interest.

Description

Protein surface remodeling

Related application

The application's case is advocated the right of priority of the U.S. Provisional Patent Application case USSN 60/810,364 of application on June 2nd, 2006 according to 35 U.S.C. § 119 (e), and its full content is incorporated herein by reference.

Government supports

Described herein study portion obtains the support of NIH (National Institutes of Health) fund (GM065400).U.S. government can have some right of the present invention.

Technical field

Do not have

Background technology

Protein is the main constituent of cell.The protein catalyzed chemical reaction, transduction signal in biosystem provides structural detail in cell and extracellular matrix, serve as courier etc.The one of the main reasons of the bad performance of protein is to assemble.This is not only the problem in the laboratory, and is the problem such as Alzheimer disease numerous diseases such as (Alzheimer ' s disease).When reaching the protein of calculation Design, gathering is especially worried problem.For instance, TOP7 is the protein with novel calculation Design that folds.The more microscler formula of TOP7 (TOP7 of prolongation (TOP7extended)) is very easily assembled.TOP7ex mainly is expressed as insoluble aggregation.

Along with greater protein matter is designed or modifies with as the instrument of research biosystem or along with more wild types or modifying protein are used as therapeutic agent, need conventionally these protein modifications for more stable and/or prevent the system of assembling.

Summary of the invention

The invention provides a kind of modifying protein so that its more stable system.Generation of the present invention is based on the understanding that can improve the extrathermodynamics character of protein to the lip-deep water repellent region of modifying protein.System of the present invention is particularly useful for improving the dissolubility of the protein of paying close attention to, and improves the anti-aggregation of protein, and/or improves the renaturation ability of protein.All these characteristics are particularly useful for protein production, protein purification and use protein as therapeutic agent and research tool.

On the one hand, the invention provides a kind of primary sequence of protein that changes to increase the method for anti-aggregation, dissolubility, refolding ability and/or the general stability of protein under the broad range of conditions.The activity of modifying protein is preferred, and roughly or substantially the protein with unmodified is identical.In certain embodiments, the activity of the wild-type protein of modifying protein reservation at least 50%, 75%, 90% or 95%.In one embodiment, said method comprising the steps of: the surface residue of (a) differentiating the protein of paying close attention to; (b) differentiate that the particular surface residue is (that is, the determining which amino acid is not indispensable concerning protein active or function) of non-high conservative in other protein relevant with concern protein; (c) hydrophobicity of definite non-conservative surface residue of being differentiated; And (e) be used in the higher or charged amino acid replacement of polarity under the physiological pH value at least one or the non-conserved residues of hydrophobicity differentiated more than one.Each above step all can use any technology known in the affiliated field, computer software, algorithm, normal form etc. to carry out.After producing modifying protein, can test its active and/or look for expection character.In certain embodiments, modifying protein is more stable.In certain embodiments, modifying protein is difficult for assembling.The inventive method is increased in the net charge (plus or minus) of protein under the physiological pH value usually.

On the other hand, the invention provides a kind of by making primary sequence that protein " super charged (supercharging) " changes protein to increase the method for anti-aggregation, dissolubility, refolding ability and/or the general stability of protein under the broad range of conditions.That is to say, compare that net charge total on the modifying protein increases (positive charge or negative charge) with wild-type protein.The activity of modifying protein is preferred, and roughly or substantially the protein with unmodified is identical.In certain embodiments, said method comprising the steps of: the surface residue of (a) differentiating the protein of paying close attention to; (b) differentiate that the particular surface residue is (that is, the determining which amino acid is not indispensable concerning protein active or function) of non-high conservative in other protein relevant with concern protein; (c) water wettability of definite non-conservative surface residue of being differentiated; And (e) be used in charged amino acid replacement charged under the physiological pH value at least one or charged or polarity, the solvent differentiated more than one and expose non-conserved residues.In certain embodiments, electronegative for preparing " super charged " protein makes the residue that is used to modify of differentiating sport aspartic acid (Asp) or glutamic acid (Glu) residue.In some other embodiment, " super charged " protein for the preparation positively charged makes the residue that is used to modify of differentiating sport lysine (Lys) or arginine (Arg) residue.Each above step can use any technology known in the affiliated field, computer software, algorithm, normal form etc. to carry out.After producing modifying protein, can test its active and/or look for expection character.In certain embodiments, modifying protein (" super charged protein ") is more stable.In certain embodiments, modifying protein is difficult for assembling.The inventive method is increased in the net charge (plus or minus) of protein under the physiological pH value usually.

(Protein Data Bank, the theoretical net charge that surpasses 80% protein in PDB) is in ± 10 scopes to enroll Protein Data Bank.The modifying protein that the present invention produced has usually less than-10 or greater than+10 net charge.In certain embodiments, modifying protein has less than-20 or greater than+20 net charge.In certain embodiments, modifying protein has less than-30 or greater than+30 net charge.In certain embodiments, modifying protein has less than-40 or greater than+40 net charge.In certain embodiments, modifying protein has less than-50 or greater than+50 net charge.Modifying protein can appropriately fold and keep its biologically active.

Can use system of the present invention to modify any protein, and the protein variants that system of the present invention produced and the polynucleotide of coding variant protein matter or the cell of carrier and expression variant protein matter all are considered as a part of the present invention.Used system of the present invention to produce several novel green fluorescent proteins (green fluorescent protein, GFP) variant.These variants keep its fluorescence; Yet its existing form than GFP under the wide region environment is more stable.Even GFP of the present invention is not through can assembling for a long time and in inducing the environment of gathering yet, even and can refolding be fluorescin by boiling after the sex change yet.Also used system of the present invention to produce novel Streptavidin (streptavidin) and glutathione-S-transferase (glutathione-S-transferase, GST) variant.These variants keep its biologically active and still solvable when heating.The present invention also comprises the polynucleotide sequence of code book invention GFP, Streptavidin and GST protein sequence; The carrier that comprises any of these nucleotide sequence; And the cell that comprises such polynucleotide sequence or carrier or expression variant of the present invention.In certain embodiments, the present invention includes bacterium or other cell of overexpression variant of the present invention.Variant of the present invention can be used in the known multiple biologicall test in affiliated field.For instance, super charged GFP can be used for using at present in any mensuration of GFP as reporter protein.

On the other hand, the invention provides other protein of having modified by system of the present invention.These modifying proteins preferably keep the initial activity of its remarkable ratio.In certain embodiments, the activity of the unmodified form of modifying protein reservation at least 99%, 98%, 95% or 90%.Modifying protein can be more solvable in multiple condition, and anti-the gathering, increase of refolding ability and/or stability are higher.The protein of being modified by system of the present invention comprises hydrophobic proteins, recombinant protein, memebrane protein, structural proteins, enzyme, extracellular protein, treatment albumen (for example, insulin, cytohormone, immunoglobulin (Ig), immunoglobulin fragment etc.), acceptor, cellular signal transduction albumen, plasmosin, nuclear protein, transcription factor etc.In some specific embodiment, protein is to be used for human or veterinary treatment albumen.In certain embodiments, protein is non-native protein, for example the protein of calculation Design.In other embodiments, protein is hybrid protein, fusion, allosteric protein, mutain, engineered protein or passes through any other protein of manual change.

Also be provided for implementing kit of the present invention.Kit can comprise modify pay close attention to protein so that its more anti-gathering, increase its renaturation ability or increase the required reagent of its general stability.These kits can comprise following each thing of all or part: polynucleotide, computer software, nucleotide, primer, carrier, clone, instructions, culture plate, nutrient culture media, damping fluid, enzyme, Ai Bende pipe (Eppendorf tube), direct mutagenesis kit etc.Kit preferably uses for lab setup through easy packing.The protein DNA coded sequence that the researcher provides the technology of the present invention to be used to modify usually.

Definition

" amino acid ": term " amino acid " is meant the basic structure subunit (subunit) of protein.A-amino acid is made up of amino, carboxyl, hydrogen atom and side chain (that is, the R group), all is binding on central carbon atom.This central carbon atom is because adjacent with carboxyl and be called α carbon.There are 20 kinds of natural amino acids, comprise glycocoll, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophane, halfcystine, methionine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamic acid and proline.Hydrophobic amino acid comprises alanine, valine, leucine, isoleucine and phenylalanine.Aromatic amino acid comprises phenylalanine, tyrosine, tryptophane and histidine.Polar amino acid comprises tyrosine, halfcystine, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine and glutamine.Sulfur-containing amino acid comprises halfcystine and methionine.Basic amino acid comprises lysine, arginine and histidine.Acid Amino acid comprises aspartic acid and glutamic acid.Also have and be inserted into alpha-non-natural amino acid in the protein.In certain embodiments, when using term " amino acid ", be meant 20 kinds of natural amino acids.

" antibody ": term " antibody " is meant the natural or synthetic wholly or in part immunoglobulin (Ig) that produces.All its derivants of keeping specific binding capacity are also included within this term.This term is also contained and is had and the immunoglobulin (Ig) binding domain homologue or any protein that combines the territory of homology basically.These protein can be derived from natural origin or partially or completely synthetic the generation.Antibody can be monoclonal antibody or polyclonal antibody.Antibody can be any immunoglobulin class member of (comprising the arbitrary classification among human classification IgG, IgM, IgA, IgD and the IgE).

" guard ": term " is guarded " corresponding nucleotide or the amino acid residue that are meant polynucleotide sequence or amino acid sequence and is not changed in the same position of two or more correlated serieses that compared.Bao Shou nucleotide or amino acid are than other place occurred in the sequence nucleotide or conservative those nucleotide or the amino acid of amino acid in correlated series relatively.

" homology ": term " homology " is the term that affiliated field is understood thoroughly as used herein, is meant that nucleic acid or protein are in nucleotide or amino acid sequence aspect height correlation.The nucleic acid of homology or protein are called homologue each other.Homology can refer to the sequence similarity degree between two sequences (that is, nucleotide sequence or amino acid).Maximum possible homology between two sequences of homology % mentioned herein numeral reflection, that is, and the homology % when comparing two sequences with coupling (homology) position with maximum number.Homology can easily be calculated by known method, method described in following document: calculate molecular biology (Computational Molecular Biology), Lai Sike (Lesk, A.M.) compile, Oxford University Press (Oxford University Press), New York (New York), 1988; Biological computation: information science and genome plan (Biocomputing:Informatics and Genome Projects), (Smith D.W.) compiles academic press (Academic Press), New York (New York), 1993 to Smith; Sequential analysis in the molecular biology (Sequence Analysis in Molecular Biology), and Fan Haiye (von Hei nje, G.), academic press (Academic Press), 1987; The Computer Analysis part i of sequence data (Computer Analysis ofSequence Data, Part I), lattice Lifei is because of (Griffin, A.M.) and lattice Lifei because of (Griffin H.G.) compiles Hu Mana publishing house (Humana Press), New Jersey (New Jersey), 1994; And sequence analysis primer (SequenceAnalysis Primer), and lucky cloth koff (Gribskov, M.) (Devereux J.) compiles, rice Stockton publishing house (M Stockton Press), New York (New York), 1991 with moral husband Rec; Be incorporated herein by reference separately.The common method of measuring homology between the sequence includes, but is not limited to the method that disclosed in the following document: Ka Liluo (Carillo, H.) and Li Piman (Lipman, D.), SIAM applied mathematics (SIAM J Applied Math.), 48:1073 (1988); It is incorporated herein by reference.The technology of measuring homology has been compiled to and discloses available computer program.The exemplary computer software of measuring homology between two sequences comprises (but being not limited to) GCG routine package (moral husband Rec (Devereux, people such as J.), nucleic acids research (Nucleic Acids Research), 12 (1), 387 (1984)), (Aunar relaxes, and (Atschul S.F.) waits people, molecular biology magazine (J Molec.Biol.) for BLASTP, BLASTN and FASTA, 215,403 (1990)).

Term " homology " necessarily means the comparison between at least two sequences (nucleotide sequence or amino acid sequence).According to the present invention, if two nucleotide sequence encoded polypeptide have at least 20 amino acid of at least one section at least about the 50-60% unanimity, preferred about 70% unanimity is considered as homology with it so.The characteristic optimization of homologous nucleotide sequence one section 4-5 the specially appointed amino acid whose ability at least that also is to encode.About waiting to consider the nucleotide sequence of homology, must consider these amino acid consistance and interval roughly each other.For the nucleotide sequence of length less than 60 nucleotide, by one section of coding at least 4-5 specially appointed amino acid whose ability determine homology.

" peptide " or " protein ": according to the present invention, " peptide " or " protein " comprises a string at least three amino acid that link together by peptide bond.Term " protein " and " peptide " are used interchangeably.Peptide of the present invention preferably only contains natural amino acid, but known alpha-non-natural amino acid in field under also can using (that is, occurring in nature does not exist but can incorporate compound in the polypeptied chain into) and/or amino acid analogue.Simultaneously, can modify one or more amino acid in the peptide of the present invention, for example by add carbohydrate group, phosphoric acid foundation, farnesyl-(farnesyl), different farnesyl-, fatty acid group, be used for connecting, chemical entities such as linking group functionalized or other modification (for example, α amidation) modify.In a preferred embodiment, peptide is modified the more stable peptide (for example, in vivo the half life period is higher) of generation.These modifications can comprise makes the peptide cyclisation, incorporate D-amino acid etc. into.These modifications all should not produce substantial interference to the expection biologically active of peptide.In certain embodiments, peptide is modified the higher peptide of generation biologic activity.

