CN108504670B - Construction method and application of escherichia coli cold shock solubilizing expression plasmid - Google Patents

Abstract

The invention relates to a construction scheme of escherichia coli solubilizing expression plasmid and a method for preparing water-soluble heterologous polypeptide by applying the same. The seamless cloning technology is utilized to construct a cold shock solubilizing expression plasmid which is expressed by the fusion of a promoter of a cold shock gene controlled small molecule ubiquitin-related modified protein (SUMO) and a foreign gene. Compared with the known pCold I and pET28 series plasmids, the water solubility, the stability and the enzyme activity of the recombinant protein are obviously improved by cloning the chimeric cysteine desulfhydrase into the plasmid. Whereas SUMO does not affect the spatial conformation of the protein of interest relative to the well-known pCold TF plasmid. According to the ratio of 1U:0.5-1mg of Ulp1 protease and recombinant protein, the enzyme digestion is carried out for 1h at 25 ℃, the cutting efficiency of the SUMO label is more than 95%, and the method is very suitable for preparing the protein with natural N end in the research fields of bioengineering and pharmacy, structural biology and the like.

Description

Construction method and application of escherichia coli cold shock solubilizing expression plasmid

Technical Field

The invention relates to a construction method of escherichia coli cold shock solubilizing expression plasmid and a specific implementation scheme of application thereof, belonging to the technical field of biological engineering.

Background

As the most common recombinant protein expression system, the Escherichia coli expression system has the characteristics of rapidness, mature technology, economy, high yield and clear genetic background of strains. Statistically, more than 60% of published documents related to recombinant protein expression are expressed in Escherichia coli, and nearly 30% of medicinal proteins are produced in Escherichia coli expression systems worldwide. However, when eukaryotic proteins are expressed in E.coli, only about 30% of the cloned genes may be expressed in soluble form in E.coli, while others are easily degraded by E.coli proteases, form inclusion bodies, or are not expressed. The inclusion body can be changed into soluble protein by adopting an in vitro renaturation method, but the method has complex operation flow, wastes time and labor and additionally increases the protein purification cost. In order to solve the above problems, researchers have explored various methods for improving the soluble expression of recombinant proteins in E.coli. The method mainly comprises the following steps:

1. co-expression with molecular chaperones, disulfide isomerases, etc. (see table 1 for details);

2. with the aid of fusion tags such as glutathione-S-transferase, thioredoxin and foldase (see Table 1 for details);

3. changing culture conditions such as cold shock expression (expression and production of cold shock-induced heterologous polypeptide. M. Ennoy, S. prader, B. summer, G. fine, H. koch. ZL 03804917.1), addition of ingredients (e.g., betaine, sorbitol, etc.) to the culture medium that contribute to correct conformational folding of the foreign protein, or control of the rate of protein synthesis by controlling the rate of feed of the feed at a later stage (a method effective for improving soluble expression of recombinant proteins in E.coli. Wei nationality, Yangxinging, Zhengchunyang. ZL 201310517277.7);

4. the solubility of the target protein is improved in a protein secretion mode (the functional construction of a SecB mediated post-translational targeting way and the application thereof. cuneiya, Zhouyai, Cheng et Shen, Roland, Flodel. ZL 201110458204.6);

5. a directed evolution technology is adopted to screen a protein mutant with strong water solubility/folding (T vector for screening protein soluble expression, a construction method and application thereof, Van Jun, Zhang Widiang, ZL 201010204681.5).

In addition, with the invention of seamless cloning and high-performance molecular cloning technologies such as Gateway, one target gene can be simultaneously inserted into a plurality of expression vectors, and then the optimal expression vector for expressing the target gene can be selected from the multiple expression vectors.

