CN108504670A - A kind of construction method of Escherichia coli cold shock hydrotropy type expression plasmid and its application - Google Patents

Abstract

The present invention relates to the methods of a kind of constructing plan of Escherichia coli hydrotropy type expression plasmid and the water-soluble heterologous polypeptide of its preparation of application.The cold shock hydrotropy type expression plasmid that a kind of promoter control small molecule ubiquitin relevant modifications albumen (SUMO) and foreign gene amalgamation and expression by cold-shock gene is built using seamless clone technology.A kind of mosaic type disulfurase is cloned into plasmid of the present invention, relatively well-known pCold I and pET28 series plasmids, water solubility, stability and the enzymatic activity of recombinant protein significantly improve.And relatively well-known pCold TF plasmids, SUMO do not interfere with destination protein space conformation.It is 1U by Ulp1 protease and recombinant protein:The ratio of 0.5 1mg, at 25 DEG C, the cutting efficiency of digestion 1h, SUMO label is more than 95%, is highly suitable for the research fields such as biotech medicine product industry and structure biology and prepares the protein with natural N end.

Description

A kind of construction method of Escherichia coli cold shock hydrotropy type expression plasmid and its application

Technical field

The present invention relates to a kind of construction method of Escherichia coli cold shock hydrotropy type expression plasmid and its specific realities of application Scheme is applied, technical field of bioengineering is belonged to.

Background technology

Escherichia expression system has quick, technology maturation, economy real as most common recombinant protein expression system Favour, high yield and the clear characteristic of bacterial strain genetic background.According to statistics, at present in world wide, recombination involved in document has been delivered Protein expression is more than that 60% use Escherichia coli are expressed, and nearly 30% pharmaceutical protein uses escherichia expression system Production.But when using Bacillus coli expression eukaryotic protein, probably only 30% clone gene can be in Escherichia coli In expressed with soluble form, it is other then be easy by the degradation of e. coli protein enzyme, formed and inclusion body or be beyond expression.Forgive Renaturation in vitro method can be used for body so that it becomes soluble protein, but this method operating process is complicated, time-consuming and laborious, additional to increase Protein purification cost.To solve the above-mentioned problems, at present researcher found out a variety of methods for improve recombination Solubility expression of the albumen in Escherichia coli body.Include mainly：

1, (referring to table 1) is co-expressed with molecular chaperones, disulfide bond isomerase etc.；

2, by the fusion tags such as glutathione-S-transferase, thioredoxin and folding enzymes (referring to table 1)；

3, change condition of culture, such as cold shock expression (expression of the heterologous polypeptide of cold shock inducible and production .M grace In Nuo Ye, S Pu Hadetalei, the B summer, G is fine, Ke H .ZL 03804917.1), add some in the medium and contribute to Composition (such as glycine betaine, sorbierite) that the correct conformation of exogenous proteins folds or by the later stage control the feed speed of feed supplement come (a kind of recombinant protein method Wei states of solubility expression in Escherichia coli that are effectively improved are auspicious, poplar for the aggregate velocity of control albumen Snow is peaceful, Zheng Chunyang .ZL 201310517277.7)；

4, the solubility that destination protein is improved in a manner of secretory protein (targets the function of approach after the translation that SecB is mediated Build and its apply Diao Liuyang, Zhou Jiahai, Yang Sheng, rowland Buddhist Lip river Dare .ZL 201110458204.6)；

5, using the strong protein mutant of directed evolution technologies screening water solubility/folding (for screening albumen solubility table The carrier T and its construction method that reach and application models army, Zhang Kuanliang, Wei Lingling .ZL 201010204681.5).

In addition, with the invention of the efficient molecule clone technology such as seamless clone and Gateway, it can be by a target gene It is inserted into multiple expression vectors simultaneously, then therefrom filters out the optimum expression carrier for expressing the target gene.

The common molecular chaperones of table 1. or hydrotropy tag fusion type colibacillus expression plasmid performance evaluation

When protein realizes solubility expression, in most cases, it is required for the hydrotropy label of excision recombinant protein, especially It is the medicinal albumen of genetic engineering, the albumen that final purifying obtains must be as native protein, and cannot be to merge egg White form uses.This can be by being inserted into one section of amino acid that can be identified by specific proteases between hydrotropy label and destination protein The method of sequence is realized.At present the protease of common excision hydrotropy label have enterokinase EK, fibrin ferment Thrombin, blood coagulation because The small molecule ubiquitin sample specific protease 1 (Ulp1) of sub- Factor Xa, tobacco etch virus (TEV) protease, saccharomyces cerevisiae Deng.In these protease, Ulp1 is very specific protease, and it also has following advantage：(1) and often The protease seen is compared, a certain specific amino acid primary sequences of Ulp1 nonrecognition, but the identification SUMO of specificity merges egg The space structure of SUMO structural domains in white, and cut off, any extra amino acid is will not leave behind after cutting, is very suitable for It is extensive to prepare the recombinant protein with natural N end；(2) cutting efficiency of Ulp1 is very high, usually can be by enzyme-to-substrate quality Than 1:1000 as starting reaction condition, and can tolerate the up to urea of 2M；(3) use coli expression system that can prepare height The Ulp1 protease of Rate activity.

Although researcher expresses multiple technologies applied to recombinant protein, which generally fits there are no a kind of The method for being used to prepare soluble homogeneous protein matter with natural N end.Therefore, a kind of foreign protein of strong applicability is built Solubility expression plasmid will have higher learning value and be widely applied foreground.

Invention content

The purpose of the present invention：One, it is directed to deficiencies of the prior art, builds a kind of new cold shock hydrotropy type table Up to plasmid, which can improve solubility of the recombinant protein inclusion body in intracellular, Activity and stabill；Two, structure is a kind of It is easily removed the cold shock hydrotropy type expression plasmid of recombinant protein N end expression companion；Three, by cold shock hydrotropy type of the present invention Expression plasmid is applied to prepare the disulfurase of high activity, to be applied to the soluble medicinal egg of industrial production its future In vain, the useful proteins matter such as enzyme preparation and antigen polypeptide, which provides, refers to model.

One of invention content：A kind of construction method of Escherichia coli cold shock hydrotropy type expression plasmid is provided

The structure of Escherichia coli cold shock hydrotropy type expression plasmid pCold-SUMOa and pCold-SUMOb of the present invention Method is as follows：

Build ammonia benzyl resistance pCold-SUMOa plasmids：

Using pE-SUMOpro Kan plasmids as template, using SUMO-F (sequence is as shown in SEQ ID N0.5) and SUMO-R (sequence is as shown in SEQ ID N0.6) primer amplification obtains small molecule ubiquitin relevant modifications albumen (SUMO) gene code frame piece Section.Using the postdigestive pCold TF plasmids of Sal I single endonuclease digestions as template, using pCold-F (sequence is as shown in SEQ ID N0.3) Being obtained without TF (Trigger factor) molecular chaperones with pCold-R (sequence is as shown in SEQ ID N0.4) primer amplification PCold plasmid backbone segments.Contain the identical homology arms of 19bp, primer in the 5 ' ends of wherein primer SUMO-F and primer pCold-R Contain the identical homology arms of 15bp in the 5 ' ends of SUMO-R and primer pCold-F.Using seamless clone technology by above-mentioned two segment Connection obtains recombinant plasmid, is named as pCold-SUMOa.Identify pCold-SUMOa plasmid multiple cloning sites (multiple Cloning site, MCS) in the restriction enzyme of single restriction enzyme site be followed successively by Nde I, Sac I, Kpn I, Xho I, BamH I,Hind III,Sal I,Xba I。

Structure card that resistance pCold-SUMOb plasmids：

Using pCold-SUMOa plasmids as template, using pCold-SUMO-F (sequence is as shown in SEQ ID N0.34) and PCold-SUMO-R (sequence is as shown in SEQ ID N0.35) primer amplification obtains plasmid backbone segment.It is with pET-28b plasmids Template, using Kana-F (sequence is as shown in SEQ ID N0.36) and Kana-R (sequence is as shown in SEQ ID N0.37) primer Amplification, which obtains, blocks that resistance gene fragment.Then above-mentioned two segment is connected using seamless clone technology and obtains recombinant plasmid, It is named as pCold-SUMOb.

The two of invention content：It provides a kind of using cold shock hydrotropy type expression plasmid of the present invention preparation high activity and steady The application implementation scheme of qualitative strong disulfurase

The application implementation scheme of the present invention for preparing the strong disulfurase of high activity and stability is as follows：

1. structure is expressed Escherichia coli disulfurase (IscS), people source disulfurase (NFS1) and is fitted into The recombinant plasmid of type disulfurase (EH-IscS).

2. structure in above-mentioned recombinant plasmid transformed to BL21 (DE3) competent cell is expressed bacterium using calcium chloride transformation Strain, using cold shock (15 DEG C of low temperature) and IPTG induction disulfurase expression.

3. purifying disulfurase using affinity chromatography.