" polynucleotide " or " oligonucleotides ": polynucleotide or oligonucleotides are meant the polymkeric substance of nucleotide.Polynucleotide comprise at least three nucleotide usually.Polymkeric substance can comprise natural nucleus glycoside (promptly, adenosine, thymidine, guanosine, cytidine, uridine, desoxyadenossine, deoxyribosylthymine, deoxyguanosine and deoxycytidine), nucleoside analog (for example, the amino adenosine of 2-, 2-sulfo-thymidine, inosine, pyrrolopyrimidine, the 3-methyladenosine, C5-propinyl cytidine, C5-propinyl uridine, the C5-Broxuridine, the C5-floxuridine, C5-ioduria glycosides, the C5-methylcytidine, 7-denitrogenation adenosine (7-deazaadenosine), 7-denitrogenation guanosine (7-deazaguanosine), 8-oxo adenosine (8-oxoadenosine), 8-oxo guanosine (8-oxoguanosine), O (6)-methyl guanine and 2-sulfo-cytidine), the chemical modification base, the bio-modification base (for example, methylated base), insert base, (for example modify sugar, 2 '-fluorine ribose, ribose, 2 '-ribodesose, arabinose (arabinose) and hexose) and/or modify phosphoric acid foundation (for example, D2EHDTPA foundation and 5 '-N-phosphoramidite binding).

" micromolecule ": term " micromolecule " is meant in the laboratory preparation or the visible non-peptide of occurring in nature, non-oligomerization organic compound as used herein.Micromolecule can refer to " natural products sample " compound as used herein, yet term " micromolecule " is not limited to " natural products sample " compound.Or rather, micromolecular feature is that usually it contains several carbon-carbon bonds and has molecular weight less than 1500, but for purposes of the present invention, and this feature does not plan to limit the present invention.In some other preferred embodiment, utilize natural products sample micromolecule.

" stablize ": any aspect that is meant protein stability when mentioning protein " stablized " in term as used herein.Compare with initial wild-type protein, stable modifying protein has one or more following feature: more solvable, more anti-gathering, more antitypy, more anti-separate folding, more anti-inappropriate or undesirable folding, the renaturation ability is stronger, thermal stability increases, multiple environment (for example, pH value, salinity, have washing agent, have denaturant etc.) down stability increase and non-aqueous environment down stability increase.In certain embodiments, stable modifying protein shows at least two above-mentioned features.In certain embodiments, stable modifying protein shows at least three above-mentioned features.These features can make reactive protein produce under higher level.For instance, can be under than the high level of protein unmodified form overexpression modifying protein and do not have gathering.These features also can make protein be used as therapeutic agent or research tool.

Description of drawings

Fig. 1: super charged green fluorescent protein (GFP).(a) protein sequence of GFP variant, form the residue of fluorophore outstanding for green, electronegative residue is outstanding for red, and the positively charged residue is outstanding for blue; (b) sfGFP (left side), GFP (+36) (in) and the surface electrostatic gesture of GFP (30) (right side), from-25kT/e (redness) to+25kT/e (blueness) is painted.

Fig. 2: character in the molecule of GFP variant.(a) dyeing of pure GFP variant and Ultraluminescence.Each swimming lane and test tube contain 0.2 μ g protein.(b) circulr dichromism spectrum of GFP variant.(c) thermodynamic stability of GFP variant is separated folding the measurement by what guanidinesalt was induced.

Fig. 3: the intermolecular character of super charged protein.(a) the ultraviolet irradiation sample of pure GFP variant (" natural ") is at 1 minute sample (" boiling ") of 100 ℃ of heating with subsequently at 2 hours sample (" cooling ") of 25 ℃ of coolings.(b) induce the gathering of GFP variant and pass through right angle light scattering monitoring with 40% TFE down at 25 ℃.(c) super charged GFP reversibly adheres to the big molecule of oppositely charged.Sample 1: 6 μ g GFP (+36) in 30 μ l 25mM Tris (pH7.0) and 100mM NaCl.Sample 2: in sample 1, add 6 μ g GFP (30).Sample 3: in sample 1, add 30 μ g salmon sperm dnas.Sample 4: in sample 1, add 20 μ g Escherichia coli (E.coli) tRNA.Sample 5: in sample 4, add NaCl and reach 1M.Sample 6-8:, identical with

sample

1,2 and 4 respectively except that using sfGFP to replace the GFP (+36).Make all samples of short duration rotation and under ultraviolet light, observing in micro centrifuge.(d) enzymatic assays of GST variant.Reaction contains 0.5mg/mL GST variant, 20mM dinitrofluorobenzene, 20mM glutathione and 100mM potassium phosphate (pH6.5).The formation of monitoring product obtains reaction rate observed value (k under 340nm _Obs), wild type GST is 6min ^-1, GST (40) is 2.2min ^-1Be 0.9min and boil and cool off back GST (40) ^-1

Fig. 4: (a) excitation spectrum of GFP variant and (b) emission spectrum.Each sample contains by the institute of chromophore absorbance under 490nm quantitatively, the protein of equivalent.

Fig. 5: the biotin of Streptavidin variant is in conjunction with activity, it is as discussed previously that (card reaches people such as (Kada), utilize fluorescent quenching or fluorescence polarization to estimate avidin and Streptavidin (Rapid estimation of avidinand streptavidin by fluorescence quenching or fluorescence polarization) fast. Acta Biochimica et Biophysica Sinica (Biochim.Biophys.Acta) 1427,44-48 (1999), it is incorporated herein by reference) measure by the combination of monitoring bio element-4-fluorescein (hero company (Invitrogen)) dependence.Titration protein example in 0.3 μ M biotin-4-fluorescein (B4F), 100mM NaCl, 1mM EDTA, 0.1mg/mL bovine serum albumin(BSA) (bovine serumalbumin BSA), 50mM potassium phosphate (pH7.5).Measure the fluorescent quenching under the 526nm on the Perkin-Elmer LS50B fluorescence spectrophotometry instrument that under 470nm, excites.With respect to the contrast titration that contains 600 times of excessive no biological elements, with measurement result normalization.Three protein that comprise the legend bottom are as negative control.

Embodiment

The invention provides a kind of modifying protein so that its more stable system.Described system moves by the non-conserved amino acid on the protein surface is become the bigger or charged amino acid residue of polarity.Amino acid residue to be finished can be hydrophobicity, water wettability or its combination.But the application of the invention system modifies any protein and produces more stable variant.Found surface residue is carried out the extrathermodynamics character that these modifications can improve protein.Along with protein day by day as therapeutic agent and along with it continues to serve as research tool, be used to change protein so that its more stable system also becomes important and suitable.By anti-usually gathering of protein that the inventive method is modified, the refolding ability increases, and is anti-inappropriate folding, and dissolubility improves and be more stable usually under the broad range of conditions that comprises sex change condition (such as heat or have washing agent).

But the application of the invention system modifies any protein and produces more stable variant.Can modify natural and non-natural protein (for example, engineered protein).Can adorned protein example comprise that acceptor, embrane-associated protein, transmembrane protein, enzyme, transcription factor, extracellular protein, treatment albumen, cell factor, courier's albumen, DNA are in conjunction with the protein of albumen, rna binding protein, participation signal transduction, structural proteins, plasmosin, nuclear protein, hydrophobic proteins, hydrophilic protein etc.Protein to be finished can be plant-derived, any species of animal or microorganism.In certain embodiments, protein is mammalian proteins matter.In certain embodiments, protein is the human protein.In certain embodiments, protein source normally used biosome in research.For instance, protein to be finished can be from primate (for example, ape, monkey), rodent (for example, rabbit, hamster, gerbil jird), pig, dog, cat, fish (for example, zebra fish (zebrafish)), nematode (for example, beautiful nematode (C.elegans)), yeast (for example, saccharomyces cerevisiae (Saccharomycescervisiae)) or bacterium (for example, Escherichia coli (E.coli)).

The protein that system of the present invention is particularly useful for modifying easy gathering or has stability problem.Native system also can be used for modifying the protein by overexpression.For instance, the treatment albumen of reorganization generation can be benefited from the modification of system of the present invention.These the not only easier production of treatment albumen and purifying through modifying, and may be more stable with regard to the preservation and the use of protein.

System of the present invention comprises the non-conservative surface residue of differentiating the protein of paying close attention to and is used under the physiological pH value to be that water wettability, polarity or charged residue are replaced some in these residues.System of the present invention not only comprises the method for modifying protein, and comprises and be applicable to modifying protein so that its more stable reagent and kit.

Use any method known in the affiliated field to differentiate the surface residue of protein to be finished.In certain embodiments, differentiate surface residue by the computer mould albuminoid.In certain embodiments, known and/or measure the three-dimensional structure of protein, and differentiate surface residue by the structure of observing protein.In other embodiments, the software prediction surface residue that uses a computer.In some specific embodiment, use average adjacent atom (Average NeighborAtoms per Sidechain Atom, AvNAPSA) exposure of prediction surface of each side chain atom.AvNAPSA is a kind of robotization surface exposure method of having implemented as computer program.Referring to appendix A.The residue that low AvNAPSA value representation surface exposes, and high value representation is positioned at the residue of protein interior.In certain embodiments, use the secondary structure of described software prediction protein and/or tertiary structure and differentiate surface residue based on this prediction.In other embodiments, the prediction of surface residue be based on the hydrophobicity of residue and water wettability with and cluster analysis in the protein primary sequence.Except that computer simulation (insilico) method, also can use multiple Measurement for Biochemistry (for example protease cracking, finishing etc.) to differentiate the surface residue of protein.

Determine then in the surface residue which be guard or be important to the function of protein.Under can using in the field known any method determine the discriminating of conserved residues.In certain embodiments, differentiate conserved residues by the primary sequence and the related protein of the comparison protein of paying close attention to.These related proteins can be from same protein family.For instance, if protein is immunoglobulin (Ig), can use other immunoglobulin sequences so.Related protein also can be the same protein from different plant species.For instance, can differentiate conserved residues from the sequence of the same protein of different plant species by comparison.As another example, can compare and have similar functions or bioactive protein.The preferred conserved amino acid that uses 2,3,4,5,6,7,8,9 or 10 different sequences to determine protein.In certain embodiments, if having same amino acid above 50%, 60%, 70%, 75%, 80% or 90% sequence at ad-hoc location, residue just is considered to be conservative so.In other embodiments, have identical or similar (for example, valine, leucine and isoleucine if surpass 50%, 60%, 70%, 75%, 80% or 90% sequence at ad-hoc location; Glycocoll and alanine; Glutamine and asparagine; Or aspartic acid and glutamic acid) amino acid, residue just is considered to be conservative so.Many software packages can be used for comparison and protein sequence more as described herein.It will be understood by one of ordinary skill in the art that and at first to determine conserved residues or can at first determine surface residue.Order is unimportant.In certain embodiments, computer packages can be determined surface residue and conserved residues simultaneously.Also can differentiate important residue in the protein by mutagenesis protein.For instance, can use the alanine of protein to scan to determine important amino acid residue in the protein.In other embodiments, can use direct mutagenesis.

In case identify the non-conservative surface residue of protein, just each residue differentiated to being hydrophobicity or water wettability.In certain embodiments, specify the hydrophobicity scoring to residue.For instance, specify the logarithm value (logP) of octanol/water partition factor can for each non-conservative surface residue.Also can use other hydrophobicity parameter.These amino acid scales have been discussed in the following document: Jian Yin (Janin), surface in the globular preteins and internal volume (＂ Surface and Inside Volumes in GlobularProteins ＂), nature (Nature) 277:491-92,1979; Wal Fenton people such as (Wolfenden), amino acid side chain is to the affinity (＂ Affinities of Amino Acid Side Chains for Solvent Water ＂) of aqueous solvent, biological chemistry (Biochemistry) 20:849-855,1981; Ka Te people such as (Kyte), the straightforward procedure (＂ A Simple Method for Displaying the Hydropathic Character of a Protein ＂) that presents protein hydrotherapy characteristic, molecular biology magazine (J.Mol.Biol.) 157:105-132,1982; Sieve silk people such as (Rose), the hydrophobicity of the amino acid residue in the globular preteins (＂ Hydrophobicity of Amino Acid Residues in Globular Proteins ＂), science (Science) 229:834-838,1985; Examine nanotesla people such as (Cornette), detect the hydrophobicity scale and the computing technique (＂ Hydrophobicity Scales and Computational Techniques for DetectingAmphipathic Structures in Proteins ＂) of the amphiphilic structure in the protein, molecular biology magazine (J.Mol.Biol.) 195:659-685,1987; Look into time (Charton) and look into time (Charton), the structure dependence of amino acid hydrophobicity parameter (＂ The StructureDependence of Amino Acid Hydrophobicity Parameters ＂), theoretical biology magazine (J.Theor.Biol.) 99:629-644,1982; Be incorporated herein by reference separately.Can use any of these hydrophobicity parameter to determine to modify which non-conserved residues in the inventive method.In certain embodiments, discriminating water wettability or charged residue are modified.

The non-conservative or non-significant surfaces residue of selecting at least one to differentiate is then modified.In certain embodiments, select hydrophobic residue to be modified.In other embodiments, select water wettability and/or charged residue to be modified.In certain embodiments, select a more than residue to be modified.In certain embodiments, select 1,2,3,4,5,6,7,8,9 or 10 residue of being differentiated to be modified.In certain embodiments, select to surpass 10, surpass 15 or surpass 20 residues and modified.It will be understood by one of ordinary skill in the art that protein is big more, the residue that then needs to modify will be many more.Simultaneously, high more or easy more gathering of protein hydrophobic or precipitation, the residue that then needs to modify will be many more.In certain embodiments, producing a plurality of protein variants that respectively have different modifying is also tested to determine with regard to biologically active and the best variant of stability.