TABLE 1 Performance analysis of common chaperone or hydrotropic tag fusion type E.coli expression plasmids

When the protein is expressed in a soluble form, it is necessary in most cases to excise the solubilizing tag of the recombinant protein, particularly a genetically engineered pharmaceutical protein, and the protein obtained by the final purification must be the same as the natural protein and cannot be used as a fusion protein. This can be achieved by inserting an amino acid sequence between the solubilizing tag and the protein of interest which is recognized by a specific protease. The protease commonly used for cutting off the hydrotropic label at present comprises enterokinase EK, Thrombin Thrombin, coagulation Factor Xa, tobacco plaque virus (TEV) protease, small molecule ubiquitin-like specific protease 1 of saccharomyces cerevisiae (Ulp1) and the like. Among these proteases, Ulp1 is a very specific protease, and it also has several advantages: (1) compared with the common protease, the Ulp1 does not recognize a specific amino acid primary sequence, but specifically recognizes the spatial structure of the SUMO domain in the SUMO fusion protein, and cuts off the spatial structure, so that no redundant amino acid is left after cutting, and the method is very suitable for preparing the recombinant protein with the natural N terminal on a large scale; (2) ulp1 has very high cleavage efficiency, can be generally used as an initial reaction condition according to the mass ratio of enzyme to substrate of 1:1000, and can tolerate urea with the mass ratio of up to 2M; (3) the Ulp1 protease with high specific activity can be prepared by adopting an Escherichia coli expression system.

Although researchers have applied a variety of techniques to recombinant protein expression, there is no method in the art that is universally applicable to the preparation of soluble, homogeneous proteins with a native N-terminus. Therefore, the construction of the foreign protein soluble expression plasmid with strong applicability has higher academic value and wide application prospect.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects in the prior art, a novel cold shock solubilizing-aid expression plasmid is constructed, and the plasmid can improve the solubility, the activity and the stability of a recombinant protein inclusion body in cells; secondly, constructing a cold shock solubilizing expression plasmid which is easy to remove the recombinant protein N-terminal expression chaperone; and thirdly, the cold shock solubilizing expression plasmid is applied to preparing the high-activity cysteine desulfhydrase, so that a reference model is provided for the future application of the cold shock solubilizing expression plasmid in industrial production of useful proteins such as soluble medicinal protein, enzyme preparation, antigen polypeptide and the like.

The invention comprises one of the following contents: provides a method for constructing escherichia coli cold shock solubilizing expression plasmid

The construction method of the escherichia coli cold shock solubilizing expression plasmids pCold-SUMOa and pCold-SUMOb comprises the following steps:

construction of ampicillin-resistant pCold-SUMOa plasmid:

pE-SUMOpro Kan plasmid is taken as a template, and SUMO-F (with a sequence shown in SEQ ID N0.5) and SUMO-R (with a sequence shown in SEQ ID N0.6) primers are adopted for amplification to obtain the small molecule ubiquitin-related modified protein (SUMO) gene coding frame fragment. The pCold TF plasmid after Sal I single digestion is taken as a template, and pCold-F (shown as SEQ ID N0.3) and pCold-R (shown as SEQ ID N0.4) primers are adopted for amplification to obtain a pCold plasmid skeleton fragment without a TF (Trigger factor) molecular chaperone. Wherein, the 5 'ends of the primer SUMO-F and the primer pCold-R contain homologous arms with 19bp being same, and the 5' ends of the primer SUMO-R and the primer pCold-F contain homologous arms with 15bp being same. The two fragments are connected by adopting a seamless cloning technology to obtain a recombinant plasmid which is named pCold-SUMOa. The restriction enzymes recognizing a single cleavage site in the Multiple Cloning Site (MCS) of pCold-SUMOa plasmid are Nde I, Sac I, Kpn I, Xho I, BamH I, Hind III, Sal I, Xba I in this order.

Construction of kanamycin-resistant pCold-SUMOB plasmid:

the pCold-SUMOa plasmid is taken as a template, and pCold-SUMO-F (the sequence is shown as SEQ ID N0.34) and pCold-SUMO-R (the sequence is shown as SEQ ID N0.35) primers are adopted for amplification to obtain a plasmid framework fragment. Using pET-28b plasmid as template, adopting Kana-F (sequence shown in SEQ ID N0.36) and Kana-R (sequence shown in SEQ ID N0.37) primer to amplify and obtain Kana resistance gene fragment. Then, the two fragments are connected by adopting a seamless cloning technology to obtain a recombinant plasmid which is named pCold-SUMOb.