4. using Hitachi, Ltd's U3900 ultraviolet-visible spectrophotometers analysis, disulfurase is active after purification And its stability.

A kind of construction method of Escherichia coli cold shock hydrotropy type expression plasmid of the present invention and its preparation of application are high living The method of property disulfurase, has following innovative point and technical advantage：

It, will well known or newfound colibacillus expression plasmid 1. the present invention provides one kind being based on seamless clone technology Promoter and well known or newfound hydrotropy label etc. are conducive to protein solubility expression and cut the matter of its fusion tag The grain element method that carries out arbitrary combination, to build a kind of or a series of foreign protein solubility expressions that are generally applicable to Plasmid provides reference scheme.

2. the present invention uses seamless clone technology by the cspA gene promoters of one of cold-shock gene and small molecule ubiquitin Relevant modifications albumen (SUMO) gene code frame merges, and builds a kind of new Escherichia coli cold shock hydrotropy type expression plasmid, should Plasmid has following technical advantage：

2.1 pCold-SUMOa and pCold-SUMOb plasmids carry cspA gene promoters and related elements, when large intestine bar For bacterium in 15 DEG C of Low- temperature cultures, low temperature can inhibit cell protein to express and inhibit the growth of thalline.But cspA promoters exist Transcriptional activity does not decline under low temperature, 5 ' UTR stable structure at low temperature, and ribosomes has a capture phenomenon, and protein is still under low temperature It can effective expression.Therefore it has been commercialized relatively, using the pET28a-SUMO plasmids of 37 DEG C of Fiber differentiations, this process can be real The expression of existing destination protein high yield (up to 60% or more cell protein) and higher purity.

2.2 small molecule ubiquitin sample specific proteases 1 (Ulp1) are the special protease of SUMO, are capable of the knowledge of specificity The space structure of SUMO structural domains in other SUMO fusion proteins, and it 100% is cut off, wherein SUMO albumen and Ulp1 protease 6 × His labels are all carried, affinity chromatography purifying can be used after endonuclease reaction and remove.Therefore, plasmid of the present invention is suitble to The recombinant protein with natural N end is prepared in extensive.

3. the preparation method of mosaic type disulfurase provided by the invention, still belong to the first time report, the mosaic type half Cystine desulfurase high characteristic active with respect to Escherichia coli disulfurase, counterpart source disulfurase With soluble good and high activity characteristic, in addition, mosaic type disulfurase will not be as the cysteine desulfurization of people source Enzyme is the same to occur self agglutination phenomenon in vitro, therefore with the good characteristic of stability.

Technique effect

1. the cold shock hydrotropy type expression plasmid (pCold-SUMOa and pCold-SUMOb) of structure can improve recombination egg Solubility of the white matter inclusion body in intracellular, Activity and stabill.

2. by way of building the chimera protein of eukaryon and protokaryon homologous protein, ensure chimera protein have with Parent protein identical function with it is active under the premise of, effectively increase solubility of the eukaryotic protein in prokaryotic expression system.

3. by mosaic type disulfurase gene cloning to cold shock hydrotropy type expression vector of the present invention, relatively Well known pET28b, pCold I and pCold-GST carriers, soluble protein expression quantity, stability and its activity significantly improve, 1L bacterium solutions are purified through one step of affinity chromatography can get 10-20mg albumen, and purity is up to 95% or more, and purifying gained mosaic type is partly Cystine desulfurase can be applied to the commercial synthesis, biological desulphurization and external preparation functionality iron-sulfur protein of sulfur-bearing biomolecule.

Description of the drawings

Fig. 1 Escherichia coli cold shock hydrotropy type expression plasmid collection of illustrative plates：(A) pCold-SUMOa plasmid maps；(B) PCold-SUMOb plasmid maps.

Fig. 2 .SDS-PAGE analysis recombination disulfurases protein expression and purifying situation in different carriers：(A) SDS-PAGE analyzes mosaic type disulfurase in pCold I, pCold-His-GST, pCold-SUMOa and pCold Protein expression profile in TF plasmids, Lane1：Full cell lysate supernatant is not induced；(B) SDS-PAGE analyzes half Guang of mosaic type Propylhomoserin desulfurase is in pET28b, pET28a-SUMO, protein expression profile in pBAD-SUMO and pET-SUMO plasmids；(C)SDS- PAGE analyzes people's source disulfurase in pCold I, pCold-His-GST, pCold-SUMOa, pCold TF, Protein expression profile in pET28b, pET28a-SUMO and pBAD-SUMO plasmid；(D) SDS-PAGE analyzes half Guang ammonia of recombinant type Sour desulfurase purity.Swimming lane 1:Escherichia coli disulfurase IscS, swimming lane 2:Mosaic type disulfurase EH- IscS, swimming lane 3:GST-EH-IscS, swimming lane 4:SUMO-EH-IscS, swimming lane 5:TF-EH-IscS, swimming lane 6:SUMO-NFS1 (55-457), swimming lane 7:TF-NFS1(55-457).M represents Protein Marker, and T represents the full cell cracking after induction Liquid, S represent the full cell lysate supernatant after induction.

The ultraviolet-visible light analysis collection of illustrative plates and its disulfurase activity point of Fig. 3 recombination disulfurases Analysis：(A) ultraviolet-visible spectrophotometer analyzes Escherichia coli after purification and mosaic type disulfurase spectroscopic properties Collection of illustrative plates.Spectrum 1：TF-EH-IscS；Spectrum 2：IscS；Spectrum 3：EH-IscS；Spectrum 4：SUMO-EH-IscS；Spectrum 5： GST- EH-IscS；(B) ultraviolet-visible spectrophotometer analysis purifying descendant source disulfurase spectroscopic properties collection of illustrative plates, light Spectrum 1：TF-NFS1(55-457)；Spectrum 2： SUMO-NFS1(55-457)；(C) activity analysis of disulfurase is recombinated Collection of illustrative plates.

The stability analysis collection of illustrative plates of Fig. 4 recombination disulfurases：(A-B) it is steady to influence protein for hydrotropy molecular chaperones Qualitative effect analysis collection of illustrative plates.

Digestion effect of Fig. 5 .SDS-PAGE analysis Ulp1 proteolytic cleavages except SUMO labels in SUMO-EH-IscS recombinant proteins Rate figure：M represents Protein Marker；Swimming lane 1:The SUMO-EH-IscS recombinant proteins that ni-sepharose purification obtains；Swimming lane 2-4: The digestion products of 100 μ g, 500 μ g and 1000 μ g SUMO-EH-IscS recombinant proteins are respectively cut in Ulp1 (3.5 μ g) protease； Swimming lane 5:The EH-IscS albumen of acquisition is purified after Ulp1 digestions；Lane-6:Ulp1 protease.

Specific implementation mode

The specific embodiment of the invention is described with reference to the accompanying drawings and embodiments.

The structure of 1. Escherichia coli cold shock hydrotropy type expression vector of embodiment

1. building ammonia benzyl resistance pCold-SUMOa plasmids

Using the pE-SUMOpro Kan plasmids of LifeSensors companies as template, using primer SUMO-F (sequence such as SEQ Shown in ID N0.3) and SUMO-R (sequence is as shown in SEQ ID N0.4) amplifications obtain Smt3 fusion proteins sequences (sequence be such as Shown in SEQ ID N0.19).Simultaneously using the pCold TF plasmids after Sal I single endonuclease digestions as template, using primer pCold-F (sequence is as shown in SEQ ID N0.1) and pCold-R (sequence is as shown in SEQ ID N0.2) amplification obtain pCold plasmid backbones Segment.Then above-mentioned two segment is connected using seamless clone technology and obtains recombinant plasmid, be named as pCold-SUMOa (such as Shown in Fig. 1).Because containing two restriction enzyme sites of EcoR I and Pst I in Smt3 antigen-4 fusion protein gene sequences, so selection is with Sal PCold TF plasmids after I single endonuclease digestions are template, and avoid pCold-SUMOa recombinant plasmids polyclonal by design of primers Contain the two restriction enzyme sites in site.Single restriction enzyme site is restricted in identification pCold-SUMOa plasmid multiple cloning sites Restriction endonuclease is followed successively by Nde I, Sac I, Kpn I, Xho I, BamH I, Hind III, Sal I, Xba I.

2. structure card that resistance pCold-SUMOb plasmids

Using pCold-SUMOa plasmids as template, using pCold-SUMO-F (sequence is as shown in SEQ ID N0.26) and PCold-SUMO-R (sequence is as shown in SEQ ID N0.27) primer amplification obtains plasmid backbone segment.It is with pET-28b plasmids Template, using Kana-F (sequence is as shown in SEQ ID N0.28) and Kana-R (sequence is as shown in SEQ ID N0.29) primer Amplification, which obtains, blocks that resistance gene fragment.Then above-mentioned two segment is connected using seamless clone technology and obtains recombinant plasmid, It is named as pCold-SUMOb (as shown in Figure 1).