In certain embodiments, the residue that selection is modified sports more hydrophilic residue (comprising charged residue).Usually make residue sport more hydrophilic natural amino acid.In certain embodiments, make residue sport amino acid charged under the physiological pH value.For instance, residue can be changed into arginine, aspartic acid, glutamic acid, histidine or lysine.In certain embodiments, all residues to be finished are become with a kind of different residues.For instance, make selected residue all become glutaminic acid residue.In other embodiments, make selected residue become different residues; Yet, can make all final residues positively charged or electronegative under the physiological pH value.In certain embodiments, for producing electronegative protein, make all residues to be suddenlyd change be converted into glutamic acid and/or asparagicacid residue.In certain embodiments, for producing the protein of positively charged, make all residues to be suddenlyd change be converted into lysine residue.For instance, all selected residues to be finished are asparagine, glutamine, lysine and/or arginine, and make these residues sport aspartic acid or glutaminic acid residue.As another example, all selected residues to be finished are aspartic acid, glutamic acid, asparagine and/or glutamine, and make these residues sport lysine.This method allows the net charge on the modifying protein to the full extent.

In other embodiments, protein can keeping on the modifying protein after the modification with unmodified protein matter on identical net charge.In other embodiments, protein can be at the total net charge that reduces after the modification on the protein, and increases the sum that charged residue is gone up on the surface.In certain embodiments, theoretical net charge increase at least+1 ,+2 ,+3 ,+4 ,+5 ,+10 ,+15 ,+20 ,+25 ,+30 or+35.In certain embodiments, theoretical net charge is reduced by at least-1 ,-2 ,-3 ,-4 ,-5 ,-10 ,-15 ,-20 ,-25 ,-30 or-35.In certain embodiments, make selected amino acid become nonionic polar residues (for example, halfcystine, serine, threonine, tyrosine, glutamine, asparagine).

Under these modifications in the protein or sudden change can be used in the field known any technology realize.Know the recombinant DNA technology of in protein sequence, introducing these variations in the affiliated field.In certain embodiments, modify by the polynucleotide of direct mutagenesis code for said proteins.Other technicism of introducing sudden change is set forth in the following document: molecular cloning: laboratory manual (Molecular Cloning:A Laboratory Manual), the 2nd edition, mountain nurse Brooker (Sambrook), good fortune Ritz (Fritsch) and Manny Ya Tisi (Maniatis) compile, (publishing house of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press): 1989); Method in the paper zymetology (Methods inEnzymology) (academic press (Academic Press, Inc.), New York (N.Y.)); Su Beier people such as (Ausubel) difficult to understand, up-to-date experimental methods of molecular biology compilation (Current Protocols in Molecular Biology) ((John Wiley of John Willie father and son publishing house; Sons, Inc.), New York (New York), 1999); Be incorporated herein by reference separately.Express and test described modifying protein.In certain embodiments, prepare a series of variants and test each variant and determine its biologically active and its stability.The variant that selection is used for follow-up use can be the most stable variant, the variant of tool activity or the active and stable overall maximum variant of combination.After preparing first group of variant, can prepare another group variant based on the knowledge of acquiring from first group of variant.Usually use recombinant technique known in the affiliated field to produce and the overexpression variant.

Used system of the present invention to produce the GFP variant.Shown that these variants are stable more and kept its fluorescence.Be described in gene pool from the GFP of Victoria jellyfish (Aequorea victoria) and deposit among the numbering P42212, it is incorporated herein by reference.The amino acid sequence of this wild type GFP is as follows:

MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW

PTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVK

FEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNI

EDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA

GITHGMDELYK(SEQ?ID?NO：1)

The theoretical net charge of wild type GFP is-7.Use system of the present invention produced theoretical net charge for-29 ,-30 ,-25 ,+36 ,+48 and+49 variant.Even after heating+36GFP to 95 ℃, 100% variant protein matter also is soluble and protein keeps 〉=70% fluorescence.

The amino acid sequence of the GFP variant that has produced comprises:

GFP-NEG25

MGHHHHHHGGASKGEELFTGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKF

ICTTGELPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD

GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGI

KAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHM

VLLEFVTAAGIDHGMDELYK(SEQ?ID?NO：2)

GFP-NEG29

MGHHHHHHGGASKGEELFDGEVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKF

ICTTGELPVPWPTLVTTLTYGVQCFSRYPDHMDQHDFFKSAMPEGYVQERTISFKDD

GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGI

KAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHM

VLLEFVTAAGIDHGMDELYK(SEQ?ID?NO：3)

GFP-NEG30

MGHHHHHHGGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKF

ICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDD

GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGI

KAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHM

VLLEFVTAAGIDHGMDELYK(SEQ?ID?NO：4)

GFP-POS36

MGHHHHHHGGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLK

FICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKK

DGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRK

NGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKR

DHMVLLEFVTAAGIKHGRDERYK(SEQ?ID?NO：5)

GFP-POS42

MGHHHHHHGGRSKGKRLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLK

FICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKK

DGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRK

NGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKR

DHMVLLEFVTAAGIKHGRKERYK(SEQ?ID?NO：6)

GFP-POS49

MGHHHHHHGGRSKGKRLFRGKVPILVKLKGDVNGHKFSVRGKGKGDATRGKLTLK

FICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKK

DGKYKTRAEVKFKGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRK

NGIKAKFKIRHNVKDGSVQLAKHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKR

DHMVLKEFVTAAGIKHGRKERYK(SEQ?ID?NO：7)

It will be understood by one of ordinary skill in the art that homologous protein is considered as within the scope of the present invention equally.For instance, any protein of one

section

20,30,40,50 or 100 amino acid and any above-mentioned sequence 60%, 70%, 80%, 90%, 95% or 100% homology that comprises is considered as a part of the present invention.In addition, interpolation and deletion mutation body are also contained in the present invention.In certain embodiments, any GFP with the sudden change residue as shown in any above-mentioned sequence is considered as a part of the present invention.In certain embodiments, sequence comprises the sudden change as shown in any above sequence more than 2,3,4,5,6,7,8,9,10 or 10.

Any dna sequence dna of above-mentioned GFP variant of encoding is also included within the scope of the invention.The exemplary dna sequence dna of above each variant of coding is as follows:

GFP-NEG25

ATGGGGCATCACCATCATCATCATGGCGGTGCGTCTAAGGGGGAGGAGTTATTTA

CGGGTGTGGTGCCGATCCTGGTGGAGCTTGATGGCGATGTTAACGGCCATGAATT

TTCTGTCCGCGGTGAAGGGGAGGGTGATGCCACGGAAGGGGAGCTGACACTTAA

ATTTATTTGCACCACCGGTGAACTCCCGGTCCCGTGGCCGACCCTGGTGACCACC

CTGACCTACGGCGTTCAATGCTTTTCACGTTATCCGGATCACATGAAGCAACACG

ACTTCTTTAAAAGCGCGATGCCTGAAGGCTATGTTCAAGAACGTACAATTAGTTT

TAAAGATGACGGCACCTACAAGACCCGTGCGGAAGTAAAATTTGAAGGGGACAC

TTTAGTGAACCGCATCGAGCTGAAAGGGATCGATTTTAAAGAAGATGGGAATAT

CCTGGGACACAAACTTGAATACAACTTTAATAGTCATGACGTCTATATCACGGCG

GACAAACAGGAAAACGGAATTAAGGCAGAATTTGAGATTCGGCATAATGTCGAA

GATGGCTCGGTACAGTTGGCTGATCACTATCAGCAGAATACGCCGATTGGAGAT

GGTCCGGTTTTATTACCAGACGATCACTATCTGTCCACCGAATCCGCCCTGAGCA

AAGATCCGAATGAAGACCGGGACCATATGGTTCTGCTGGAATTTGTTACGGCGG

CTGGTATTGACCATGGCATGGATGAGCTGTATAAGTAG(SEQ?ID?NO：8)

GFP-NEG29

ATGGGGCATCACCATCATCATCATGGCGGTGCGTCTAAGGGGGAGGAGTTATTTG

ATGGTGAAGTGCCGATCCTGGTGGAGCTTGATGGCGATGTTAACGGCCATGAATT

TTCTGTCCGCGGTGAAGGGGAGGGTGATGCCACGGAAGGGGAGCTGACACTTAA

ATTTATTTGCACCACCGGTGAACTCCCGGTCCCGTGGCCGACCCTGGTGACCACC

CTGACCTACGGCGTTCAATGCTTTTCACGTTATCCGGATCACATGGACCAACACG

ACTTCTTTAAAAGCGCGATGCCTGAAGGCTATGTTCAAGAACGTACAATTAGTTT

TAAAGATGACGGCACCTACAAGACCCGTGCGGAAGTAAAATTTGAAGGGGACAC

TTTAGTGAACCGCATCGAGCTGAAAGGGATCGATTTTAAAGAAGATGGGAATAT

CCTGGGACACAAACTTGAATACAACTTTAATAGTCATGACGTCTATATCACGGCG

GACAAACAGGAAAACGGAATTAAGGCAGAATTTGAGATTCGGCATAATGTCGAA

GATGGCTCGGTACAGTTGGCTGATCACTATCAGCAGAATACGCCGATTGGAGAT

GGTCCGGTTTTATTACCAGACGATCACTATCTGTCCACCGAATCCGCCCTGAGCA

AAGATCCGAATGAAGACCGGGACCATATGGTTCTGCTGGAATTTGTTACGGCGG

CTGGTATTGACCATGGCATGGATGAGCTGTATAAGTAG(SEQ?ID?NO：9)

GFP-NEG30

ATGGGGCATCACCATCATCATCATGGCGGTGCGTCTAAGGGGGAGGAGTTATTTG

ATGGTGTGGTGCCGATCCTGGTGGAGCTTGATGGCGATGTTAACGGCCATGAATTTTCTGTCCGCGGTGAAGGGGAGGGTGATGCCACGGAAGGGGAGCTGACACTTAA

ATTTATTTGCACCACCGGTGAACTCCCGGTCCCGTGGCCGACCCTGGTGACCACC

CTGACCTACGGCGTTCAATGCTTTTCAGATTATCCGGATCACATGGACCAACACG

ACTTCTTTAAAAGCGCGATGCCTGAAGGCTATGTTCAAGAACGTACAATTAGTTTTAAAGATGACGGCACCTACAAGACCCGTGCGGAAGTAAAATTTGAAGGGGACAC

TTTAGTGAACCGCATCGAGCTGAAAGGGATCGATTTTAAAGAAGATGGGAATAT

CCTGGGACACAAACTTGAATACAACTTTAATAGTCATGACGTCTATATCACGGCG

GACAAACAGGAAAACGGAATTAAGGCAGAATTTGAGATTCGGCATAATGTCGAA

GATGGCTCGGTACAGTTGGCTGATCACTATCAGCAGAATACGCCGATTGGAGAT

GGTCCGGTTTTATTACCAGACGATCACTATCTGTCCACCGAATCCGCCCTGAGCA

AAGATCCGAATGAAGACCGGGACCATATGGTTCTGCTGGAATTTGTTACGGCGG

CTGGTATTGACCATGGCATGGATGAGCTGTATAAGTAG(SEQ?ID?NO：10)

GFP-POS36

ATGGGGCATCATCATCATCACCACGGCGGGGCGTCTAAGGGAGAGCGCTTGTTTC

GCGGCAAAGTCCCGATTCTTGTGGAGCTCAAAGGTGATGTAAATGGTCATAAATT

TAGTGTGCGCGGGAAAGGGAAAGGAGATGCTACGCGGGGCAAGCTCACCCTGAA

ATTTATTTGCACAACCGGCAAACTGCCAGTGCCGTGGCCTACATTAGTCACTACT

CTGACGTACGGTGTTCAGTGCTTTTCTCGCTATCCCAAACACATGAAACGCCATG

ATTTCTTCAAGAGCGCGATGCCAAAAGGTTATGTGCAGGAACGCACCATCAGCTT

TAAAAAAGACGGCAAATATAAAACCCGTGCAGAAGTTAAATTCGAAGGCCGCAC

CCTGGTCAACCGCATTAAACTGAAAGGTCGTGACTTCAAAGAGAAAGGTAATAT

TCTTGGTCACAAACTGCGCTATAATTTCAACTCTCACAAAGTTTATATTACGGCG

GATAAACGTAAAAACGGGATTAAAGCGAAATTTAAGATTCGTCATAATGTTAAA

GACGGCAGTGTGCAGTTAGCGGATCATTATCAGCAGAATACCCCAATTGGTCGC

GGTCCAGTGCTGCTGCCGCGTAACCATTATCTGTCGACCCGCAGCAAACTCAGCA

AAGACCCGAAAGAAAAACGTGACCACATGGTATTACTGGAATTTGTGACCGCAG

CAGGCATTAAACATGGCCGCGATGAACGTTACAAATAG(SEQ?ID?NO：11)