The second invention is: provides an application embodiment of the cold shock solubilizing expression plasmid for preparing the cysteine desulfhydrase with high activity and strong stability

The application embodiment of the preparation of the cysteine desulfhydrase with high activity and strong stability is as follows:

1. constructing recombinant plasmids expressing the cysteine desulfhydrase (IscS) of Escherichia coli, the humanized cysteine desulfhydrase (NFS1) and the chimeric cysteine desulfhydrase (EH-IscS).

2. The recombinant plasmid is transformed into BL21(DE3) competent cells by a calcium chloride transformation method to construct an expression strain, and the cysteine desulfhydrase expression is induced by cold shock (at a low temperature of 15 ℃) and IPTG.

3. Purifying cysteine desulfhydrase by nickel column affinity chromatography.

4. The activity and stability of purified cysteine desulfhydrase were analyzed by Hitachi U3900 UV-visible spectrophotometer.

The construction method of the escherichia coli cold shock solubilizing expression plasmid and the method for preparing the high-activity cysteine desulfhydrase by applying the escherichia coli cold shock solubilizing expression plasmid have the following innovation points and technical advantages:

1. the invention provides a method for randomly combining known or newly-discovered escherichia coli expression plasmid promoters, known or newly-discovered solubilizing labels and other plasmid elements which are beneficial to protein soluble expression and cutting fusion labels thereof based on a seamless cloning technology, thereby providing a reference scheme for constructing one or a series of plasmids generally suitable for exogenous protein soluble expression.

2. The invention uses seamless cloning technology to fuse the cspA gene promoter of one of cold shock genes with the coding frame of small molecule ubiquitin-related modified protein (SUMO) gene to construct a new escherichia coli cold shock solubilizing-aid expression plasmid, which has the following technical advantages:

2.1 pCold-SUMOa and pCold-SUMOb plasmids carry cspA gene promoter and related elements, and when Escherichia coli is cultured at 15 ℃ low temperature, the low temperature can inhibit cell protein expression and inhibit the growth of thalli. However, the cspA promoter has no reduction of transcription activity at low temperature, the 5' UTR has stable structure at low temperature, the ribosome has a capture phenomenon, and the protein can still be effectively expressed at low temperature. Therefore, compared with the pET28a-SUMO plasmid which is commercially available and is induced and cultured at 37 ℃, the process can realize the expression of the target protein with high yield (up to 60 percent and more of cellular protein) and higher purity.

2.2 Small molecule ubiquitin-like specific protease 1(Ulp1) is a SUMO specific protease, can specifically recognize the spatial structure of the SUMO domain in SUMO fusion protein, and can cut off 100% of the SUMO domain, wherein both the SUMO protein and the Ulp1 protease have 6 XHis tags, and can be purified and removed by nickel column affinity chromatography after enzyme digestion reaction. Therefore, the plasmid of the present invention is suitable for large-scale production of recombinant proteins having a native N-terminus.

3. The preparation method of the chimeric cysteine desulfhydrase provided by the invention is reported for the first time, the chimeric cysteine desulfhydrase has the characteristic of high activity relative to Escherichia coli cysteine desulfhydrase, has the characteristics of good solubility and high activity relative to humanized cysteine desulfhydrase, and in addition, the chimeric cysteine desulfhydrase does not generate self-aggregation phenomenon in vitro like humanized cysteine desulfhydrase, so the chimeric cysteine desulfhydrase has the characteristic of good stability.

Technical effects

1. The constructed cold shock solubilizing-aid expression plasmids (pCold-SUMOa and pCold-SUMOb) can improve the solubility, activity and stability of the recombinant protein inclusion body in cells.

2. By constructing the chimeric protein of the eukaryotic and prokaryotic homologous proteins, the solubility of the eukaryotic protein in a prokaryotic expression system is effectively improved on the premise of ensuring that the chimeric protein has the same function and activity as the parent protein.

3. The chimeric cysteine desulfhydrase gene is cloned to the cold shock solubilizing expression vector, compared with the known pET28b, pCold I and pCold-GST vectors, the expression quantity, stability and activity of soluble protein are obviously improved, 10-20mg of protein can be obtained by one-step purification of 1L bacterial liquid through nickel column affinity chromatography, the purity is more than 95%, and the chimeric cysteine desulfhydrase obtained by purification can be applied to industrial synthesis, biological desulfurization and in-vitro preparation of functional iron-sulfur protein of sulfur-containing biomolecules.