PCold-SUMOa plasmid resistances are transform as to the purpose of that resistance of card by ammonia benzyl resistance：

Ampicillin is a kind of lactam antibiotics, interferes the crosslinking of peptide glycan to inhibit the synthesis of cell wall. Gram negative strain whole cell peptidoglycan content is low, insensitive to ammonia benzyl mycin, after overnight incubation around positive colony bacterium colony Easily growth satellite colony, is not easy picking single bacterium colony；Kanamycins is a kind of inhibition of protein biosynthesis agent, is pressed down to Escherichia coli Bacterium effect is good.

Embodiment 2. expresses the structure of disulfurase recombinant plasmid

1. the recombinant plasmid of structure expression Escherichia coli disulfurase

Using E. coli MC4100 genomes as template, using primer I scS-pCold-F (sequence such as SEQ Shown in ID N0.9) and IscS-pCold-R (sequence is as shown in SEQ ID N0.10) amplification acquisition Escherichia coli IscS encoder blocks Gene.Using Kpn I single endonuclease digestion pCold I plasmids, and glue recycles carrier segments after digestion.Then seamless Cloning Kit is used Structure obtains recombinant plasmid I scS-pCold I, and the recombinant plasmid is sent to Hua Da gene sequencing and is identified.

2. the recombinant plasmid of structure expression people source disulfurase

Using NFS1 (55-457)-pET28b plasmids as template, using primer m-NFS1-pCold-F (sequence such as SEQ ID Shown in N0.11)/SUMO-m-NFS1-F (sequence is as shown in SEQ ID N0.13) and m-NFS1-pCold-R (sequence such as SEQ Shown in ID N0.12)/SUMO-m-NFS1-F (sequence is as shown in SEQ ID N0.24) amplification obtain contain half Guang of encoding human source The gene order of propylhomoserin desulfurase 55-457 amino acids, the 1-54 amino acids sequences wherein in NFS1 albumen are to mediate it Into Intramitochondrial signal peptide sequence.Using Kpn I single endonuclease digestions pCold I, pCold-GST and pCold TF plasmids, and glue Carrier segments after recycling digestion.Using Nde I single endonuclease digestion pCold-SUMOa plasmids, and glue recycles carrier segments after digestion.Then NFS1 genes are connected to after above three single endonuclease digestion in carrier segments respectively using seamless Cloning Kit, obtain NFS1 (55- 457)-pCold I, NFS1 (55-457)-pCold-SUMOa, NFS1 (55-457)-pCold-GST and NFS1 (55-457)- PCold TF recombinant plasmids, and three recombinant plasmids are sent to Hua Da gene sequencing and are identified.

3. the recombinant plasmid of structure expression mosaic type disulfurase

3.1 amplification IscS albumen n end structural domain nucleic acid sequences

3.1.1 PCR amplification IscS albumen n ends structural domain (1-263 amino acids) nucleic acid sequence system and condition are as follows：

95℃,3min；95 DEG C, 10sec, 51.6 DEG C, 30sec, 72 DEG C, 30sec, 35 cycles； 72℃,10min,4℃ It preserves.

3.1.2 Dpn I digest the IscS-pCold I plasmid templates to methylate

Digestion system and condition are as follows：

37 DEG C be incubated 1 hour or longer time.

3.1.3 the glue recycling of digestion products：

Product carries out 1% low melting-point agarose gel electrophoresis after endonuclease reaction, and target DNA piece is cut under long-wave ultra violet lamp Section, with TaKaRa

Company's plastic recovery kit is recycled.It is finally quantitative to recovery product with Du-800 nucleic acid/protein analyzer.

3.2 amplification NFS1 PROTEIN C terminal domains nucleic acid sequences

3.2.1 PCR amplification NFS1 PROTEIN Cs terminal domains (316-457 amino acids) nucleic acid sequence system and condition be such as Under：

95℃,3min；95 DEG C, 10sec, 52 DEG C, 15sec, 72 DEG C, 30sec, 35 cycles；72 DEG C, 10min, 4 DEG C guarantors It deposits.

3.2.2 Dpn I digest NFS1 (the 55-457)-pCold I plasmid templates to methylate

Digestion system and condition are as follows：

37 DEG C be incubated 1 hour or longer time.

3.2.3 the glue recycling of digestion products：

Product carries out 1% low melting-point agarose gel electrophoresis after endonuclease reaction, and target DNA piece is cut under long-wave ultra violet lamp Section, is recycled with TaKaRa companies plastic recovery kit.It is finally fixed to recovery product with Du-800 nucleic acid/protein analyzer Amount.

3.3 structure Escherichia coli and people source disulfurase dna chimeric gene

3.3.1 PCR reaction systems and condition are as follows：

95℃,3min；95 DEG C, 10sec, 53 DEG C, 45sec, 72 DEG C, 30sec, 35 cycles；72 DEG C, 10min, 4 DEG C guarantors It deposits.

3.3.2 the glue recycling of PCR product：

PCR product is subjected to 1% low melting-point agarose gel electrophoresis, target DNA fragment is cut under long-wave ultra violet lamp, with TaKaRa companies plastic recovery kit is recycled.It is finally quantitative to recovery product with Du-800 nucleic acid/protein analyzer.

The recombinant plasmid of 3.4 structure expression EH-IscS

3.4.1 PCR amplification EH-IscS genes

PCR reaction systems and condition are as follows：

95℃,3min；95 DEG C, 10sec, 53 DEG C, 45sec, 72 DEG C, 30sec, 35 cycles；72 DEG C, 10min, 4 DEG C guarantors It deposits.

PCR product is subjected to 1% low melting-point agarose gel electrophoresis, target DNA fragment is cut under long-wave ultra violet lamp, with TaKaRa companies plastic recovery kit is recycled.It is finally quantitative to recovery product with Du-800 nucleic acid/protein analyzer.

3.4.2 pCold I, pCold-GST, pCold TF, pCold-SUMOa, pET28a-SUMO, pET-SUMO and The preparation of pBAD-SUMO linearized vector segments

PET28a-SUMO, pET-SUMO and pBAD-SUMO plasmid are studied by Wenzhou Medical University's enzyme engineering and medical diagnosis It is provided.

PCold I, pCold-GST and pCold TF plasmid single endonuclease digestion systems are as follows：

37 DEG C, digestion 2h.

PCold-SUMOa, pET-SUMO and pBAD-SUMO plasmid single endonuclease digestion system are as follows：

37 DEG C, digestion 2h.

PET28a-SUMO plasmid single endonuclease digestion systems are as follows：

37 DEG C, digestion 2h.

Digestion products carry out 1% low melting-point agarose gel electrophoresis after 10 × Loading buffer are added, in long wave purple Purpose carrier segments are cut under outer lamp, are recycled with TaKaRa companies plastic recovery kit.Finally with Du-800 nucleic acid/albumen Analyzer is quantitative to recovery product.

3.5 express the recombination matter of EH-IscS using seamless Cloning Kit (and first bio tech ltd) structure Grain

3.5.1 seamless cloning reaction system is as follows：

37 DEG C of incubation 30min, are then transferred into and place 5min on ice.DH5 α competence is converted using the above-mentioned reaction solutions of 10 μ l Cell.

3.5.2 conversion：Pass through cold CaCl₂The above-mentioned seamless cloning reaction liquid of 10 μ l is converted DH5 competent cells by method.

3.5.3 bacterium colony PCR identifies positive colony

With the above-mentioned LB/Amp of transfer needle picking⁺It is sterile in one that the single bacterium colony grown on tablet distinguishes streak inoculation a little LB/Amp⁺Or LB/Kana⁺Culture 10h is inverted on tablet, in 37 DEG C of constant incubators.It then, should with sterile pipette tips difference picking The thalline grown in each small lattice on tablet is a little, is mixed in the EP pipes containing 50 μ l sterile waters, boiling water boiling 10min, 12000rpm, from After heart 5min, takes 9.2 μ l of supernatant to be added in 200 μ l PCR reaction tubes and identify template as PCR.And take following system that the pipe is added In, mixing.

94℃,5min；94 DEG C, 30sec, 50.3 DEG C, 30sec, 72 DEG C, 1min 30sec, 25 cycles；72℃, 10min, 4 DEG C of preservations.5 μ l PCR products are taken to be identified with 1.0% Agarose gel electrophoresises.Select correct two sun of identification Property clone send to Hua Da gene sequencing identify.

The induction of 3. disulfurase of embodiment and expression condition optimization

The above-mentioned recombinant plasmid built is passed through into cold CaCl₂Method is transformed into BL21 (DE3) competent cell.The present invention The induction and expression of the recombination disulfurase, remove special declaration, are all operated by following experimental procedure：

(1) transformant of screening is stayed overnight into bacterium and presses 1:50 volume ratio is seeded to the LB liquid containing 100 μ g/ml Amp In culture medium, 37 DEG C, 250rpm shaken cultivations；

(2) when bacterium solution OD600 values are about 0.4-0.5, bacterium solution is set place 5min in ice-water bath immediately, then move to 15 DEG C stand 30min；

(3) add IPTG to its final concentration of 100 μM/L, 15 DEG C, 250rpm shaken cultivations for 24 hours.