GFP-POS44

ATGGGCCATCATCATCACCACCACGGCGGCCGCTCAAAAGGTAAACGCTTGTTCC

GTGGTAAAGTACCGATCTTAGTGGAGCTCAAAGGGGATGTGAATGGCCATAAGT

TCTCGGTTCGTGGCAAAGGTAAGGGAGATGCGACGCGCGGCAAATTAACGCTGA

AATTCATTTGTACTACAGGTAAACTGCCGGTGCCATGGCCTACTCTCGTCACCAC

GTTGACCTATGGGGTTCAATGCTTCAGCCGGTACCCTAAACACATGAAGCGCCAC

GATTTCTTCAAATCGGCGATGCCAAAGGGGTATGTCCAGGAACGCACTATCAGCT

TCAAAAAAGACGGTAAGTATAAAACTCGTGCTGAAGTTAAATTCGAAGGACGCA

CACTGGTAAATCGCATTAAATTGAAGGGGCGCGACTTTAAGGAAAAAGGTAATA

TCTTAGGTCACAAATTGCGCTACAACTTCAACTCTCATAAAGTTTACATTACAGC

AGATAAGCGTAAAAATGGCATCAAAGCGAAATTCAAAATTCGTCACAATGTGAA

AGATGGTAGCGTGCAATTAGCCGATCATTACCAGCAGAATACGCCGATCGGTCG

CGGCCCAGTACTGTTGCCGCGCAAACATTACTTATCTACCCGGAGTAAACTGTCT

AAAGACCCAAAAGAGAAGCGCGACCATATGGTTCTCCTGGAGTTTGTCACCGCC

GCCGGAATTAAACACGGCCGCAAAGAGCGCTATAAATAG(SEQ?ID?NO：12)

GFP-POS49

ATGGGCCACCATCATCATCACCACGGGGGACGCTCTAAAGGTAAACGTCTGTTTC

GTGGAAAGGTGCCCATTCTGGTTAAACTCAAAGGTGATGTCAACGGCCATAAGTT

TTCGGTTCGTGGCAAAGGTAAAGGTGATGCGACGCGCGGGAAATTAACACTGAA

ATTTATTTGCACAACCGGAAAACTCCCTGTGCCGTGGCCGACTTTGGTGACCACA

TTAACCTATGGTGTTCAATGCTTCTCACGTTATCCGAAGCATATGAAACGTCATG

ATTTTTTCAAATCGGCTATGCCGAAAGGTTACGTCCAGGAGCGCACCATCTCATT

TAAGAAAGACGGTAAGTATAAAACCCGTGCTGAAGTAAAATTCAAAGGACGCAC

CCTGGTGAATCGCATTAAACTGAAAGGTCGTGATTTCAAAGAAAAGGGAAATAT

TTTAGGGCATAAGCTCCGTTATAATTTTAACAGTCATAAGGTGTATATTACCGCT

GATAAACGCAAAAACGGAATCAAAGCGAAATTTAAGATCCGTCATAATGTAAAA

GATGGCTCAGTCCAACTGGCAAAACATTACCAGCAGAATACCCCGATCGGCCGC

GGTCCTGTGCTTCTGCCGGGTAAACACTACTTGTCGACCCGGTCAAAATTGAGTA

AAGATCCGAAGGAAAAGCGTGATCACATGGTCTTGAAGGAATTTGTAACTGCAG

CAGGTATTAAACACGGGCGCAAAGAACGTTACAAATAG(SEQ?ID?NO：13)

With the polynucleotide sequence of above-mentioned sequence homology also within the scope of the present invention.In certain embodiments, polynucleotide sequence comprises one section 50,100 or 150 nucleotide with any one above-mentioned sequence 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% homology.The present invention also comprises the sequence of inserting or lack one or more nucleotide in the above-mentioned sequence.Any polynucleotide sequence with the sudden change shown in any above-mentioned sequence all is considered as a part of the present invention.In certain embodiments, sequence comprises the sudden change as shown in any above sequence more than 2,3,4,5,6,7,8,9,10 or 10.

The present invention also provides to comprise and uses sequence any of the present invention herein that system of the present invention modifies or the carrier (for example, plastid, clay, virus etc.) of any other sequence (DNA or protein).In certain embodiments, carrier comprises element (such as promoter, enhancer, the ribosome bind site etc.) sequence that is applicable to overexpression GFP variant of the present invention in cell.The present invention also comprises the cell that comprises sequence of the present invention or carrier.In certain embodiments, cell transition is expressed variation GFP.Cell can be bacterial cell (for example, Escherichia coli), fungal cell's (for example, pichia pastoris phaff (P.pastoris)), yeast cells (for example, saccharomyces cerevisiae), mammalian cell (for example, Chinese hamster ovary celI) or human cell.

Used system of the present invention to produce the Streptavidin variant.Shown that these variants form the solubility tetramer in conjunction with biotin.The amino acid sequence of this wild type Streptavidin is as follows:

AAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATD

GSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWK

STLVGHDTFTKVKPSAAS(SEQ?ID?NO：XX)

The theoretical net charge of wild type Streptavidin is-4.Use system of the present invention to produce theoretical net charge and be-40 and+52 variant.Even behind the heating variant to 100 ℃, protein remains is solvable.

The amino acid sequence of the Streptavidin variant that has produced comprises:

SAV-NEG40

MGHHHHHHGGAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGDAESEYVLT

GRYDSAPATDGSGTALGWTVAWKNDYENAHSATTWSGQYVGGAEARINTQWLLT

SGTTEADAWKSTLVGHDTFTKVEPSAAS(SEQ?ID?NO：XX)

SAV-POS52

MGHHHHHHGGAKAGITGTWYNQLGSTFIVTAGAKGALTGTYESAVGNAKSRYVLT

GRYDSAPATKGSGTALGWTVAWKNKYRNAHSATTWSGQYVGGAKARINTQWLLT

SGTTKAKAWKSTLVGHDTFTKVKPSAAS(SEQ?ID?NO：XX)

section

20,30,40,50 or 100 amino acid and any above-mentioned sequence 60%, 70%, 80%, 90%, 95% or 100% homology that comprises is considered as a part of the present invention.In addition, interpolation and deletion mutation body are also contained in the present invention.In certain embodiments, any Streptavidin with the sudden change residue as shown in any above-mentioned sequence is considered as a part of the present invention.In certain embodiments, sequence comprises the sudden change as shown in any above sequence more than 2,3,4,5,6,7,8,9,10 or 10.

Any dna sequence dna of above-mentioned Streptavidin variant of encoding is also included within the scope of the invention.The exemplary dna sequence dna of above-mentioned each variant of coding is as follows:

SAV-NEG40

GGTTCAGCCATGGGTCATCACCACCACCATCACGGTGGCGCCGAAGCAGGTATT

ACCGGTACCTGGTATAACCAGTTAGGCTCAACCTTTATTGTGACCGCGGGAGCGG

ACGGCGCCTTAACCGGTACCTACGAATCAGCTGTAGGTGACGCGGAATCAGAGT

ACGTATTAACCGGTCGTTATGATAGCGCGCCGGCGACTGACGGTAGCGGTACTGC

TTTAGGTTGGACCGTAGCGTGGAAGAATGATTATGAAAACGCACATAGCGCAAC

AACGTGGTCAGGGCAGTACGTTGGCGGAGCTGAGGCGCGCATTAACACGCAGTG

GTTATTAACTAGCGGCACCACTGAAGCTGATGCCTGGAAGAGCACGTTAGTGGG

TCATGATACCTTCACTAAAGTGGAACCTTCAGCTGCGTCATAATAATGACTCGAG

ACCTGCA(SEQ?ID?NO：XX)

SAV-POS52

GGTTCAGCCATGGGTCATCAGCACCACCATCACGGTGGCGCCAAAGCAGGTATT

ACCGGTACCTGGTATAACCAGTTAGGCTCAACCTTTATTGTGACCGCGGGAGCGA

AAGGCGCCTTAACCGGTACCTACGAATCAGCTGTAGGAAACGCAAAATCACGCT

ACGTATTAACCGGTCGTTATGATAGCGCGCCGGCGACTAAAGGTAGCGGTACTG

CTTTAGGTTGGACCGTAGCGTGGAAGAATAAGTATCGTAATGCGCACAGTGCTAC

CACTTGGTCAGGGCAGTACGTAGGGGGAGCCAAAGCACGTATCAACACGCAGTG

GTTATTAACATCAGGTACCACCAAAGCGAAAGCCTGGAAGAGCACGTTAGTGGG

TCATGATACCTTCACTAAAGTGAAACCTTCAGCTGCGTCATAATAATGACTCGAG

ACCTGCA(SEQ?ID?NO：XX)

The present invention also provides to comprise and uses sequence any of the present invention herein that system of the present invention modifies or the carrier (for example, plastid, clay, virus etc.) of any other sequence (DNA or protein).In certain embodiments, carrier comprises element (such as promoter, enhancer, the ribosome bind site etc.) sequence that is applicable to overexpression Streptavidin variant of the present invention in cell.The present invention also comprises the cell that comprises sequence of the present invention or carrier.In certain embodiments, cell transition is expressed the variation Streptavidin.Cell can be bacterial cell (for example, Escherichia coli), fungal cell's (for example, pichia pastoris phaff), yeast cells (for example, saccharomyces cerevisiae), mammalian cell (for example, Chinese hamster ovary celI) or human cell.

Used system of the present invention to produce glutathione-S-transferase (glutathione-S-transferase, variant GST).Shown that these variants keep the catalytic activity of wild type GST.The amino acid sequence of this wild type GST is as follows:

MGHHHHHHGGPPYTITYFPVRGRCEAMRMLLADQDQSWKEEVVTMETWPPLKPSC

LFRQLPKFQDGDLTLYQSNAILRHLGRSFGLYGKDQKEAALVDMVNDGVEDLRCKY

ATLIYTNYEAGKEKYVKELPEHLKPFETLLSQNQGGQAFVVGSQISFADYNLLDLLRI

HQVLNPSCLDAFPLLSAYVARLSARPKIKAFLASPEHVNRPINGNGKQ(SEQ?ID?NO：

XX)

The theoretical net charge of wild type GST is+2.Use system of the present invention to produce theoretical net charge and be-40 variant.This variant catalysis glutathione is added into dinitrofluorobenzene, and its specific activity only is 1/2.7 of wild type GST.Even behind the heating variant to 100 ℃, protein remains is solvable, and protein recovers its catalytic activity of 40% when cooling.

The amino acid sequence of GST variant comprises:

GST-NEG40

MGHHHHHHGGPPYTITYFPVRGRCEAMRMLLADQDQSWEEEVVTMETWPPLKPSC

LFRQLPKFQDGDLTLYQSNAILRHLGRSFGLYGEDEEEAALVDMVNDGVEDLRCKY

ATLIYTDYEAGKEEYVEELPEHLKPFETLLSENEGGEAFVVGSEISFADYNLLDLLRIH

QVLNPSCLDAFPLLSAYVARLSARPEIEAFLASPEHVDRPINGNGKQ(SEQ?ID?NO：

XX)

GST-POS50

MGHHHHHHGGPPYTITYFPVRGRCEAMRMLLADQKQSWKEEVVTMKTWPPLKPSC

LFRQLPKFQDGKLTLYQSNAILRHLGRSFGLYGKKQKEAALVDMVNDGVEDLRCKY

ATLIYTKYKAGKKKYVKKLPKHLKPFETLLSKNKGGKAFVVGSKISFADYNLLDLLR

IHQVLNPSCLKAFPLLSAYVARLSARPKIKAFLASPEHVKRPINGNGKQ(SEQ?ID?NO：

XX)

section

Any dna sequence dna of above-mentioned GST variant of encoding is also included within the scope of the invention.The exemplary dna sequence dna of above-mentioned each variant of coding is as follows:

GST-NEG40

GGTTCAGCCATGGGTCATCACCACCACCATCACGGTGGCCCGCCGTACACCATTA

CATACTTTCCGGTACGTGGTCGTTGTGAAGCGATGCGTATGTTATTAGCGGACCA

GGACCAATCATGGGAAGAAGAAGTAGTGACAATGGAAACCTGGCCGCCGTTAAA

GCCTAGCTGTTTATTCCGTCAATTACCGAAGTTTCAGGATGGTGATTTAACCTTAT

ACCAGTCTAACGCGATCTTACGTCATTTAGGTCGCTCATTTGGTTTATACGGTGA

AGATGAAGAAGAAGCAGCCTTAGTGGATATGGTGAATGATGGCGTGGAAGACTT

ACGTTGTAAATACGCGACGTTAATTTACACTGATTATGAAGCCGGTAAAGAGGA

GTACGTGGAAGAATTACCTGAACACCTGAAGCCGTTTGAAACATTACTGAGCGA

AAATGAAGGAGGTGAGGCGTTCGTAGTTGGTAGCGAAATTAGCTTCGCTGATTAT

AACTTATTAGACTTATTACGCATTCACCAGGTTTTAAATCCTAGCTGTTTAGACGC

TTTCCCGTTACTGAGCGCATATGTAGCGCGCCTGAGCGCCCGTCCGGAAATTGAA

GCTTTCTTAGCGTCACCTGAACACGTAGACCGCCCGATTAACGGAAACGGCAAG

CAGTAATAATGAGGTACCACCTGCA(SEQ?ID?NO：XX)

GST-POS50

GGTTCAGCCATGGGTCATCACCACCACCATCACGGTGGCCCGCGGTACACCATTA

CATACTTTCCGGTACGTGGTCGTTGTGAAGCGATGCGTATGTTATTAGCGGACCA

GAAACAATCATGGAAAGAAGAAGTAGTGACAATGAAGACCTGGCCGCCGTTAAA

GCCTAGCTGTTTATTCCGTCAATTACCGAAGTTTCAGGATGGTAAATTAACCTTAT

ACCAGTCTAACGCGATCTTACGTCATTTAGGTCGCTCATTTGGTTTATACGGTAA

GAAGCAGAAAGAAGCAGCCTTAGTGGATATGGTGAATGATGGCGTGGAAGACTT

ACGTTGTAAATACGCGACGTTAATTTACACTAAATATAAAGCCGGTAAAAAGAA

GTACGTGAAAAAATTACCTAAACACCTGAAGCCGTTTGAAACATTACTGAGCAA

AAATAAAGGAGGTAAGGCGTTCGTAGTTGGTAGCAAGATTAGCTTCGCTGATTAT

AACTTATTAGACTTATTACGCATTCACCAGGTTTTAAATCCTAGCTGTTTAAAGGC

TTTCCCGTTACTGAGCGCATATGTAGCGCGCCTGAGCGCCCGTCCGAAGATCAAA

GCTTTCTTAGCGTCACCTGAACACGTGAAGCGCCCGATTAACGGAAACGGCAAG

CAGTAATAATGAGGTACCACCTGCA(SEQ?ID?NO：XX)

The present invention also provides to comprise and uses sequence any of the present invention herein that system of the present invention modifies or the carrier (for example, plastid, clay, virus etc.) of any other sequence (DNA or protein).In certain embodiments, carrier comprises element (such as promoter, enhancer, the ribosome bind site etc.) sequence that is applicable to overexpression GST variant of the present invention in cell.The present invention also comprises the cell that comprises sequence of the present invention or carrier.In certain embodiments, cell transition is expressed variation GST.Cell can be bacterial cell (for example, Escherichia coli), fungal cell's (for example, pichia pastoris phaff), yeast cells (for example, saccharomyces cerevisiae), mammalian cell (for example, Chinese hamster ovary celI) or human cell.