Drawings

FIG. 1. Escherichia coli cold shock solubilizing expression plasmid map: (A) pCold-SUMOa plasmid map; (B) pCold-SUMOB plasmid map.

FIG. 2 SDS-PAGE analysis of protein expression and purification of recombinant cysteine desulfhydrase in different vectors: (A) SDS-PAGE analysis of protein expression profiles of chimeric cysteine desulfhydrases in pCold I, pCold-His-GST, pCold-SUMOa and pCold TF plasmids, Lane 1: uninduced whole cell lysate supernatant; (B) SDS-PAGE analysis of protein expression maps of the chimeric cysteine desulfhydrase in pET28b, pET28a-SUMO, pBAD-SUMO and pET-SUMO plasmids; (C) SDS-PAGE analysis of protein expression maps of the humanized cysteine desulfhydrase in pCold I, pCold-His-GST, pCold-SUMOa, pCold TF, pET28b, pET28a-SUMO and pBAD-SUMO plasmids; (D) the purity of the recombinant cysteine desulfhydrase was analyzed by SDS-PAGE. Lane1, E.coli cysteine desulfurization enzyme IscS, lane 2, chimeric cysteine desulfurization enzyme EH-IscS, lane 3, GST-EH-IscS, lane 4, SUMO-EH-IscS, lane 5, TF-EH-IscS, lane 6, SUMO-NFS1(55-457), lane 7, TF-NFS1 (55-457). M represents protein molecular weight standard, T represents induced whole cell lysate, and S represents induced whole cell lysate supernatant.

FIG. 3 is an ultraviolet-visible light analysis spectrum of the recombinant cysteine desulfurization enzyme and the analysis of the activity of the cysteine desulfurization enzyme: (A) and analyzing the purified escherichia coli and chimeric cysteine desulfurization enzyme spectroscopy property spectrum by using an ultraviolet-visible spectrophotometer. Spectrum 1: TF-EH-IscS; spectrum 2: IscS; spectrum 3: EH-IscS; spectrum 4: SUMO-EH-IscS; spectrum 5: GST-EH-IscS; (B) analyzing the spectral property spectrum of the purified human cysteine desulfhydrase by an ultraviolet-visible spectrophotometer, wherein the spectrum 1: TF-NFS1 (55-457); spectrum 2: SUMO-NFS1 (55-457); (C) and (3) an activity analysis map of the recombinant cysteine desulfurization enzyme.

FIG. 4. stability analysis of recombinant cysteine desulfhydrase: (A-B) analysis of the effect of solubilizing chaperones on protein stability.

FIG. 5 is a graph showing the efficiency of cleavage of SUMO tag in SUMO-EH-IscS recombinant protein by Ulp1 protease analyzed by SDS-PAGE: m represents a protein molecular weight standard; lane 1-SUMO-EH-IscS recombinant protein obtained by nickel column purification; lanes 2-4 Ulp1 (3.5. mu.g) protease cleaved cleavage products of 100. mu.g, 500. mu.g and 1000. mu.g of SUMO-EH-IscS recombinant protein, respectively; lane 5 EH-IscS protein purified after digestion with Ulp 1; lane-6: Ulp1 protease.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings and examples.

Example 1 construction of E.coli Cold shock solubilization type expression vector