(4) after the completion of cultivating, the presence or absence of destination protein, expression quantity and solubility are analyzed using SDS-PAGE.

3.1 stay overnight the optimization of bacterium initial inoculation amount

The transformant of screening is stayed overnight into bacterium and presses 1 respectively:50,1:500,1:1000 volume ratio is seeded to containing 100 μ g/ml In the LB liquid medium of Amp, 37 DEG C, 250rpm shaken cultivations.Subsequent experimental procedure is the same as described in embodiment 4.

Influence of the 3.2 IPTG inducers concentration to recombinant protein expression quantity

0,100,200,400,800 μM/five IPTG inducer concentration of L are respectively set, other experimental procedures are the same as embodiment 4 It is described.

3.3 experimental result：(1) bacterium initial inoculation amount is stayed overnight 1:1000-1:To recombinating half Guang ammonia in 50 (v/v) ranges Sour desulfurase expression quantity has no significant effect, therefore in order to save thalline incubation time, determines 1:50 be optimum inoculation amount；(2) SDS-PAGE is not analysis shows IPTG inducers concentration has notable shadow to IscS expressing quantities within the scope of 100-800 μM/L It rings, therefore in order to cost-effective, determines that 100 μM/L IPTG are best inducer concentration；(3) as shown in Fig. 2, half Guang ammonia of people source Sour desulfurase NFS1 (55-457) is expressed all in pET28b, pCold I, pCold-GST and pBAD-SUMO plasmids to forgive Body form exists.And it is similar with commercialized pET28a-SUMO plasmids effect, pCold-SUMOa plasmids can be with fraction The solubility expression of NFS1 (55-457) albumen is improved, and TF molecular chaperones can make NFS1 (55-457) albumen completely with solvable Property form expression.When mosaic type disulfurase EH-IscS uses pET28b plasmid expressions, exist with inclusion bodies, And pCold I, pCold-GST, pCold-SUMOa/pET28a-SUMO/pET-SUMO/pBAD-SUMO and pCold TF matter Grain can improve the solubility of EH-IscS albumen successively, and pCold-SUMOa plasmids are with respect to pET28a-SUMO, pET- SUMO and pBAD-SUMO plasmids can improve the yield of destination protein and its account for the ratio of full total protein of cell amount, induce egg Full cell after white expression can be directly used for nuclear magnetic resonance spectroscopy.In conclusion Escherichia coli cold shock hydrotropy of the present invention Type expression vector has good application potential in terms of promoting heterologous polypeptide solubility expression.

Embodiment 4. recombinates the isolation and purification of disulfurase

4.1 isolate and purify recombination disulfurase using affinity chromatography

IscS of the present invention, EH-IscS, SUMO-EH-IscS, GST-EH-IscS, TF-EH-IscS, SUMO-NFS1 (55-457) TF-NFS1 (55-457) albumen uses affinity chromatography isolation and purification.That steps are as follows is described for specific experiment：

4.1.1 the preparation of separation and purification of protein buffer solution：

A liquid：Also known as DB (Desalting Buffer) or Binding Buffer buffer solutions, pH=8.0 are used for albumen In conjunction with the buffer solution of chromatographic column, 4 DEG C of preservations when preserving, purifying.

B liquid：Also known as Ellution Buffer, the albumen combined on elution chromatography column, 4 DEG C are kept in dark place.

4.1.2 4 DEG C, 6,000rpm centrifugation 6min, thalline after 15 DEG C of collection culture 24 hours；

4.1.3 thalline is resuspended with Desalting Buffer and centrifuges, repeated washing twice, then with 30ml precoolings Thalline is resuspended in Desalting Buffer；

4.1.4 clasmatosis：With high pressure cell cracker at 4 DEG C, smudge cells 4 times, make brokenly under the conditions of 1000-1500w Final volume is 35ml after broken；

4.1.5 suspension purifies after 4 DEG C, 24,000rpm centrifugation 45min, 0.45 μM of membrane filtration of supernatant after being crushed.

4.1.6 computer is opened, protein purification instrument starts operation program, instrument is waited for be connect with computer；

4.1.7 ddH₂O cleanings flow through A1 the and B1 pipelines of A pumps and B pumps, and pump wash three times, are used in combination A liquid and B liquid clear A1 and B1 pipelines are washed,

Pump wash are twice；

4.1.8 Ni column of the connection for purifying rinses Ni with 100%Elution Buffer with the flow velocity of 3ml/min Column 3min uses the A liquid of 20 nickel column volumes instead and balances Ni column about 10-15min with the flow velocity of 3ml/min, makes baseline stability, ddH₂O cleans loading column；

4.1.9 filtered albumen supernatant is injected in loading column, starts affinity chromatography purifying procedure, wait for instrument Device is automatically performed protein purification

Process, i.e. balance, loading, elution and collection.

Affinity chromatography purifying procedure design parameter is as follows：

Sample introduction：35ml samples, 3ml/min, 2%Elution Buffer

Elution：Elute foreign protein 10cv 3ml/min, 6%Elution Buffer

Elute destination protein：15cv 3ml/min, 100%Elution Buffer

It collects：Destination protein 2ml is collected according to the corresponding sample cell in the absorbing proteins peak shown in program；

4.1.10 albumen desalination：After the completion of protein purification, desalting column Desalting tube are changed, after pillar balance, By ni-sepharose purification albumen sample

Product carry out desalination successively.

It collects：Destination protein 2ml is collected according to the corresponding sample cell in the absorbing proteins peak shown in program, is placed on ice.

4.1.11 the measurement of albumen concentration

250-700nm full wavelength scanners are carried out to albumen with ultraviolet-uisible spectrophotometer (U3900).Since albumen exists There is specific absorption peak, therefore (each albumen extinction coefficient is by institute for specific extinction coefficient at 280 nm by it at 280nm The amino acid contained is calculated), the concentration of corresponding albumen is calculated as follows.

Albumen concentration calculation formula：C_pro=(OD₂₈₀-OD₇₀₀) * 1000/ extinction coefficient (unit：μM)

4.2 experimental result：(1) half Guang of recombination obtained through SDS-PAGE and the purifying of coomassie brilliant blue staining identification and analysis The purity of protein of propylhomoserin desulfurase is all higher than 90%, can be directly used for follow-up zymetology experimental analysis (as shown in Figure 2)；(2) to pure Albumen after change carries out full wavelength scanner analysis, it can be found that mosaic type EH-IscS, GST-EH-IscS, SUMO-EH-IscS with Escherichia coli IscS is consistent, occurs disulfurase prothetic group phosphopyridoxal pyridoxal phosphate (PLP) characteristic absorption peak at 395nm, Albumen is in faint yellow or yellow.People source disulfurase NFS1 (55-457) and SUMO-NFS1 (55-457) exists Occurs PLP characteristic absorptions peak at 420nm, albumen is equally in faint yellow or yellow.TF-EH-IscS is in faint yellow after purification, but It is that its disulfurase activity is very weak.TF-NFS1 (55-457) albumen is without color after purification, and the spy at 420nm Sign property absorption peak is very faint, and illustration purpose albumen is not associated with PLP, this is because Trigger factor labels are too big (52kDa) may influence destination protein space structure, and SUMO labels are relatively small (16kDa), have as molecular chaperones Help the correct folding of destination protein, and does not interfere with destination protein space structure (as shown in Figure 3).

The active measurement of 5. disulfurase of embodiment

The measurement of iron in 5.1 Nth protein samples, sulfur content

5.1.1 in Nth protein samples iron content measurement

The measurement of iron content is using coffee Lip river piperazine method, basic principle in protein sample：Under the action of L-cysteine, Iron in protein sample releases, and is combined with coffee Lip river piperazine (Ferrozine), forms Fe-Ferrozine complex compounds.The network Close object has specific absorption peak, extinction coefficient 27.9mM at 564nm^-1cm^-1, by measuring the value of the absorption peak, i.e., The content of iron in albumen is calculated using formula.

After upper table plus good system, 85 DEG C of heating 20min being placed in, 30min is placed at room temperature for, 12,000 rpm centrifuge 5min, Supernatant is taken to carry out 450-700nm full wavelength scanners, and using Desalting Buffer as negative control.

Iron content is calculated according to formula：C_Fe=(OD₅₆₄-OD₇₀₀) * 1000/27.9 (unit：μM)

5.1.2 in Nth protein samples sulfur content measurement

The measurement of sulfur content is using DPD methods, basic principle in protein sample：Sulfide and FeCl₃Interaction, DPD can form characteristic absorption peak as color developing agent at 669nm, and albumen can be calculated to obtain using the extinction coefficient of formula and sulphur In sulfur content.