The present invention also comprise be used to modify the protein of paying close attention to produce the kit of more stable protein variants.These kits generally include required all of the more stable variant that produces protein or most of reagent.In certain embodiments, kit comprises the computer software of support study dies person based on the more stable variant protein matter of the inventive method design.Kit also can comprise following all or part: reagent, primer, oligonucleotides, nucleotide, enzyme, damping fluid, cell, nutrient culture media, culture plate, test tube, instructions, carrier etc.Use the research of this kit to be provided for usually suddenling change to produce the dna sequence dna of more stable variant.These inclusions of conventional packing are so that use in the laboratory.

After considering that hereinafter example is partly, will further understand these and other aspect of the present invention, these examples are intended to illustrate some specific embodiment of the present invention, but do not plan restriction as the defined scope of the present invention of claims.

Example

Example 1-makes the super charged great restoring force (Resilience) of giving of protein

Protein aggregation, be well-known reason of human diseases (examine the Chinese (Cohen, F.E.); Kai Li (Kelly, J.W.), nature (Nature) 2003,426, (6968), 905-9; Chi Di (Chiti, F.); Many uncles gloomy (Dobson, C.M), biological chemistry yearbook (Annu Rev Biochem) 2006,75,333-66; Be incorporated herein by reference separately), also be to use protein as treatment or subject matter that diagnosticum faced (Fu Luokejie (and Frokjaer, S.); Ao Tezhen (Otzen, D.E.), comment naturally: drug discovery (Nat Rev Drug Discov) 2005,4, (4), 298-306; The method Waller (Fowler, S.B.); Pu En (Poon, S.); Mo Fo (Muff, R.); Chi Di (Chiti, F.); Many uncles gloomy (Dobson, C.M.); Help many (Zurdo, J.), institute of NAS periodical (Proc Natl Acad Sci USA) 2005,102, (29), 10105-10; Be incorporated herein by reference separately).From to obtaining understanding the research of native protein to protein aggregation.We know, protein is solubility minimum (Li Bai (Loeb, J.), general physiology magazine (J Gen Physiol) 1921,4,547-555 is incorporated herein by reference) when carrying the isoelectric point of zero net charge.Recently, the fine difference (± 3 unit of charge) that has shown net charge can predict between the globular preteins variant (Chi Di (and Chiti, F.); Si Taifani (Stefani, M); Thailand wear (Taddei, N.); The general Buddhist nun of rem (Ramponi, G.); Between gloomy (Dobson, C.M.), nature (Nature) 2003,424, (6950), 805-8 is incorporated herein by reference) of many uncles and intrinsic unordered peptide (Pa Wa (Pawar, A.P.); Superintend and direct and visit (Dubay, K.F.); Help many (Zurdo, J.); Chi Di (Chiti, F.); Literary virtue Si Keluo (Vendruscolo, M.); Many uncles' gloomy (Dobson, C.M), molecular biology magazine (J Mol Biol) 2005,350, (2), 379-92 is incorporated herein by reference) gathering tendency.The nearest evidence that these observationss can be stood the net charge marked change together with some protein (for example; find that carbonic anhydrase keeps catalytic activity (ancient Dixon people such as (Gudiksen) after the thorough chemical acetylation of its surperficial lysine; JACS (J Am Chem Soc) 2005; 127; (13); 4707-14; be incorporated herein by reference)) together, we are inferred by the surface is suddenlyd change on a large scale may obviously strengthen the dissolubility of some protein and anti-aggregation and not eliminate its folding or function with remarkable increase net charge (we are referred to as the process of " super charged " in this article).

We with nearest report aspect folding efficiency and the denaturant resistance the prior art variant of the green fluorescent protein of height optimization (GFP) (being called " super folding GFP " (sfGFP)) (send moral clarke people such as (Pedelacq), country's biotechnology (Nat Biotechnol) 2006,24, (1), 79-88 is incorporated herein by reference) beginning.The net charge of super folding GFP is-7, and GFP is similar with wild type.According to calculating the simple algorithm (referring to " material and method " part) that amino acid whose solvent exposes, the amino acid that we sport positively charged by (most solvent-exposed) residue that 29 solvents are exposed is designed theoretical net charge and is+36 the super charged variant of GFP (Fig. 1).The expression of gene of coding sfGFP or GFP (+36) produces the green fluorescence bacterium densely.Behind the protein purification, the photoluminescent property of measurement GFP (+36) and the photoluminescent property of discovery and sfGFP are very similar.Encouraged by this discovery, our design and purifying net charge be other super charged GFP of+48 ,-25 and-30, (Fig. 2 is a) for the same fluorescence of finding the similar sfGFP of whole demonstrations.All super charged GFP variants show the circulr dichromism spectrum that is similar to sfGFP, show that protein has similar secondary structure content (Fig. 2 b).Though there is nearly 36 places sudden change in super charged GFP variant, its thermodynamic stability is only than sfGFP low slightly (1.0-4.1kcal/mol, Fig. 2 c and table 1).

Though sfGFP is time-honored product (graceful this people such as (Giepmans) of jeep, science (Science) 2006,312, (5771), 217-24 that GFP optimizes; Be incorporated herein by reference), but it still is subjected to heat or chemistry easily and separates and folding induce and assemble.Heat sfGFP to 100 ℃ induce its quantitatively precipitation and the irreversible forfeiture of fluorescence (Fig. 3 a).On the contrary, super charged GFP (+36) and GFP (30) are still solvable when being heated to 100 ℃, and (Fig. 3 a) and tangible fluorescence is recovered in the cooling back.Importantly, 40% 2,2,2 tfifluoroethyl alcohol (TFE) just induces sfGFP to assemble fully in 25 ℃ of following a few minutes, and+36 and-30 super charged GFP variant under the same conditions a few hours all experience significantly assemble or fluorescence forfeiture (Fig. 3 b).

Except that this significant anti-aggregation, super charged GFP variant shows that the highly charged big molecule with opposite charges is had powerful reversible affinity (Fig. 3 c).When GFP (+36) and GFP (30) mix with the 1:1 stoichiometry, form the green fluorescence co-precipitation immediately, show that unfolded protein associates.GFP (+36) similarly with high concentration RNA or DNA co-precipitation.Adding NaCl is enough to dissolve these compounds, and this point is consistent with the static basis of its formation.On the contrary, sfGFP is not added GFP (30), RNA or DNA influence (Fig. 3 c).

We need then to determine whether super charged principle may be applicable to the protein except that GFP, and GFP is monomer and has the good fluorophore of shielding.For this purpose, we use super charged process to two kinds of protein that have nothing to do with GFP.Streptavidin is that total net charge is-4 the tetramer.Use solvent to expose algorithm, we design two kinds of net charges for-40 or+52 super charged Streptavidin variant.Two kinds of super charged Streptavidin variants can both form the solubility tetramer in conjunction with biotin, but affinity reduces.

Make the super charged net charge that produces of glutathione-S-transferase (GST) (total net charge for+2 dimer) be-40 dimer, its catalysis glutathione is to the addition of dinitrofluorobenzene, and specific activity only is 1/2.7 (Fig. 3 d) of wild type GST.In addition, super charged Streptavidin and super charged GST are still solvable when being heated to 100 ℃, and this is opposite with its wild type counterparts, and the latter is similar to sfGFP quantitative and irreversible precipitation (table 1) takes place.In addition, 40% (Fig. 3 d) of its catalytic activity recovered in GST (40) cooling back.

In a word, we are verified can be by making monomer and the polyprotein " super charged " with different structure and function with the residue that exposes with the most of solvents of the amino acid replacement of similar electric charge simply.The super charged intermolecular character that can change protein greatly, the ability of giving tangible anti-aggregation and associating with the big molecule (as " molecule Velcro (molecular Velcro) ") of folded form and oppositely charged.We notice that these uncommon intermolecular character are to come from high net charge, rather than come from charged amino acid whose sum, and charged amino acid whose sum does not obviously change (table 1) because of super charged process.

Opposite with these significant intermolecular effects, character in the molecule of Yan Jiu 7 kinds of super charged protein (comprise folding, fluorescence, part in conjunction with and enzyme-catalyzed change) is still intact basically herein.Therefore, super charged may be that a kind of the reduction assembles tendency and improve protein solubility and do not eliminate the effective ways of its function.These principles may be particularly useful for the design effort of novelty (de novo) protein, are still a significant challenge comprising the uncertain protein handling properties in accumulating in.According to making the super charged The above results of native protein, the anti-aggregation that people can't help inferring designed protein also may improve in the locational frequency that prediction is positioned at the unfolded protein outside by the amino acid that design process is partial to increase with similar electric charge.

The remarkable plasticity of the super charged explanation protein surface of protein and the outstanding chance that comes from the sudden change tolerance (mutational tolerance) of solvent exposed residue.For instance, the thermodynamic stability that shows some protein recently can strengthen (this Qu Kele people such as (Strickler), biological chemistry (Biochemistry) 2006 by engineered electric charge-charge interaction reasonably, 45, (9), 2761-6 is incorporated herein by reference).Thereby how this plasticity of the super charged proof of protein can be used in a different manner and give extremely strong protein aggregation resistance.Our discovery is consistent with the result of Supplementary Study, in Supplementary Study, remove all electric charges from ubiquitin, its folding property remain intact and its dissolubility obviously impaired (Laura is people such as (Loladze) now, protein science (Protein Sci) 2002,11, (1), 174-7 is incorporated herein by reference).

These observationss also can illustrate appropriateness (modest) net charge of native protein distribute (Ka Naite (Knight) people of etc.ing., institute of NAS prints (Proc Natl Acad Sci USA) 2004,101, (22), 8390-5; Ji Telin people such as (Gitlin), German applied chemistry magazine (Angew Chem Int Ed Engl) 2006,45, (19), 3022-60 is incorporated herein by reference separately): for example, the net charge of 84% Protein Data Bank (PDB) polypeptide is in ± 10 scopes.Thereby our result and high net charge produce enough electrostatic repulsions and force and separate folding hypothesis and conflict mutually.Really, GFP (+48) has the positive net charge of any polypeptide that is higher than among the present PDB, but still keeps folding and fluorescigenic ability.In fact, adhere to the evolution of the too many high electric charge native protein of possibility disapprove between our discovery prompting non-specific molecules.Nearly all native protein with high net charge (such as in conjunction with the ribosomal protein L 3 (+36) of RNA and L15 (+44) or in conjunction with the cationic calsequestrin of calcium (80)) all material with oppositely charged associates, as its must cell function a part.