1. Construction of ampicillin-resistant pCold-SUMOa plasmid

A pE-SUMOpro Kan plasmid of Lifesensors company is taken as a template, and a Smt3 fusion protein sequence (the sequence is shown as SEQ ID N0.19) is obtained by amplification by adopting a primer SUMO-F (the sequence is shown as SEQ ID N0.3) and a SUMO-R (the sequence is shown as SEQ ID N0.4). Meanwhile, a pCold TF plasmid subjected to Sal I single enzyme digestion is used as a template, and primers pCold-F (with a sequence shown as SEQ ID N0.1) and pCold-R (with a sequence shown as SEQ ID N0.2) are adopted for amplification to obtain a pCold plasmid skeleton fragment. The two fragments were then ligated using the seamless cloning technique to obtain a recombinant plasmid, designated pCold-SUMOa (shown in FIG. 1). Because the gene sequence of the Smt3 fusion protein contains two enzyme cutting sites of EcoR I and Pst I, a pCold TF plasmid after Sal I single enzyme cutting is selected as a template, and the two enzyme cutting sites contained in the multiple cloning sites of the pCold-SUMOa recombinant plasmid are avoided through primer design. The restriction enzymes for recognizing a single restriction site in the multiple cloning site of pCold-SUMOa plasmid are Nde I, Sac I, Kpn I, Xho I, BamH I, Hind III, Sal I and Xba I in sequence.

2. Construction of kanamycin-resistant pCold-SUMOB plasmid

The pCold-SUMOa plasmid is taken as a template, and pCold-SUMO-F (the sequence is shown as SEQ ID N0.26) and pCold-SUMO-R (the sequence is shown as SEQ ID N0.27) primers are adopted for amplification to obtain a plasmid framework fragment. Using pET-28b plasmid as template, adopting Kana-F (sequence shown in SEQ ID N0.28) and Kana-R (sequence shown in SEQ ID N0.29) primer to amplify and obtain Kana resistance gene fragment. The two fragments were then ligated using the seamless cloning technique to obtain a recombinant plasmid, designated pCold-SUMOB (shown in FIG. 1).

The pCold-SUMOa plasmid resistance was engineered from ampicillin to kanamycin resistance for the purpose:

ampicillin is a lactam antibiotic that interferes with the cross-linking of peptidoglycan to inhibit cell wall synthesis. The gram-negative bacterial strain has low cell wall peptidoglycan content and is insensitive to ampicillin, satellite colonies are easy to grow around the positive clone colonies after overnight culture, and single colonies are not easy to pick; kanamycin is a protein biosynthesis inhibitor and has a good bacteriostatic effect on escherichia coli.

Example 2 construction of recombinant plasmid expressing cysteine desulfurization enzyme

1. Construction of recombinant plasmid for expression of cysteine desulfurization enzyme of Escherichia coli

Coli E.coli MC4100 genome is used as a template, and primers IscS-pCold-F (shown in SEQ ID N0.9) and IscS-pCold-R (shown in SEQ ID N0.10) are adopted for amplification to obtain the E.coli IscS coding frame gene. The pCold I plasmid is digested by Kpn I single enzyme, and the digested vector fragment is recovered by gel. Then, a seamless cloning kit is adopted to construct and obtain a recombinant plasmid IscS-pCold I, and the recombinant plasmid is sent to Huada gene sequencing for identification.

2. Construction of recombinant plasmid expressing humanized cysteine desulfurization enzyme

A gene sequence containing 55-457 amino acids for encoding the human cysteine desulfurization enzyme is obtained by amplification by using NFS1(55-457) -pET28b plasmid as a template and primers m-NFS1-pCold-F (the sequence is shown as SEQ ID N0.11)/SUMO-m-NFS 1-F (the sequence is shown as SEQ ID N0.13) and m-NFS1-pCold-R (the sequence is shown as SEQ ID N0.12)/SUMO-m-NFS 1-F (the sequence is shown as SEQ ID N0.24), wherein the 1-54 amino acid sequence in NFS1 protein is a signal peptide sequence for mediating the NFS1 protein to enter the mitochondria. The pCold I, pCold-GST and pCold TF plasmids were digested with Kpn I alone, and the digested vector fragment was recovered by gel digestion. The pCold-SUMOa plasmid is digested by Nde I single enzyme, and the digested vector fragment is recovered. Then, the NFS1 gene is respectively connected to the three single-enzyme-digested vector fragments by using a seamless cloning kit to 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 the three recombinant plasmids are sent to Huada gene sequencing and identification.

3. Construction of recombinant plasmid expressing chimeric cysteine desulfurization enzyme

3.1 amplification of IscS protein N-terminal Domain nucleic acid sequences

3.1.1 PCR amplification of IscS protein N-terminal domain (amino acids 1-263) nucleic acid sequence system and conditions as follows:

at 95 ℃ for 3 min; 95 ℃,10sec,51.6 ℃,30sec,72 ℃,30sec,35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.