After upper table plus good system, it is placed at room temperature for 30min, 12,000rpm centrifugation 5min take supernatant to carry out 450-800nm Full wavelength scanner, using Desalting Buffer as negative control, and with the extinction coefficient of Nth calculating sulphur.

Sulfur content is calculated according to formula：C_S=(OD₆₆₉-OD₈₀₀) * 1000/S extinction coefficient (units：μM)

S extinction coefficients=(OD₆₆₉-OD₈₀₀)*1000/Nth C_S

Nth C_S=Nth C_Fe=(OD₅₆₄-OD₇₀₀)*1000/27.9

5.2 IscS,EH-IscS,GST-EH-IscS,SUMO-EH-IscS,TF-EH-IscS, SUMO-NFS1(55- And the active measurement of TF-NFS1 (55-457) protein sample disulfurase 457)

The reaction system of 1ml is prepared before determination of activity：The albumen for taking known concentration is diluted to final concentration of with A liquid (DB) 10 μM, 3 μ L (final concentration of 3mM) of 1M DTT are added, are incubated 5min for 37 DEG C after mixing, and the L- of 20 μ l 0.1M is added immediately Cysteine (final concentration of 2mM) takes the sulfur content of 160 μ L measurement at this time after overturning mixing, and this moment is defined as immediately 0min takes a sample (160 μ L) to survey sulfur content, is terminated when to 15min, totally 4 times every 5min later.

Catalystic converter system is as follows：

37 DEG C of incubation 5min, are added 20 μ l 0.1M L-cysteine, 160 μ L are taken to survey this moment after above-mentioned system mixing Sulfur content, hereafter surveyed every 10min primary.

Sulphur content determination system：

Above-mentioned survey sulfur system is statically placed on 37 DEG C of dry-type thermostats and heats 20min, after 12,000 rpm centrifuge 5min, is used U3900 carries out 500nm-800nm full wavelength scanners, it can be seen that the characteristic absorption peak at 669nm.

According to formula C_S=(OD₆₆₉-OD₈₀₀1000 (the units of extinction coefficient * of)/S：μM) sulfur content is calculated, sulfur content How much the size of IscS proteinase activity is directly reacted.Using DB as negative control, a kind of albumen Nth (very stable [4Fe- 4S]) it is used as positive control, the extinction coefficient of sulphur to be calculated by the content of iron in positive control Nth.(note：Fe, S contain in Nth Quantity measuring method is with described in 5.1)

M- sulfur content curve graph when being drawn after the completion of measuring：M- sulphur contains when according to how much draftings of calculated sulfur content Curve graph is measured, the size of the enzymatic activity of each albumen is analyzed.

5.3 experimental result：(1) as shown in Figure 3 C, SUMO-EH-IscS protein cysteines desulfurization enzymatic activity highest, and And it is higher than Escherichia coli IscS protein actives, and EH-IscS protein cysteine desulfurization enzymatic activitys are less than IscS albumen, explanation On the one hand SUMO molecular chaperones can improve EH-IscS albumen solubilities, on the other hand can significantly improve the work of EH-IscS albumen Property, reason may can enhance EH-IscS protein stabilities with SUMO molecular chaperones, prevent it from occurring self agglutination related, Specific molecular mechanism needs further to be studied；(2) TF-EH-IscS albumen has faint disulfurase activity, this It is that destination protein space structure may be influenced, with Such analysis result since Trigger factor labels are too big (52kDa) Unanimously；(3) people source disulfurase NFS1 needs are combined just active with ISD11, therefore, individually detect SUMO- NFS1 (55-457) and when TF-NFS1 (55-457) albumen, does not have disulfurase activity.

Influence of the different hydrotropy labels of embodiment 6. to recombination disulfurase stability

Using DB by purified IscS, EH-IscS, GST-EH-IscS, SUMO-EH-IscS and TF-EH-IscS eggs White concentration is diluted to 1 μM respectively, and 30min is then incubated at 37 DEG C, and extinction of the protein sample at 320nm is measured every 5min Value.If self agglutination can occur for protein sample, light absorption value will be incremented by with the extension of time at 320nm.It is with the time Horizontal axis, light absorption value is longitudinal axis mapping at 320nm.As shown in Figure 4：(1) though mosaic type disulfurase EH-IscS have compared with Good disulfurase activity, but the protein stability is poor, and albumen concentration is higher, and self agglutination phenomenon is brighter It is aobvious；(2) EH-IscS and the recombinant protein expressed after SUMO and TF tag fusions, have good stability.According to another document Report, NFS1 are one and are easy to the protein for occurring self to be aggregated, but SUMO-NFS1 (55-457) and TF- in this experiment NFS1 (55-457) albumen has good stability, illustrates hydrotropy label other than the solubility with enhancing protein, It can also prevent self agglutination of protein.

Embodiment 7. expresses the structure of Ulp1 protease recombinant plasmids

The expression and purification of 7.1 Ulp1 protease

Using the cDNA of saccharomyces cerevisiae BY4743 as template, the small molecule ubiquitin sample that saccharomyces cerevisiae is expanded using round pcr is special The positions the 404-621 active fragment of foreign preteins enzyme 1, and it is cloned into using seamless clone technology the Nde I of pET28b plasmids In restriction enzyme site, Ulp1-pET28b recombinant plasmids are built.

Using Calcium Chloride Method by Ulp1-pET28b recombinant plasmid transformeds to BL21 (DE3) competent cell, build E.coli Ulp1-pET28b/BL21 engineering strains.

The preparation of embodiment 8.Ulp1 protease

The expression and purification of 8.1 Ulp1 protease

Take the E.coli Ulp1-pET28b/BL21 inoculations that 5 μ l are preserved in -30 DEG C to containing 50 μ g/ with liquid-transfering gun In the LB culture mediums of ml kanamycins, 37 DEG C, 250rpm shaken cultivations are stayed overnight.Expand culture to OD₆₀₀Close to 1.0, IPTG is added To final concentration of 200 μM, 37 DEG C, 250rpm shaken cultivations receive bacterium after 3 hours.

The isolation and purification of Ulp1 protease is the same as described in the embodiment of the present invention 4.

The enzymatic activity of 8.2 Ulp1 protease defines

In 50mM Tris-HCl, pH 7.5,150mM NaCl, 25 DEG C of reaction 1h, 100 μ g reference proteins have more than 95% enzyme amount of Ulp1 needed for cutting is defined as 1U.

The agent prescription of 8.3 Ulp1 protease

3.5mg/ml Ulp1 protease is stored in comprising 7.5,150 mM NaCl of 50mM Tris-HCl pH, and 20% is sweet Oil, 1mM benzenecarboximidamides, 0.2mM phenylmethylsulfonyl fluorides, 0.1mM ethyleneglycol bistetraacetic acids, 0.1%2- mercaptoethanols, 0.03% ten In the biological reagent of dialkyl group polyglycol ether.

The storage of 8.4 Ulp1 protease and its stability

The Ulp1 protease preserved using in the present invention 8.3 biological reagents, is calculated since the date of manufacture, can in- 70 DEG C of 1 years of preservation.

The removal of SUMO labels in 9. recombinant protein of embodiment

9.1 prepare the recombinant protein with natural N end using Ulp1 protease

According to 1U：The ratio of 500-1000 μ g albumen, the digestion 1-2h in 25 DEG C of water-baths carry out digestion preliminary experiment.Choosing It takes cutting efficiency to be more than 90% digestion ratio, carries out the extensive recombinant protein for preparing and there is natural N end.Wherein SUMO albumen 6 × His labels are all carried with Ulp1 protease, are removed using affinity chromatography purifying after endonuclease reaction.Ulp1 protease Digesting efficiency is as shown in Figure 5.

The foregoing is only a preferred embodiment of the present invention, is not intended to limit the scope of the present invention.