Material and method

Design program and super charged protein sequence: from disclosed structured data differentiate AvNAPSA＜150 (wherein AvNAPSA be each side chain atom average adjacent atom (

In the scope)) solvent exposed residue (following show) (weber (Weber with grey, P.C.), Orlando husband (Ohlendorf, D.H.), Wen Duoluosiqi (Wendoloski, J.J.) and Sha Lemai (Salemme, F.R.), the structure cause of the high-affinity biotin that combines with Streptavidin (Structural origins of high-affinity biotin binding to streptavidin). science (Science) 243,85-88 (1989); Deere (Dirr, H.), Rui Namo (Reinemer, P.) and recklessly primary (Huber, R.) the refinement crystal structure of Swine π glutathione S-transferase (pGST PI-1) (Refined crystal structureof porcine class Pi glutathione S-transferase (pGST PI-1) at 2.1 under 2.1 resolution

Resolution), molecular biology magazine (J Mol Biol) 243,72-92 (1994); General moral clarke (Pedelacq, J.D.), Ka Bantesi (Cabantous, S.), bent grace (Tran, T.), Te Weierge (Terwilliger, T.C.) and Wa Erduo (Waldo, G.S.) the engineered and sign (Engineering and characterization of a superfoldergreen fluorescent protein) of super folding green fluorescent protein. Nature Biotechnol (Nat Biotechnol) 24,79-88 (2006) is incorporated herein by reference separately).The solvent exposed residue (DERKNQ) of charged or the high polarity of suddenling change:, sport Asp or Glu for super electronegative (redness); Or, sport Lys or Arg for super positively charged (blueness).Other surperficial exposure position to be suddenlyd change in green fluorescent protein (GFP) variant is selected according to the sequence variations of these positions in the GFP homologue.The super charged design process of Streptavidin (SAV) and glutathione-S-transferase (GST) is full automatic: at first expose the sorting residue by solvent, the charged or high polar residues that the solvent that suddenlys change then exposes: for super positively charged, sport Lys; Or, sport Glu (, under this situation, sporting Asp) unless the beginning residue is Asn for super electronegative.

Protein expression and purifying: buy at e. coli codon from DNA 2.0 and to use the synthetic gene be optimized, be cloned into pET expression vector (the outstanding company (Novagen) of Nova) and under 15 ℃ in e. coli bl21 (DE3) pLysS overexpression 5-10 hour.Dissolve by centrifugal collecting cell and by the ultrasound wave effect.(Kai Jie company (Qiagen) protein purification changes damping fluid into 100mM NaCl, 50mM potassium phosphate (pH7.5), and concentrates by ultrafiltration (Millipore Corp. (Millipore)) by the Ni-NTA agarose chromatography.All GFP variants of purifying under natural condition.Buy the wild type Streptavidin from Pu Luomaige company (Promega).Under the sex change condition the super charged Streptavidin variant of purifying and as before about the wild type Streptavidin report carry out refolding (Sai Mupusen people such as (Thompson). synthesize structure and the expression (Construction and expression of a synthetic streptavidin-encoding gene in Esche richia coli) of Streptavidin encoding gene in Escherichia coli. gene (Gene) 136,243-246 (1993), be incorporated herein by reference), super charged GST also is like this.Purifying wild type GST under natural or sex change condition, but the protein of specific activity had.

The surface electrostatic gesture is calculated (Fig. 1 b) :-30 and+model of 48 super charged GFP variant is based on the crystal structure of super folding GFP (general moral clarke people such as (Pedelacq), engineered and the sign (Engineering and characterization of a superfolder green fluorescent protein) of super folding green fluorescent protein. Nature Biotechnol (Nat Biotechnol) 24,79-88 (2006) is incorporated herein by reference).Use-25kT/e (redness) is to the scale of+25kT/e (blueness), use APBS (Bake people such as (Baker), the electrostatics of nanosystems: to tubulin and ribosomal application (Electrostatics of nanosystems:application to microtubules and theribosome). periodical (the Proc Natl Acad Sci USA) 98 of institute of NAS, 10037-10041 (2001), be incorporated herein by reference) calculate electrostatic potential and use PyMol (Draenor (Delano, W.L), PyMOL molecule drawing system (The PyMOL Molecular Graphics System) Www.pymol.org(2002), be incorporated herein by reference) performance.

Protein staining and UV light-induced fluorescence are (Fig. 2 a): each GFP variant by electrophoretic analysis 0.2 microgram in 10% denaturing polyacrylamide gel and with Coomassie brilliant blue (Coomassie brilliant blue) dyeing.In 0.2mL Chinese mugwort Bender pipe (Eppendorf tube), put into 0.2 microgram in the same protein quality sample of the 25mM Tris (pH8.0) with 100mM NaCl and under ultraviolet light (360nm), take pictures.

Thermal denaturation and gathering are (Fig. 3 a): pure GFP variant diluted in 25mM Tris (pH8.0), 100mM NaCl and 10mM beta-mercaptoethanol (BME) be 2mg/mL, take pictures then (" natural ") under UV-irradiation.Heated sample to 100 ℃ continues 1 minute, and then take pictures under UV-irradiation (" boiling ").At last, at room temperature cool off sample 2 hours and under UV-irradiation, take pictures once more (" cooling ").

The gathering of chemical induction (Fig. 3 b): add 2,2,2 tfifluoroethyl alcohol (TFE) has 1.5mg/mL protein, 25mM Tris (pH7.0), 10mM BME and 40% TFE with generation solution.Assemble by right angle light scattering monitoring down at 25 ℃.

Size exclusion chromatography (SEC) (table 1): by on Xiu Podaikesi 75 (Superdex 75) solvent resistant column, analyzing the poly state that the 20-50 micrograms of protein is determined SAV and GST variant.Damping fluid is 100mM NaCl, 50mM potassium phosphate (pH7.5).By with the one group of monomeric protein reference material with known molecular amount of separate analysis definite molecular weight of making comparisons under the same conditions.

Table 1: the protein properties definite of calculating with experiment

Title MW length n _Justn _Negativen _ChargedQ _OnlyThe natural MW of pI Δ G (kD) ^bAfter boiling

(kD) (aa) (kcal/mol) ^aSolvable

Percent ^c

GFP(-30) 27.8248?19?49?68?-30?4.8 10.2 n.d. 98

GFP(-25) 27.8 248 21 46 67 -25 5.0 n.d. n.d. n.d.

sfGFP 27.8 248 27 34 61 -7 6.6 11.2 n.d. 4

GFP(+36) 28.5 248 56 20 76 +36 10.4 8.8 n.d. 97

GFP(+48) 28.6 248 63 15 78 +48 10.8 7.1 n.d. n.d.

SAV (40) 14.3 137 51 5 20-10 5.1 n.d. 55 ± 5 (tetramer) 99

WtSAV 13.3 128 89 17-1 6.5 n.d. 50 ± 5 (tetramer) 7

SAV (+52) 14.5 137 16 3 19+13 10.3 n.d. 55 ± 5 (tetramer) 97

GST (40) 24.7 217 17 37 54-20 4.8 n.d. 50 ± 5 (dimer) 96

WtGST 24.6 217 24 23 47+1 7.9 n.d. 50 ± 5 (dimer) 3

GST(+50)d 24.7 217 39 14 53 +25 10.0 n.d. n.d. n.d.

n _JustAmino acid whose number (each monomer) for positively charged

n _NegativeBe electronegative amino acid whose number

n _ChargedBe charged amino acid whose sum

Q _OnlyBe the theoretical net charge under the pH neutral

PI is for calculating isoelectric point

N.d. represent that undetermined arrives

^aMeasure (Fig. 2 c) by the guanidinesalt sex change.

^bMeasure by size exclusion chromatography (SEC).

^c100 ℃ following 5 minutes, be cooled to the protein percent that is kept in 25 ℃ and the of short duration centrifugal back supernatants.

^dFail protein at expression in escherichia coli.

Other embodiment

One of ordinary skill in the art should easily understand aforementioned content and only represent some preferred embodiment of the present invention.Under the situation of the spirit or scope of the present invention of in not breaking away from the claims of enclosing, being stated, can carry out multiple change and correction to said procedure and composition.

Appendix A

#！/usr/local/bin/perl

####################################################################

#

#?avnapsa

#

#?prints?list?of?AvNAPSA?values?for?the?specified?PDB

#

#?Mike?Lawrence/Kevin?Phillips?3/17/2006

#

####################################################################

sub?show_usage

{

print＂\n＂，

＂Usage：avnapsa<start_pdb>[params]\n＂，

＂-3?use?3-letter?aa?abbreviations(default)\n＂，

＂-1?use?1-letter?aa?abbreviations\n＂，

＂-onecol?print?one?column?only(i.e.only?the?AvNAPSA?results)\n\n＂；

}

#################global?variables################################

@atoms；

#?fields?loaded?from?PDB：

#?type

#?atomNum

#?atomName

#?resName

#?chain

#?resNum

#?x，y，z

#?computed?fields

#?neighborCount

@distances；

@residues；

#?fields?copied?from?PDB

#?resNum(PDB?numbering)

#?resName

#?computed?fields

#?avNapsa

######################################################################

##?parse?command?line

Suse3or1＝3；

Sonecol_flag＝0；

Sstart_pdb＝SARGV[0]；

for(my?$a＝1；$a<@ARGV；++$a)

{

if($ARGV[$a]eq＂-1＂){$use3or1＝1；}

elsif($ARGV[$a]eq＂-3＂){$use3or1＝3；}

elsif(SARGV[$a]eq＂-onecol＂){$onecol_flag＝1；}

else{show_usage()；die＂Invalid?argument?$ARGV[$a]\n＂；}

}

unless(lc?$start_pdb＝～/\.pdb/){show_usage()；die＂No?starting?pdb

specified.\n＂；}

##?read?PDB?and?compute?molecular?parameters

read_PDB($start_pdb)；

tabulate_residues()；

$nres＝@residues；

compute_distances()；

compute_neighbor_counts()；

compute_residue_avNapsa()；

print_residues()；

exit；

#

#?print_res?idues

#

sub?print_residues

{

for?{my?$r＝0；$r<@residues；$r++)

{

my?$name＝$residues[$r]{resName}；

Sname＝toggle31(Sname)if(Suse3or1＝＝1)；

printf?＂％d?％s?AvNAPSA＂，$residues[$r]{resNum}，$name?unless

$onecol_flag；

printf＂％.of\n″，$residues[$r]{avNapsa}；

}

print＂\nNum?residues＝＂，$#residues+1，＂\n\n＂unless?$onecol_flag；

}

#

#?tabulate_residues

#

#?goesthrough?listof?atoms?andmakes?a?list?of?aminoacid?residues

#?and?stores?it?in?global?variable?@residues

#

sub?tabulate_residues

{

for(Sa＝0；$a<@atoms；$a++)

{

$resNum＝$atoms[$a]{resNum}；

if?(！resNum_exists($resNum))

{

push?@residues，

{

resNum?=>$resNum，

resName＝>$atoms[$a]{resName}

}；

}

#

#?resNum_exists

#

#?returns?1?if?resNum?is?contained?in?@residues

#

sub?resNum_exists($)

{

my?($resNum)＝@_；

for?($r＝0；$r<@residues；$r++)

{

return?1?if($residues[$r]{resNum}＝＝$resNum)；

return?0；

}

#

#?resNum_to_resindex

#

#?converts?PDB?numbering?to?index?in?@residues

#

sub?resNum_to_resindex($)

{

my(SresNum)＝@_；

for($r＝0；$r<@residues；$r++)

{

return?$r?if($residues[$r]{resNum}＝＝$resNum)；

}

return＂none＂；

}

#

#?readPDB(filename)

#

#?reads?the?atoms?from?a?PDB?and?returns?them?as?anarray?of?hashes

#

sub?read_PDB($)

{

my($filename)＝@_；

open(PDB，$filename)or?die(＂Could?not?open?$filename\n＂)；

$#atoms=-1； #?clear?atoms?storage

#?read?the?file

foreach(<PDB>){

my?$type＝trim{substr($_，0，6))； #?RTyp?field?is?columns?1-6

next?unless($type?eq＂ATOM＂||$type?eq＂HETATM＂)；

my?$resName＝trim(substr{$_，17，3))； #?Res?field?is?columns?18-

20

my?$atomName＝trim(substr($_，12，4))； #?Atm?field?is?columns

13-16

next?if?uc?$resName?eq＂HOH＂； #?omit?waters

next?if?uc?$atomName＝-/^[0-9]＊H/； #?omit?protons

#?add?a?hash?to?the?array，containing?data?fromthis?record?of?the?PDB

push?@atoms，{

type?=> $type，

resName?=> $resName，

atomName?=>?$atomName，

atomNum?=> trim(substr($_，6，5))，?#?Num?field?is?columns?7-11

chain?=> trim(substr($_，21，1))，#?Chain?field?is?column?22

resNum?=> trim(substr($_，22，4))，#?ResNo?field?is?columns?23-26

x?=> trim(substr($_，30，8))，#?X?field?is?columns?31-38

y?=> trim(substr($_，38，8))，#?Y?field?is?columns?39-46

z?=> trim(substr{$_，46，8)) #?Z?field?is?columns?37-54

}；

}

close(PDB)；

}

#

#?trim

#

#?removes?whitespace?from?start?and?end?of?string

#

sub?trim($)

{

my($string)＝@_； #?retrieve?the?passed?argument

$string＝-s/^\s+//； #?remove?leading?whitespace

$string＝～s/\s+$//； #?remove?trailing?whitespace

return?$string；

}

#

#?is_number

#

#?returns?1?if?passed?argumentis?a?number(allows?whitespace，negative，and

decimal?point)

#?returns?0?if?passed?argument?isblankor?not?a?number

#

sub?is_number($)

{

$_＝shift；

s/^\s+//；

s/\s+$//；

return?1?if/^-？[0-9]+$/||/^-？[0-9]＊\.[0-9]+$/||/^-？[0-9]+\.[0-9]＊$/；

return?0；

}

#

#?inter_residue_distance

#

#?returns?the?minimum?distance?between?any?atoms?of?the?specified?residues

#?(residues?are?specified?according?to?index?in?@residues)

#

sub?inter_residue_distance($，$)

{

my($r1，$r2)＝@_；

##?convert?to?PDB?numbering

my?$resNum1?＝?$residues{$r1}{resNum}；

my?$resNum2?＝?$residues{$r2}{resNum}；

my?$min_dist?＝?1000000；

for?($al＝0；$al<@atoms；++$al)

{

next?unless($atoms[$al]{resNum}＝＝$resNuml)；

for?($a2＝0；$a2<@atoms；++$a2)

{

next?unless($atoms[$a2]{resNum}＝＝$resNum2)；

my?$dist＝$distances[$a1][$a2]；

$min_dist＝$dist?if($dist<$min_dist)；

}

return?$min_dist；

}

#

#?compute_distances

#

#?computes?the?distances?between?all?atoms

#

sub?compute_distances

{

for(my?$atoml＝0；$atoml<@atoms；$atoml++)

{

for(my?$atom2＝$atoml；$atom2<@atoms；$atom2++)

{

my($x1，$y1，$z1)＝($atoms[$atoml]->{x}，$atoms[$atoml]->{y}，

$atoms[$atoml]->{z})；

my($x2，$y2，$z2)＝($atoms[$atom2]->{x}，$atoms[$atom2]->{y}，

$atoms[$atom2]->{z})；

my?$distance＝sqrt(($x1-$x2)＊＊2+($y1-$y2)＊＊2+($z1-$z2)＊＊2)；

$distances[$atoml][$atom2]＝$distance；

$distances[$atom2][$atoml]＝$distance；

}

#

#?compute_neighbor_counts

#

#?computes?the?number?of?neighbors?that?each?atom?has.