3.1.2 Dpn I digestion of methylated IscS-pCold I plasmid template

The enzyme digestion system and conditions were as follows:

incubation at 37 ℃ for 1 hour or more.

3.1.3 gel recovery of the cleavage product:

performing 1% low melting point agarose gel electrophoresis on the product after the enzyme digestion reaction, cutting a target DNA fragment under a long-wave ultraviolet lamp, and performing TaKaRa

The company glue recovery kit is used for recovery. Finally, the recovered product was quantified using a Du-800 nucleic acid/protein analyzer.

3.2 amplification of the C-terminal Domain nucleic acid sequence of the NFS1 protein

3.2.1 PCR amplification of the C-terminal domain (amino acid 457 at position 316-:

at 95 ℃ for 3 min; 95 ℃,10sec,52 ℃,15sec,72 ℃,30sec,35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.

3.2.2 Dpn I digestion of methylated NFS1(55-457) -pCold I plasmid template

The enzyme digestion system and conditions were as follows:

incubation at 37 ℃ for 1 hour or more.

3.2.3 gel recovery of the cleavage product:

and (3) performing 1% low-melting point agarose gel electrophoresis on the product after the enzyme digestion reaction, cutting a target DNA fragment under a long-wave ultraviolet lamp, and recovering by using a TaKaRa gel recovery kit. Finally, the recovered product was quantified using a Du-800 nucleic acid/protein analyzer.

3.3 construction of chimeric Gene of Escherichia coli and humanized cysteine desulfurization enzyme

3.3.1 PCR reaction System and conditions were as follows:

at 95 ℃ for 3 min; 95 ℃,10sec,53 ℃,45sec,72 ℃,30sec,35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.

3.3.2 gel recovery of PCR products:

the PCR product was subjected to 1% low melting point agarose gel electrophoresis, and the target DNA fragment was cut under a long-wave ultraviolet lamp and recovered with a TaKaRa gel recovery kit. Finally, the recovered product was quantified using a Du-800 nucleic acid/protein analyzer.

3.4 construction of recombinant plasmids expressing EH-IscS

3.4.1 PCR amplification of EH-IscS Gene

The PCR reaction system and conditions were as follows:

at 95 ℃ for 3 min; 95 ℃,10sec,53 ℃,45sec,72 ℃,30sec,35 cycles; preserving at 72 deg.C for 10min and 4 deg.C.

The PCR product was subjected to 1% low melting point agarose gel electrophoresis, and the target DNA fragment was cut under a long-wave ultraviolet lamp and recovered with a TaKaRa gel recovery kit. Finally, the recovered product was quantified using a Du-800 nucleic acid/protein analyzer.

Preparation of 3.4.2 pCold I, pCold-GST, pCold TF, pCold-SUMOa, pET28a-SUMO, pET-SUMO and pBAD-SUMO linearized vector fragments

The pET28a-SUMO, pET-SUMO and pBAD-SUMO plasmids were provided by the institute for enzyme engineering and medical diagnostics, Wenzhou university of medical science.

The pCold I, pCold-GST and pCold TF plasmid single cleavage system is as follows:

the enzyme was cleaved at 37 ℃ for 2 h.

The single digestion system of pCold-SUMOa, pET-SUMO and pBAD-SUMO plasmids is as follows:

the enzyme was cleaved at 37 ℃ for 2 h.

The pET28a-SUMO plasmid single enzyme digestion system is as follows:

the enzyme was cleaved at 37 ℃ for 2 h.

Adding 10 XLoading buffer into the enzyme digestion product, performing 1% low melting point agarose gel electrophoresis, cutting the target vector fragment under a long-wave ultraviolet lamp, and recovering by using a TaKaRa gel recovery kit. Finally, the recovered product was quantified using a Du-800 nucleic acid/protein analyzer.

3.5 construction of recombinant plasmids expressing EH-IscS Using seamless cloning kit (and Biotech Ltd.)

3.5.1 seamless cloning reaction System:

incubate at 37 ℃ for 30min, then transfer to ice and stand for 5 min. DH 5. alpha. competent cells were transformed with 10. mu.l of the above reaction solution.