<110>Wenzhou Medical University

<120>A kind of construction method of Escherichia coli cold shock hydrotropy type expression plasmid and its application

<160> 32

<170> PatentIn version 3.3

<210> 1

<211> 55

<212> DNA

<213> pCold-F

<400> 1

cctcgaggga tccaagcttg tcgactctag ataggtaatc tctgcttaaa agcac 55

<210> 2

<211> 27

<212> DNA

<213> pCold-R

<400> 2

gtgatgatga tgatgatgca ctttgtg 27

<210> 3

<211> 38

<212> DNA

<213> SUMO-F

<400> 3

gcatcatcat catcatcacg ggtccctgca ggactcag 38

<210> 4

<211> 55

<212> DNA

<213> SUMO-R

<400> 4

ttggatccct cgagggtacc gagctccata tgacctccaa tctgttcgcg gtgag 55

<210> 5

<211> 21

<212> DNA

<213> Chimeric IscS-F

<400> 5

atgaaattac cgatttatct c 21

<210> 6

<211> 40

<212> DNA

<213> Chimeric IscS-R

<400> 6

gattcgcttg tggtcatact ccatctcttc ttttgcgatg 40

<210> 7

<211> 40

<212> DNA

<213> Chimeric NFS1-F

<400> 7

catcgcaaaa gaagagatgg agtatgacca caagcgaatc 40

<210> 8

<211> 19

<212> DNA

<213> Chimeric NFS1-R

<400> 8

ctagtgttgg gtccacttg 19

<210> 9

<211> 46

<212> DNA

<213> IscS-pCold-F

<400> 9

aggcatatgg agctcatgaa attaccgatt tatctcgact actccg 46

<210> 10

<211> 44

<212> DNA

<213> IscS-pCold-R

<400> 10

attcggatcc ctcgagttaa tgatgagccc attcgatgct gttc 44

<210> 11

<211> 40

<212> DNA

<213> m-NFS1-pCold-F

<400> 11

aggcatatgg agctcgtgct gcgacctctc tatatggatg 40

<210> 12

<211> 41

<212> DNA

<213> m-NFS1-pCold-R

<400> 12

attcggatcc ctcgagctag tgttgggtcc acttgatgct c 41

<210> 13

<211> 41

<212> DNA

<213> SUMO-m-NFS1-F

<400> 13

cgaacagatt ggaggtgtgc tgcgacctct ctatatggat g 41

<210> 14

<211> 42

<212> DNA

<213> SUMO-m-NFS1-R

<400> 14

tcgagggtac cgagctccta gtgttgggtc cacttgatgc tc 42

<210> 15

<211> 46

<212> DNA

<213> EH-IscS-pCold-F

<400> 15

aggcatatgg agctcatgaa attaccgatt tatctcgact actccg 46

<210> 16

<211> 39

<212> DNA

<213> EH-IscS-pCold-R

<400> 16

attcggatcc ctcgagctag tgttgggtcc acttgatgc 39

<210> 17

<211> 47

<212> DNA

<213> SUMO-EH-IscS-F

<400> 17

cgaacagatt ggaggtatga aattaccgat ttatctcgac tactccg 47

<210> 18

<211> 40

<212> DNA

<213> SUMO-EH-IscS-R

<400> 18

tcgagggtac cgagctccta gtgttgggtc cacttgatgc 40

<210> 19

<211> 306

<212> DNA

<213> Smt3 Fusion Protein

<400> 19

atggggtccc tgcaggactc agaagtcaat caagaagcta agccagaggt caagccagaa 60

gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc 120

aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt 180

aaggaaatgg actccttaag attcttgtac gacggtatta gaattcaagc tgatcaggcc 240

cctgaagatt tggacatgga ggataacgat attattgagg ctcaccgcga acagattgga 300

ggttag 306

<210> 20

<211> 4683

<212> DNA

<213> pCold-SUMOa

<400> 20

aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa 60

tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat 120

ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt 180

cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt 240

aagaggtaat acaccatgaa tcacaaagtg catcatcatc atcatcacgg gtccctgcag 300

gactcagaag tcaatcaaga agctaagcca gaggtcaagc cagaagtcaa gcctgagact 360

cacatcaatt taaaggtgtc cgatggatct tcagagatct tcttcaagat caaaaagacc 420

actcctttaa gaaggctgat ggaagcgttc gctaaaagac agggtaagga aatggactcc 480

ttaagattct tgtacgacgg tattagaatt caagctgatc aggcccctga agatttggac 540

atggaggata acgatattat tgaggctcac cgcgaacaga ttggaggtca tatggagctc 600

ggtaccctcg agggatccaa gcttgtcgac tctagatagg taatctctgc ttaaaagcac 660

agaatctaag atccctgcca tttggcgggg atttttttat ttgttttcag gaaataaata 720

atcgatcgcg taataaaatc tattattatt tttgtgaaga ataaatttgg gtgcaatgag 780

aatgcgcagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 840

gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg 900

tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga 960

gcagattgta ctgagagtgc accataaaat tgtaaacgtt aatattttgt taaaattcgc 1020

gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc 1080

ttataaatca aaagaatagc ccgagatagg gttgagtgtt gttccagttt ggaacaagag 1140

tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 1200

tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc 1260

actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa 1320

cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 1380

agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 1440

gtactatggt tgctttgacg tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 1500

taccgcatca ggcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 1560

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 1620

ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 1680

ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 1740

aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 1800

gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 1860

ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 1920

gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1980

cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 2040

cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 2100

acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 2160

caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 2220

taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 2280

ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 2340

aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 2400

agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 2460

atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 2520

tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 2580

tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 2640

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 2700

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 2760

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 2820

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 2880

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 2940

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 3000

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 3060

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 3120

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 3180

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 3240

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 3300

ccttttgctg gccttttgct cacatagtca tgccccgcgc ccaccggaag gagctgactg 3360

ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta 3420

cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 3480

attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt 3540

tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag 3600

agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg 3660

gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata 3720

tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga 3780

tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt 3840

tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg 3900

cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg 3960

cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc 4020

gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga 4080

aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc 4140

ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta 4200

caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg 4260

gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg 4320

gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg 4380

taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg 4440

gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg 4500

tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat 4560

gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt 4620

atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc 4680

cgc 4683

<210> 21

<211> 4690

<212> DNA

<213> pCold-SUMOb

<400> 21

aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa 60

tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat 120

ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt 180

cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt 240

aagaggtaat acaccatgaa tcacaaagtg catcatcatc atcatcacgg gtccctgcag 300

gactcagaag tcaatcaaga agctaagcca gaggtcaagc cagaagtcaa gcctgagact 360

cacatcaatt taaaggtgtc cgatggatct tcagagatct tcttcaagat caaaaagacc 420

actcctttaa gaaggctgat ggaagcgttc gctaaaagac agggtaagga aatggactcc 480

ttaagattct tgtacgacgg tattagaatt caagctgatc aggcccctga agatttggac 540

atggaggata acgatattat tgaggctcac cgcgaacaga ttggaggtca tatggagctc 600

ggtaccctcg agggatccaa gcttgtcgac tctagatagg taatctctgc ttaaaagcac 660

agaatctaag atccctgcca tttggcgggg atttttttat ttgttttcag gaaataaata 720

atcgatcgcg taataaaatc tattattatt tttgtgaaga ataaatttgg gtgcaatgag 780

aatgcgcagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 840

gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg 900

tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga 960

gcagattgta ctgagagtgc accataaaat tgtaaacgtt aatattttgt taaaattcgc 1020

gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc 1080

ttataaatca aaagaatagc ccgagatagg gttgagtgtt gttccagttt ggaacaagag 1140

tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga 1200

tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc 1260

actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa 1320

cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt 1380

agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc 1440

gtactatggt tgctttgacg tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 1500