#?paramter?is?the?cutoff，in?Angstroms，for?atomic?neighborhood

#

sub?compute_neighbor_counts

{

$DISTANCE_CUTOFF＝10； #?criterion?for?neighborhood，in?Angstroms

for?($atoml＝0；$atoml<@atoms；$atoml++)

{

my?$count＝0；

for?($atom2＝0；$atom2<@atoms；$atom2++)

{

$count++?if($distances[$atoml][$atom2]<＝$DISTANCE_CUTOFF

&&?$atoml?|＝$atom2)；

}

$atoms[$atoml]{neighborCount}＝$count；

}

#

#?compute_residue_avNapsa

#

#?for?each?residue，compute

#?Average?Neighbor?Atoms?Per?Sidechain?Atom(AvNAPSA)

#?(sidechain?atoms?are?all?those?except?N，C，O，CA)

#?for?glycines，just?use?CA

#

sub?compute_resi?due_avNapsa

{

for?(my?$r＝0；$r<@residues；$r++)

{

my?$numSideChainAtoms＝0；

my?$totalNeighbors＝0；

my?$resName＝$residues[$r]{resName}；

my?$resNum＝$residues[$r]{resNum}；

for?(my?$a＝0；$a<@atoms；$a++>

{

if?($atoms[$a]{resNum}＝＝$resNum)

{

my?$atomName＝$atoms[$a]{atomName}；

if?(

( $atomName?ne＂C＂

&&?$atomName?ne＂O＂

&&?$atomName?ne＂N＂

&&?$atomName?ne＂CA＂

)

||($atomName?eq＂CA＂&&?$resName?eq＂GLY＂)

}

{

$numSideChainAtoms++；

$totalNeighbors+＝$atoms[$a]{neighborCount}；

}

my?$avNapsa＝$totalNeighbors/$numSideChainAtoms；

$residues[$r]{avNapsa}＝$avNapsa；

}

#

#?toggle?31

#

#?converts?3-letter?abbrev?to?1-letter

#?or?1-letter?abbrev?to?3-letter

#

sub?toggle?31($)

{

％conv3to1＝(＂ALA＂＝>＂A＂，＂CYS＂＝>＂C＂，＂SER＂＝>＂S＂，＂LEU＂＝>＂L＂，

＂ILE＂＝>＂I＂，＂PHE＂＝>＂F＂，＂ARG＂＝>＂R＂，＂ASN＂＝>＂N＂，＂GLN＂＝>＂Q＂，

＂TYR＂＝>＂Y＂，＂LYS＂＝>＂K＂，＂ASP＂＝>＂D＂，＂GLU＂＝>＂E＂，＂VAL＂＝>＂V＂，

＂TRP＂＝>＂W＂，＂MET＂＝>＂M＂，＂HIS＂＝>＂H＂，＂GLY＂＝>＂G＂，＂PRO＂＝>＂P＂，

＂THR＂＝>＂T＂)；

％convlto3＝reverse?％conv3to1；

my?($abbrev)＝@_；

$abbrev＝uc?$abbrev；

return?$convlto3{$abbrev}if?length($abbrev)＝＝1；

return?$conv3to1{$abbrev}if?length($abbrev)＝＝3；

die＂in?toggle31()：invalid?amino?acid?abbreviation?$abbrev\n＂；

}

#

#?is_aa

#

#?returns?1?if?passed?argument?is?a?1-letter?amino?acid

#

sub?is_aa($)

{

my?($string)＝@_；

return?1?if(length?toggle31($string)＝＝3)；

return?0；

}

Sequence table

＜110〉The President and Fellows of Harvard College

＜120〉protein surface remodeling

<130>034291-0223(HU?2772)

<140>PCT/US2007/070254

<141>2007-06-01

<160>23

<170>PatentIn?version?3.5

<210>1

<211>238

<212>PRT

＜213〉Victoria jellyfish

<400>1

<210>2

<211>248

<212>PRT

＜213〉artificial sequence

<220>

＜223〉from the finishing green fluorescent protein (GFP) of Victoria jellyfish

<400>2

<210>3

<211>248

<212>PRT

＜213〉artificial sequence

<220>

<400>3

<210>4

<211>248

<212>PRT

＜213〉artificial sequence

<220>

<400>4

<210>5

<211>248

<212>PRT

＜213〉artificial sequence

<220>

<400>5

<210>6

<211>248

<212>PRT

＜213〉artificial sequence

<220>

<400>6

<210>7

<211>248

<212>PRT

＜213〉artificial sequence

<220>

<400>7

<210>8

<211>747

<212>DNA

＜213〉artificial sequence

<220>

<400>8

<210>9

<211>747

<212>DNA

＜213〉artificial sequence

<220>

<400>9

<210>10

<211>747

<212>DNA

＜213〉artificial sequence

<220>

<400>10

<210>11

<211>747

<212>DNA

＜213〉artificial sequence

<220>

<400>11

<210>12

<211>747

<212>DNA

＜213〉artificial sequence

<220>

<400>12

<210>13

<211>747

<212>DNA

＜213〉artificial sequence

<220>

<400>13

<210>14

<211>128

<212>PRT

＜213〉artificial sequence

<220>

＜223〉wild type Streptavidin

<400>14

<210>15

<211>137

<212>PRT

＜213〉artificial sequence

<220>

＜223〉finishing Streptavidin (SAV-NEG40)

<400>15

<210>16

<211>137

<212>PRT

＜213〉artificial sequence

<220>

＜223〉finishing Streptavidin (SAV-POS52)

<400>16

<210>17

<211>442

<212>DNA

＜213〉artificial sequence

<220>

＜223〉finishing Streptavidin (SAV-NEG40)

<400>17

<210>18

<211>442

<212>DNA

＜213〉artificial sequence

<220>

＜223〉finishing Streptavidin (SAV-POS52)

<400>18

<210>19

<211>217

<212>PRT

＜213〉artificial sequence

<220>

＜223〉wild type glutathione-S-transferase (GST)

<400>19

<210>20

<211>217

<212>PRT

＜213〉artificial sequence

<220>

＜223〉finishing glutathione-S-transferase (GST-NEG40)

<400>20

<210>21

<211>217

<212>PRT

＜213〉artificial sequence

<220>

＜223〉finishing glutathione-S-transferase (GST-POS50)

<400>21

<210>22

<211>682

<212>DNA

＜213〉artificial sequence

<220>

＜223〉finishing glutathione-S-transferase (GST-NEG40)

<400>22

<210>23

<211>682

<212>DNA

＜213〉artificial sequence

<220>

＜223〉finishing glutathione-S-transferase (GST-POS50)

<400>23

Claims

1. method that improves the stability of the protein of paying close attention to, described method comprises following steps:

The surface residue of differentiating the protein of paying close attention to is non-high conservative in other protein relevant with the described protein of being paid close attention to; With

Be used in a plurality of non-conservative surface residues of radical amino acid replacement of positively charged under the physiological pH value.

2. method that improves the stability of the protein of paying close attention to, described method comprises following steps:

Be used in a plurality of non-conservative surface residues of electronegative radical amino acid replacement under the physiological pH value.

3. method according to claim 1 and 2, wherein said non-conservative surface residue is hydrophobic.

4. method according to claim 1, wherein said non-conservative surface residue is hydrophilic or electronegative.

5. method according to claim 2, wherein said non-conservative surface residue is hydrophilic or positively charged.

6. method that improves the stability of the protein of paying close attention to, described method comprises following steps:

Differentiate the surface residue of the protein of paying close attention to;

The surface residue of differentiating the described protein of being paid close attention to is non-high conservative in other protein relevant with the described protein of being paid close attention to;

Specify the hydrophobicity value for each described non-conservative surface residue of differentiating; With

Be used at least one surface residue of radical amino acid replacement charged under the physiological pH value, wherein said residue of being replaced all sports the residue of same type, and the residue of wherein said type is positively charged residue or electronegative residue.

7. method according to claim 6, wherein said displacement step comprise at least one hydrophobic surface residue of displacement.

8. method according to claim 6, wherein said displacement step comprise at least one hydrophilic surface residue of displacement.

9. method according to claim 6, wherein said displacement step comprise at least one powered surfaces residue of displacement.

10. method according to claim 6, wherein said displacement step comprise with lysine residue replaces at least one surface residue.

11. comprising with aspartic acid or glutaminic acid residue, method according to claim 6, wherein said displacement step replace at least one surface residue.

12. according to claim 1,2 or 6 described methods, wherein said displacement step comprises at least two surface residues of displacement.

13. according to claim 1,2 or 6 described methods, wherein said displacement step comprises at least five surface residues of displacement.

14. according to claim 1,2 or 6 described methods, wherein said displacement step comprises at least ten surface residues of displacement.

15. according to claim 1,2 or 6 described methods, wherein said displacement step comprises at least two ten surface residues of displacement.

16. according to claim 1,2 or 6 described methods, wherein said displacement step comprises at least three ten surface residues of displacement.

17. according to claim 1,2 or 6 described methods, wherein said method is created in that net charge compares the high modifying protein of being paid close attention to of initial protein under the physiological pH value.

18. according to claim 2 or 6 described methods, wherein said method is created in following electronegative modifying protein of being paid close attention to of Duoing than the initial protein of being paid close attention to of physiological pH value.

19. according to claim 2 or 6 described methods, wherein said method is created in that following of physiological pH value is electronegative lacks-5 the modifying protein of being paid close attention to than the initial protein as many as of being paid close attention to.

20. according to claim 2 or 6 described methods, wherein said method is created in that following of physiological pH value is electronegative lacks-10 the modifying protein of being paid close attention to than the initial protein as many as of being paid close attention to.

21. according to claim 2 or 6 described methods, wherein said method is created in that following of physiological pH value is electronegative lacks-15 the modifying protein of being paid close attention to than the initial protein as many as of being paid close attention to.

22. according to claim 2 or 6 described methods, wherein said method is created in that following of physiological pH value is electronegative lacks-20 the modifying protein of being paid close attention to than the initial protein as many as of being paid close attention to.

23. according to claim 1 or 6 described methods, wherein said method is created in following positively charged modifying protein of being paid close attention to of Duoing than the initial protein of being paid close attention to of physiological pH value.

24. according to claim 1 or 6 described methods, wherein said method is created in that following of physiological pH value is positively charged to be lacked+5 the modifying protein of being paid close attention to than the initial protein as many as of being paid close attention to.

25. according to claim 1 or 6 described methods, wherein said method is created in that following of physiological pH value is positively charged to be lacked+10 the modifying protein of being paid close attention to than the initial protein as many as of being paid close attention to.

26. according to claim 1 or 6 described methods, wherein said method is created in that following of physiological pH value is positively charged to be lacked+15 the modifying protein of being paid close attention to than the initial protein as many as of being paid close attention to.

27. according to claim 1 or 6 described methods, wherein said method is created in that following of physiological pH value is positively charged to be lacked+20 the modifying protein of being paid close attention to than the initial protein as many as of being paid close attention to.

28. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is the protein of easily assembling.

29. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is hydrophobic protein.

30. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is memebrane protein.

31. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is the protein that is difficult to overexpression.

32. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is the protein that is difficult to purifying.

33. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is acceptor.

34. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is transcription factor.

35. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is enzyme.

36. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is structural proteins.

37. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is fluorescin.

38. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is green fluorescent protein (GFP).

39. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is extracellular protein.

40. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is Streptavidin.

41. according to claim 1,2 or 6 described methods, the wherein said protein of paying close attention to is glutathione-S-transferase.

42. according to claim 1,2 or 6 described methods, the step of the described surface residue of wherein said discriminating comprises the three-dimensional structure of the described protein of computer simulation.

43. according to claim 1,2 or 6 described methods, the step of the described surface residue of wherein said discriminating comprises the surface of using the algorithm predicts residue whether to be found in protein.

44. according to claim 1,2 or 6 described methods, the step of the described surface residue of wherein said discriminating comprises differentiates the residue of AvNAPSA value less than threshold value.

45. according to claim 1,2 or 6 described methods, the step of the surface residue of the described non-high conservative of wherein said discriminating comprises the amino acid sequence of comparing described protein and at least one other protein from same protein family.

46. according to claim 1,2 or 6 described methods, the step of the surface residue of the described non-high conservative of wherein said discriminating comprises the amino acid sequence of comparing described protein and at least one other amino acid sequence from the described protein of different plant species.

47. according to claim 45 or 46 described methods, wherein said comparison is carried out with at least two other protein sequences.

48. according to claim 45 or 46 described methods, wherein said comparison is carried out with at least three other protein sequences.

49. according to claim 45 or 46 described methods, wherein said comparison is carried out with at least five other protein sequences.

50. comprising, method according to claim 6, the step of wherein said appointment hydrophobicity value use octanol/water partition factor value.

51. according to claim 1,2 or 6 described methods, wherein said displacement step comprises the sequence of the described protein of mutagenesis and replaces the hydrophobic surface residue of described discriminating to be used in natural amino acid residue charged under the physiological pH value.

52. according to the described method of claim 51, wherein said natural amino acid residue is selected from the group that is made up of lysine, glutamic acid, aspartic acid, histidine and arginine.

53. according to the described method of claim 51, wherein said natural amino acid residue is selected from the group that is made up of lysine, histidine and arginine.

54. according to the described method of claim 51, wherein said natural amino acid residue is a lysine.

55. according to the described method of claim 51, wherein said natural amino acid residue is selected from the group that is made up of glutamic acid and aspartic acid.

56. according to claim 1,2 or 6 described methods, wherein said displacement step comprises the surface residue of the described discriminating of direct mutagenesis.

57. according to claim 1,2 or 6 described methods, wherein said displacement step comprises the surface residue of the described discriminating of PCR mutagenesis.

58. a green fluorescent protein (+36GFP), it has following amino acid sequence:

MGHHHHHHGGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLK

FICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKK

DGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRK

NGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKR

DHMVLLEFVTAAGIKHGRDERYK.

。

59. polynucleotide, it is encoded according to the described green fluorescent protein of claim 58.

60. according to the described polynucleotide of claim 59, it has following sequence:

ATGGGGCATCATCATCATCACCACGGCGGGGCGTCTAAGGGAGAGCGCTTGTTTC

GCGGCAAAGTCCCGATTCTTGTGGAGCTCAAAGGTGATGTAAATGGTCATAAATT

TAGTGTGCGCGGGAAAGGGAAAGGAGATGCTACGCGGGGCAAGCTCACCCTGAA

ATTTATTTGCACAACCGGCAAACTGCCAGTGCCGTGGCCTACATTAGTCACTACT

CTGACGTACGGTGTTCAGTGCTTTTCTCGCTATCCCAAACACATGAAACGCCATG

ATTTCTTCAAGAGCGCGATGCCAAAAGGTTATGTGCAGGAACGCACCATCAGCTT

TAAAAAAGACGGCAAATATAAAACCCGTGCAGAAGTTAAATTCGAAGGCCGCAC

CCTGGTCAACCGCATTAAACTGAAAGGTCGTGACTTCAAAGAGAAAGGTAATAT

TCTTGGTCACAAACTGCGCTATAATTTCAACTCTCACAAAGTTTATATTACGGCG

GATAAACGTAAAAACGGGATTAAAGCGAAATTTAAGATTCGTCATAATGTTAAA

GACGGCAGTGTGCAGTTAGCGGATCATTATCAGCAGAATACCCCAATTGGTCGC

GGTCCAGTGCTGCTGCCGCGTAACCATTATCTGTCGACCCGCAGCAAACTCAGCA

AAGACCCGAAAGAAAAACGTGACCACATGGTATTACTGGAATTTGTGACCGCAG

CAGGCATTAAACATGGCCGCGATGAACGTTACAAATAG.

。

61. a green fluorescent protein (+49GFP), it has following amino acid sequence:

MGHHHHHHGGRSKGKRLFRGKVPILVKLKGDVNGHKFSVRGKGKGDATRGKLTLK

FICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKK

DGKYKTRAEVKFKGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRK

NGIKAKFKIRHNVKDGSVQLAKHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKR

DHMVLKEFVTAAGIKHGRKERYK.

。

62. polynucleotide, it is encoded according to the described green fluorescent protein of claim 61.

63. according to the described polynucleotide of claim 62, it has following sequence:

ATGGGCCACCATCATCATCACCACGGGGGACGCTCTAAAGGTAAACGTCTGTTTC

GTGGAAAGGTGCCCATTCTGGTTAAACTCAAAGGTGATGTCAACGGCCATAAGTT

TTCGGTTCGTGGCAAAGGTAAAGGTGATGCGACGCGCGGGAAATTAACACTGAA

ATTTATTTGCACAACCGGAAAACTCCCTGTGCCGTGGCCGACTTTGGTGACCACA

TTAACCTATGGTGTTCAATGCTTCTCACGTTATCCGAAGCATATGAAACGTCATG

ATTTTTTCAAATCGGCTATGCCGAAAGGTTACGTCCAGGAGCGCACCATCTCATT

TAAGAAAGACGGTAAGTATAAAACCCGTGCTGAAGTAAAATTCAAAGGACGCAC

CCTGGTGAATCGCATTAAACTGAAAGGTCGTGATTTCAAAGAAAAGGGAAATAT

TTTAGGGCATAAGCTCCGTTATAATTTTAACAGTCATAAGGTGTATATTACCGCT

GATAAACGCAAAAACGGAATCAAAGCGAAATTTAAGATCCGTCATAATGTAAAA

GATGGCTCAGTCCAACTGGCAAAACATTACCAGCAGAATACCCCGATCGGCCGC

GGTCCTGTGCTTCTGCCGCGTAAACACTACTTGTCGACCCGGTCAAAATTGAGTA

AAGATCCGAAGGAAAAGCGTGATCACATGGTCTTGAAGGAATTTGTAACTGCAG

CAGGTATTAAACACGGGCGCAAAGAACGTTACAAATAG.

。

64. a green fluorescent protein (29GFP), it has following amino acid sequence:

MGHHHHHHGGASKGEELFDGEVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKF

ICTTGELPVPWPTLVTTLTYGVQCFSRYPDHMDQHDFFKSAMPEGYVQERTISFKDD

GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGI

KAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHM

VLLEFVTAAGIDHGMDELYK.

。

65. polynucleotide, it is encoded according to the described green fluorescent protein of claim 64.

66. according to the described polynucleotide of claim 65, it has following sequence:

ATGGGGCATCACCATCATCATCATGGCGGTGCGTCTAAGGGGGAGGAGTTATTTG

ATGGTGAAGTGCCGATCCTGGTGGAGCTTGATGGCGATGTTAACGGCCATGAATT

TTCTGTCCGCGGTGAAGGGGAGGGTGATGCCACGGAAGGGGAGCTGACACTTAA

ATTTATTTGCACCACCGGTGAACTCCCGGTCCCGTGGCCGACCCTGGTGACCACC

CTGACCTACGGCGTTCAATGCTTTTCACGTTATCCGGATCACATGGACCAACACG

ACTTCTTTAAAAGCGCGATGCCTGAAGGCTATGTTCAAGAACGTACAATTAGTTT

TAAAGATGACGGCACCTACAAGACCCGTGCGGAAGTAAAATTTGAAGGGGACAC

TTTAGTGAACCGCATCGAGCTGAAAGGGATCGATTTTAAAGAAGATGGGAATAT

CCTGGGACACAAACTTGAATACAACTTTAATAGTCATGACGTCTATATCACGGCG

GACAAACAGGAAAACGGAATTAAGGCAGAATTTGAGATTCGGCATAATGTCGAA

GATGGCTCGGTACAGTTGGCTGATCACTATCAGCAGAATACGCCGATTGGAGAT

GGTCCGGTTTTATTACCAGACGATCACTATCTGTCCACCGAATCCGCCCTGAGCA

AAGATCCGAATGAAGACCGGGACCATATGGTTCTGCTGGAATTTGTTACGGCGG

CTGGTATTGACCATGGCATGGATGAGCTGTATAAGTAG.

。

67. a Streptavidin (40SAV), it has following amino acid sequence:

MGHHHHHHGGAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGDAESEYVLT

GRYDSAPATDGSGTALGWTVAWKNDYENAHSATTWSGQYVGGAEARINTQWLLT

SGTTEADAWKSTLVGHDTFTKVEPSAAS.

。

68. polynucleotide, it is encoded according to the described Streptavidin of claim 67.

69. according to the described polynucleotide of claim 68, it has following sequence:

GGTTCAGCCATGGGTCATCACCACCACCATCACGGTGGCGCCGAAGCAGGTATT

ACCGGTACCTGGTATAACCAGTTAGGCTCAACCTTTATTGTGACCGCGGGAGCGG

ACGGCGCCTTAACCGGTACCTACGAATCAGCTGTAGGTGACGCGGAATCAGAGT

ACGTATTAACCGGTCGTTATGATAGCGCGCCGGCGACTGACGGTAGCGGTACTGC

TTTAGGTTGGACCGTAGCGTGGAAGAATGATTATGAAAACGCACATAGCGCAAC

AACGTGGTCAGGGCAGTACGTTGGCGGAGCTGAGGCGCGCATTAACACGCAGTG

GTTATTAACTAGCGGCACCACTGAAGCTGATGCCTGGAAGAGCACGTTAGTGGG

TCATGATACCTTCACTAAAGTGGAACCTTCAGCTGCGTCATAATAATGACTCGAG

ACCTGCA.

。

70. a Streptavidin (+52SAV), it has following amino acid sequence:

MGHHHHHHGGAKAGITGTWYNQLGSTFIVTAGAKGALTGTYESAVGNAKSRYVLT

GRYDSAPATKGSGTALGWTVAWKNKYRNAHSATTWSGQYVGGAKARINTQWLLT

SGTTKAKAWKSTLVGHDTFTKVKPSAAS.

。

71. polynucleotide, it is encoded according to the described Streptavidin of claim 70.

72. according to the described polynucleotide of claim 71, it has following sequence:

GGTTCAGCCATGGGTCATCACCACCACCATCACGGTGGCGCCAAAGCAGGTATT

ACCGGTACCTGGTATAACCAGTTAGGCTCAACCTTTATTGTGACCGCGGGAGCGA

AAGGCGCCTTAACCGGTACCTACGAATCAGCTGTAGGAAACGCAAAATCACGCT

ACGTATTAACCGGTCGTTATGATAGCGCGCCGGCGACTAAAGGTAGCGGTACTG

CTTTAGGTTGGACCGTAGCGTGGAAGAATAAGTATCGTAATGCGCACAGTGCTAC

CACTTGGTCAGGGCAGTACGTAGGGGGAGCCAAAGCACGTATCAACACGCAGTG

GTTATTAACATCAGGTACCACCAAAGCGAAAGCCTGGAAGAGCACGTTAGTGGG

TCATGATACCTTCACTAAAGTGAAACCTTCAGCTGCGTCATAATAATGACTCGAG

ACCTGCA.

。

73. a glutathione-S-transferase (40GST), it has following amino acid sequence:

MGHHHHHHGGPPYTITYFPVRGRCEAMRMLLADQDQSWEEEVVTMETWPPLKPSC

LFRQLPKFQDGDLTLYQSNAILRHLGRSFGLYGEDEEEAALVDMVNDGVEDLRCKY

ATLIYTDYEAGKEEYVEELPEHLKPFETLLSENEGGEAFVVGSEISFADYNLLDLLRIH

QVLNPSCLDAFPLLSAYVARLSARPEIEAFLASPEHVDRPINGNGKQ.

。

74. polynucleotide, it is encoded according to the described Streptavidin of claim 73.

75. according to the described polynucleotide of claim 74, it has following sequence:

GGTTCAGCCATGGGTCATCACCACCACCATCACGGTGGCCCGCCGTACACCATTA

CATACTTTCCGGTACGTGGTCGTTGTGAAGCGATGCGTATGTTATTAGCGGACCA

GGACCAATCATGGGAAGAAGAAGTAGTGACAATGGAAACCTGGCCGCCGTTAAA

GCCTAGCTGTTTATTCCGTCAATTACCGAAGTTTCAGGATGGTGATTTAACCTTAT

ACCAGTCTAACGCGATCTTACGTCATTTAGGTCGCTCATTTGGTTTATACGGTGA

AGATGAAGAAGAAGCAGCCTTAGTGGATATGGTGAATGATGGCGTGGAAGACTT

ACGTTGTAAATACGCGACGTTAATTTACACTGATTATGAAGCCGGTAAAGAGGA

GTACGTGGAAGAATTACCTGAACACCTGAAGCCGTTTGAAACATTACTGAGCGA

AAATGAAGGAGGTGAGGCGTTCGTAGTTGGTAGCGAAATTAGCTTCGCTGATTAT

AACTTATTAGACTTATTACGCATTCACCAGGTTTTAAATCCTAGCTGTTTAGACGC

TTTCCCGTTACTGAGCGCATATGTAGCGCGCCTGAGCGCCCGTCCGGAAATTGAA

GCTTTCTTAGCGTCACCTGAACACGTAGACCGCCCGATTAACGGAAACGGCAAG

CAGTAATAATGAGGTACCACCTGCA.

。

76. a protein, it is by modifying according to the described method of arbitrary claim among the claim 1-57.

77. polynucleotide, it is encoded according to the described protein of claim 76.

78. a kit, it is used for carrying out according to the described method of the arbitrary claim of claim 1-57.