taccgcatca ggcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 1560

gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 1620

catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata 1680

ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat 1740

atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt 1800

atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg 1860

atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca 1920

tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga 1980

aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc 2040

tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg 2100

atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga 2160

gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata 2220

aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc 2280

ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag 2340

accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac 2400

agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc 2460

atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt 2520

atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggg 2580

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 2640

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 2700

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 2760

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 2820

ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 2880

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 2940

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 3000

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 3060

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 3120

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 3180

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 3240

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 3300

ttcctggcct tttgctggcc ttttgctcac atagtcatgc cccgcgccca ccggaaggag 3360

ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc 3420

taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 3480

cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag 3540

ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc 3600

ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt 3660

gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac 3720

cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc 3780

catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat 3840

ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat 3900

ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact 3960

taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc 4020

cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac 4080

atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc 4140

atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc 4200

cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag 4260

ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact 4320

ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt 4380

gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac 4440

gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc 4500

gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg 4560

ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct 4620

ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca 4680

ccgccgccgc 4690

<210> 22

<211> 1215

<212> DNA

<213> IscS

<400> 22

atgaaattac cgatttatct cgactactcc gcaaccacgc cggtggaccc gcgtgttgcc 60

gagaaaatga tgcagtttat gacgatggac ggaacctttg gtaacccggc ctcccgttct 120

caccgtttcg gctggcaggc tgaagaagcg gtagatatcg cccgtaatca gattgccgat 180

ctggtcggcg ctgatccgcg tgaaatcgtc tttacctctg gtgcaaccga atctgacaac 240

ctggcgatca aaggtgcagc caacttttat cagaaaaaag gcaagcacat catcaccagc 300

aaaaccgaac acaaagcggt actggatacc tgccgtcagc tggagcgcga aggttttgaa 360

gtcacctacc tggcaccgca gcgtaacggc attatcgacc tgaaagaact tgaagcagcg 420

atgcgtgacg acaccatcct cgtgtccatc atgcacgtaa ataacgaaat cggcgtggtg 480

caggatatcg cggctatcgg cgaaatgtgc cgtgctcgtg gcattatcta tcacgttgat 540

gcaacccaga gcgtgggtaa actgcctatc gacctgagcc agttgaaagt tgacctgatg 600

tctttctccg gtcacaaaat ctatggcccg aaaggtatcg gtgcgctgta tgtacgtcgt 660

aaaccgcgcg tacgcatcga agcgcaaatg cacggcggcg gtcacgagcg cggtatgcgt 720

tccggcactc tgcctgttca ccagatcgtc ggaatgggcg aggcctatcg catcgcaaaa 780

gaagagatgg cgaccgagat ggaacgtctg cgcggcctgc gtaaccgtct gtggaacggc 840

atcaaagata tcgaagaagt ttacctgaac ggtgacctgg aacacggtgc gccgaacatt 900

ctcaacgtca gcttcaacta cgttgaaggt gagtcgctga ttatggcgct gaaagacctc 960

gcagtttctt caggttccgc ctgtacgtca gcaagcctcg aaccgtccta cgtgctgcgc 1020

gcgctggggc tgaacgacga gctggcacat agctctatcc gtttctcttt aggtcgtttt 1080

actactgaag aagagatcga ctacaccatc gagttagttc gtaaatccat cggtcgtctg 1140

cgtgaccttt ctccgctgtg ggaaatgtac aagcagggcg tggatctgaa cagcatcgaa 1200

tgggctcatc attaa 1215

<210> 23

<211> 1374

<212> DNA

<213> NFS1

<400> 23

atgctgctcc gagtcgcttg gaggcgggcg gcagtggcgg tgacagcggc tccagggccg 60

aagcccgcgg cgcccactcg ggggctgcgc ctgcgcgttg gagaccgtgc tcctcagtct 120

gcggttcccg cagatacaac cgctgccccg gaggtggggc cagtgctgcg acctctctat 180

atggatgtgc aagctacaac tcctctggac ccccgggtgc ttgatgccat gctcccttac 240

ctaatcaact actatgggaa cccacactcc cggacacatg cttatggctg ggagagtgag 300

gcagccatgg aacgtgctcg tcagcaagta gcatctctga ttggagctga tcctcgtgag 360

atcattttta ctagtggtgc tactgaatcc aacaacatag caattaaggg ggtggcccga 420

ttctacaggt cacggaaaaa gcacttgatc accacccaga cagaacacaa atgtgtcttg 480

gactcctgcc gttcactgga agctgagggc tttcaggtca cctacctccc agtgcagaag 540

agtgggatca ttgacctaaa ggaactagag gctgctatcc agccagatac tagcctggtg 600

tcagtcatga ctgtgaacaa tgagattgga gtgaagcagc ctattgcaga aatagggcgg 660

atttgcagtt ccagaaaggt atatttccat actgatgcag cccaggctgt tggaaaaatc 720

ccacttgatg tcaatgacat gaaaattgat ctcatgagca ttagtggtca caaaatctac 780

ggtcccaaag gggttggtgc catctacatc cgtcgccggc cccgtgtgcg tgtggaggcc 840

ctgcagagtg gaggggggca ggagcggggt atgcggtctg ggacagtgcc cacaccctta 900

gtggtggggt tgggggctgc gtgtgaggtg gcacagcaag agatggagta tgaccacaag 960

cgaatctcaa agttgtcaga gcggctgata cagaatataa tgaagagcct tccagatgtg 1020

gtgatgaatg gggaccctaa gcaccattat cccggctgta tcaacctctc ctttgcatat 1080

gtggaagggg aaagtctgct gatggcactg aaggacgttg ccttatcctc agggagtgcc 1140

tgcacctctg catccctgga gccctcttat gtgcttagag caattggcac tgatgaggat 1200

ttagcgcact cttctatcag gtttggaatt ggcgctttca ctacagagga ggaagtggac 1260

tacacagtgg agaaatgcat tcagcatgtg aatcgtcttc gagaaatgag ccctctctgg 1320

gagatggttc aggatggcat tgacctcaag agcatcaagt ggacccaaca ctag 1374

<210> 24

<211> 276

<212> DNA

<213> ISD11

<400> 24

atggcagcct ccagtcgcgc acaagtgtta tctctgtacc gggcgatgct gagagagagc 60

aagcgtttca gcgcctacaa ttacagaaca tatgctgtca ggaggataag agatgccttc 120

agagaaaata aaaatgtaaa ggatcctgta gaaattcaaa ccctagtgaa taaagccaag 180

agagaccttg gagtaattcg tcgacaggtc cacattggcc aactgtattc aactgacaag 240

ctgatcattg agaatcgaga catgcccagg acctag 276

<210> 25

<211> 1218

<212> DNA

<213> Chimeric EH-IscS

<400> 25

atgaaattac cgatttatct cgactactcc gcaaccacgc cggtggaccc gcgtgttgcc 60

gagaaaatga tgcagtttat gacgatggac ggaacctttg gtaacccggc ctcccgttct 120

caccgtttcg gctggcaggc tgaagaagcg gtagatatcg cccgtaatca gattgccgat 180

ctggtcggcg ctgatccgcg tgaaatcgtc tttacctctg gtgcaaccga atctgacaac 240

ctggcgatca aaggtgcagc caacttttat cagaaaaaag gcaagcacat catcaccagc 300

aaaaccgaac acaaagcggt actggatacc tgccgtcagc tggagcgcga aggttttgaa 360

gtcacctacc tggcaccgca gcgtaacggc attatcgacc tgaaagaact tgaagcagcg 420

atgcgtgacg acaccatcct cgtgtccatc atgcacgtaa ataacgaaat cggcgtggtg 480

caggatatcg cggctatcgg cgaaatgtgc cgtgctcgtg gcattatcta tcacgttgat 540

gcaacccaga gcgtgggtaa actgcctatc gacctgagcc agttgaaagt tgacctgatg 600

tctttctccg gtcacaaaat ctatggcccg aaaggtatcg gtgcgctgta tgtacgtcgt 660

aaaccgcgcg tacgcatcga agcgcaaatg cacggcggcg gtcacgagcg cggtatgcgt 720

tccggcactc tgcctgttca ccagatcgtc ggaatgggcg aggcctatcg catcgcaaaa 780

gaagagatgg agtatgacca caagcgaatc tcaaagttgt cagagcggct gatacagaat 840

ataatgaaga gccttccaga tgtggtgatg aatggggacc ctaagcacca ttatcccggc 900

tgtatcaacc tctcctttgc atatgtggaa ggggaaagtc tgctgatggc actgaaggac 960

gttgccttat cctcagggag tgcctgcacc tctgcatccc tggagccctc ttatgtgctt 1020

agagcaattg gcactgatga ggatttagcg cactcttcta tcaggtttgg aattggccgc 1080

ttcactacag aggaggaagt ggactacaca gtggagaaat gcattcagca tgtgaagcgt 1140

cttcgagaaa tgagccctct ctgggagatg gttcaggatg gcattgacct caagagcatc 1200

aagtggaccc aacactag 1218

<210> 26

<211> 32

<212> DNA

<213> pCold-SUMO-F

<400> 26

ggatctaggt gaagatcctt tttgataatc tc 32

<210> 27

<211> 27

<212> DNA

<213> pCold-SUMO-R

<400> 27

aacaaatagg ggttccgcgc acatttc 27

<210> 28

<211> 41

<212> DNA

<213> Kana-F

<400> 28

cggaacccct atttgttgat cttttctacg gggtctgacg c 41

<210> 29

<211> 34

<212> DNA

<213> Kana-R

<400> 29

tcttcaccta gatcccaggt ggcacttttc gggg 34

<210> 30

<211> 5633

<212> DNA

<213> pET28a-SUMO

<400> 30

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100

agcagcggcc tggtgccgcg cggcagccat atggctagca tgtcggactc agaagtcaat 5160

caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag 5220

gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg 5280

ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac 5340

gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat 5400

attattgagg ctcacagaga acagattggt ggatccgaat tcgagctccg tcgacaagct 5460

tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg 5520

aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 5580

ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gat 5633

<210> 31

<211> 5603

<212> DNA

<213> pET-SUMO

<400> 31

agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 60

ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg ggtcatcacc 120

atcatcatca cgggtccctg caggactcag aagtcaatca agaagctaag ccagaggtca 180

agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga tcttcagaga 240

tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg ttcgctaaaa 300

gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga attcaagctg 360

atcaggcccc tgaagatttg gacatggagg ataacgatat tattgaggct caccgcgaac 420

agattggagg tcatatggga tccgaattcg agctccgtcg acaagcttgc ggccgcactc 480

gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag 540

ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc 600

ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct 660

gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 720

ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 780

gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 840

ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 900

gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 960

tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 1020

tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 1080

ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa 1140

cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct 1200

tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata 1260

ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat 1320

aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct 1380

attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact 1440

gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag 1500

ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc 1560

gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa 1620

tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat 1680

tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca 1740

tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt 1800

agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac 1860

aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca 1920

ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc 1980

ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg 2040

taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 2100

agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 2160

aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 2220

agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 2280

tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 2340

atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 2400

taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 2460

gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 2520

gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 2580

aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 2640

tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 2700

gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 2760

cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 2820

ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 2880

cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct 2940

gtgcggtatt tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca 3000

tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac 3060

acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 3120

gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 3180

aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt 3240

ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc 3300

tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt 3360

aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga 3420

tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg 3480

cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta 3540

atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca 3600

taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga 3660

ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct 3720

cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc 3780

tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct 3840

gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca 3900

agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct 3960

cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga 4020

cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt 4080

tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat 4140

taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 4200

aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt 4260

cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt 4320

tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt 4380

aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc 4440

gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg 4500

ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga 4560

aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga 4620

gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc 4680

gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta 4740

ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat 4800

aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga 4860

tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag 4920

gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg 4980

cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca 5040

acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa 5100

ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc 5160

tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat 5220

aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc 5280

ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg 5340

cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc 5400

aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac 5460

catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc 5520

ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac 5580

gatgcgtccg gcgtagagga tcg 5603

<210> 32

<211> 4333

<212> DNA

<213> pBAD-SUMO

<400> 32

aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60

tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120

aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180

attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240

atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc 300

taacaggagg aattaaccat gaatcacaaa gtgcatcatc atcatcatca cgggtccctg 360

caggactcag aagtcaatca agaagctaag ccagaggtca agccagaagt caagcctgag 420

actcacatca atttaaaggt gtccgatgga tcttcagaga tcttcttcaa gatcaaaaag 480

accactcctt taagaaggct gatggaagcg ttcgctaaaa gacagggtaa ggaaatggac 540

tccttaagat tcttgtacga cggtattaga attcaagctg atcaggcccc tgaagatttg 600

gacatggagg ataacgatat tattgaggct caccgcgaac agattggagg tcatatggag 660

ctcggtaccc tcgagggatc caagcttgtc gactctagat aggctgtttt ggcggatgag 720

agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga 780

atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga 840

aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg 900

catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg 960

tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag 1020

caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag 1080

cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt 1140

ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 1200

atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 1260

tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 1320

tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 1380

ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 1440

atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca 1500

ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 1560

catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 1620

cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 1680

ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 1740

cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 1800

cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 1860

tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 1920

agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 1980

ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 2040

gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 2100

atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 2160

cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 2220

agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 2280

ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 2340

accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 2400

tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 2460

cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 2520

gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 2580

gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 2640

gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 2700

cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 2760

tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 2820

ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 2880

ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 2940

taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 3000

agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg 3060

tatttcacac cgcagctggt gcactctcag tacaatctgc tctgatgccg catagttaag 3120

ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca 3180

acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 3240

gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 3300

aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg cataatgtgc ctgtcaaatg 3360

gacgaagcag ggattctgca aaccctatgc tactccgtca agccgtcaat tgtctgattc 3420

gttaccaatt atgacaactt gacggctaca tcattcactt tttcttcaca accggcacgg 3480

aactcgctcg ggctggcccc ggtgcatttt ttaaataccc gcgagaaata gagttgatcg 3540

tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc gggtggtgct caaaagcagc 3600

ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc taactgctgg 3660

cggaaaagat gtgacagacg cgacggcgac aagcaaacat gctgtgcgac gctggcgata 3720

tcaaaattgc tgtctgccag gtgatcgctg atgtactgac aagcctcgcg tacccgatta 3780

tccatcggtg gatggagcga ctcgttaatc gcttccatgc gccgcagtaa caattgctca 3840

agcagattta tcgccagcag ctccgaatag cgcccttccc cttgcccggc gttaatgatt 3900

tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta 3960

ttggcaaata ttgacggcca gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa 4020

acccactggt gataccattc gcgagcctcc ggatgacgac cgtagtgatg aatctctcct 4080

ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg atttttcacc 4140

accccctgac cgcgaatggt gagattgaga atataacctt tcattcccag cggtcggtcg 4200

ataaaaaaat cgagataacc gttggcctca atcggcgtta aacccgccac cagatgggca 4260

ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccatact tttcatactc 4320

ccgccattca gag 4333

Claims (13)

1. a kind of method of foreign protein solubility expression in Escherichia coli, it is characterised in that：By building cold shock hydrotropy Type expression plasmid improves solubility of the inclusion body in Bacillus coli cells, Activity and stabill.

2. the method for foreign protein according to claim 1 solubility expression in Escherichia coli, the plasmid used is Fig. 1 Constructed colibacillus expression plasmid pCold-SUMOa and pCold-SUMOb, it is characterised in that：With after Sal I single endonuclease digestions PCold TF plasmids be to set out carrier, SUMO gene code frames, which are inserted into the cold of the carrier that sets out, using seamless clone technology stops Between the promoter and multiple cloning sites of gram gene cspA.

3. the method for foreign protein according to claim 2 solubility expression in Escherichia coli, it is characterised in that：It is described Restriction endonuclease sites are followed successively by Nde I, Sac I in the multiple cloning sites of pCold-SUMOa and pCold-SUMOb plasmids, Kpn I,Xho I,BamH I,Hind III,Sal I,Xba I。

4. the method for foreign protein according to claim 2 solubility expression in Escherichia coli, it is characterised in that：It is described PCold-SUMOa and pCold-SUMOb plasmids carry cspA gene promoters and related elements.

5. the method for foreign protein according to claim 2 solubility expression in Escherichia coli, it is characterised in that：It is described The SUMO that pCold-SUMOa and pCold-SUMOb plasmids carry is that hydrotropy chaperone and foreign gene carry out fusion table together It reaches, the recombinant protein of expression can be used affinity chromatography and carry out a step isolation and purification.

6. the method for foreign protein according to claim 2 solubility expression in Escherichia coli, it is characterised in that：Ulp1 Can in the identification SUMO fusion proteins of specificity SUMO structural domains space structure, and it 100% is cut off, is suitable for advising greatly Mould prepares the recombinant protein with natural N end.

7. the method for foreign protein according to claim 6 solubility expression in Escherichia coli, it is characterised in that： In 50mM Tris-HCl, pH 7.5,150mM NaCl, 25 DEG C of reaction 1h, 100 μ g reference proteins have more than 95% by cutting institute The enzyme amount of Ulp1 is needed to be defined as 1U；According to 1U：The ratio of 100-1000 μ g albumen, digestion 1h, cleavable in 25 DEG C of water-baths 95% or more SUMO fusion proteins, wherein SUMO albumen and Ulp1 protease all carry 6 × His labels, are adopted after endonuclease reaction Purify removing with affinity chromatography.

8. a kind of biological reagent formula preserving Ulp1 protease, it is characterised in that：Including 50mM Tris-HCl pH 7.5, 150mM NaCl, 20% glycerine, 1mM benzenecarboximidamides, 0.2mM phenylmethylsulfonyl fluorides, 0.1mM ethyleneglycol bistetraacetic acids, 0.1%2- mercaptos Base ethyl alcohol and 0.03% Brij-35.

9. a kind of selectable marker gene by cold shock hydrotropy type expression plasmid by ampicillin resistance gene is changed to card, that is mould The method of plain resistant gene, it is characterised in that：Using pCold-SUMOa plasmids as template, using pCold-SUMO-F and pCold- SUMO-R primer amplifications obtain plasmid backbone segment, the gene order of pCold-SUMO-F as shown in SEQ ID N0.26, The gene order of pCold-SUMO-R is as shown in SEQ ID N0.27；Using pET-28b plasmids as template, using Kana-F and Kana-R primer amplifications, which obtain, blocks that resistance gene fragment, and the gene order of Kana-F is as shown in SEQ ID N0.28, Kana-R Gene order as shown in SEQ ID N0.29；Then above-mentioned two segment is connected using seamless clone technology and obtains recombination matter Grain, is named as pCold-SUMOb.

10. a kind of construction method of mosaic type disulfurase gene, it is characterised in that：It, will be big using round pcr is intersected N-terminal structural domain (1-263 amino acids) nucleic acid sequence of enterobacteria disulfurase IscS albumen and people source cysteine Desulfurase NFS1 PROTEIN Cs terminal domains (316-457 amino acids) nucleic acid sequence is merged, and a kind of half Guang ammonia of mosaic type is built Sour desulfurase expressing gene.

11. the mosaic type disulfurase of method structure according to claim 10, it is characterised in that：Half Guang of mosaic type The stability of propylhomoserin desulfurase is higher than people source disulfurase, and activity is slightly below Escherichia coli disulfurase.

12. a kind of method of foreign protein solubility expression in Escherichia coli biotech medicine product industry, structure biology, point The productions such as sub- biology and the application in research field.

13. a kind of method of foreign protein solubility expression in Escherichia coli biotech medicine product industry, structure biology, point The productions such as sub- biology and the application in research field, it is characterised in that：Target gene is cloned into the large intestine bar constructed by Fig. 1 In bacterium expression plasmid pCold-SUMOa and pCold-SUMOb plasmid.