3.5.2 transformation: by means of cold CaCl₂Method 10. mu.l of the above seamless cloning reaction was transformed into DH5 competent cells.

3.5.3 colony PCR identification of Positive clones

Picking the LB/Amp with an inoculating needle⁺Single colonies grown on the plates were streaked a few times onto a sterile LB/Amp⁺Or LB/Kana⁺On the plate, the plate was cultured in an inverted state at 37 ℃ for 10 hours. Then, a small amount of the cells grown in each cell on the plate were picked up with a sterile pipette tip, mixed in an EP tube containing 50. mu.l of sterile water, boiled in boiling water for 10min, centrifuged at 12000rpm for 5min, and 9.2. mu.l of the supernatant was taken out and added to a 200. mu.l PCR reaction tube as a PCR identification template. And the following systems were added to the tube and mixed well.

94 ℃ for 5 min; 94 ℃,30sec,50.3 ℃,30sec,72 ℃,1min, 30sec,25 cycles; preserving at 72 deg.C for 10min and 4 deg.C. Mu.l of the PCR product was identified by electrophoresis on a 1.0% Agarose gel. Two positive clones which are correctly identified are selected and sent to Huada gene sequencing for identification.

Example 3 Induction of cysteine desulfhydrase and optimization of expression conditions

Passing the constructed recombinant plasmid through cold CaCl₂The method is transformed into BL21(DE3) competent cells. The induction and expression of the recombinant cysteine desulfurization enzyme are operated according to the following experimental steps except for special statement:

(1) inoculating overnight bacteria of the selected transformant into LB liquid culture medium containing 100 mu g/ml Amp according to the volume ratio of 1:50, and performing shaking culture at 37 ℃ and 250 rpm;

(2) when the OD600 value of the bacterial liquid is about 0.4-0.5, immediately placing the bacterial liquid in an ice-water bath for 5min, and then moving to 15 ℃ for standing for 30 min;

(3) IPTG was added to a final concentration of 100. mu.M/L, and shaking culture was carried out at 15 ℃ and 250rpm for 24 hours.

(4) After the completion of the culture, the presence or absence, expression level and solubility of the target protein were analyzed by SDS-PAGE.

3.1 optimization of initial inoculum size of overnight bacteria

The selected overnight-transformants were inoculated into LB liquid medium containing 100. mu.g/ml Amp at a volume ratio of 1:50,1:500, and 1:1000, respectively, and cultured at 37 ℃ with shaking at 250 rpm. The subsequent experimental procedures were as described in example 4.

3.2 Effect of IPTG inducer concentration on recombinant protein expression

Five IPTG inducer concentrations were set at 0, 100, 200, 400, 800. mu.M/L, respectively, and the other experimental procedures were as described in example 4.

3.3 Experimental results: (1) the initial inoculation amount of the overnight bacteria is within the range of 1:1000-1:50(v/v), the expression amount of the recombinant cysteine desulfhydrase is not obviously influenced, and therefore, in order to save the culture time of the bacteria, 1:50 is determined as the optimal inoculation amount; (2) SDS-PAGE analysis shows that IPTG inducer concentration has no significant influence on IscS protein expression in the range of 100-800. mu.M/L, so that 100. mu.M/L IPTG is determined as the optimal inducer concentration for saving cost; (3) as shown in FIG. 2, the expression of NFS1(55-457) as a human cysteine desulfurization enzyme in pET28b, pCold I, pCold-GST and pBAD-SUMO plasmids was observed as inclusion bodies. And similar to the effect of pET28a-SUMO plasmid which is already commercialized, pCold-SUMOa plasmid can improve the soluble expression of NFS1(55-457) protein in a small part, and TF molecular chaperones can make NFS1(55-457) protein be completely expressed in a soluble form. When the chimeric cysteine desulfurization enzyme EH-IscS is expressed by using pET28b plasmid, the chimeric cysteine desulfurization enzyme EH-IscS exists in the form of inclusion body, while pCold I, pCold-GST, pCold-SUMOa/pET28a-SUMO/pET-SUMO/pBAD-SUMO and pCold TF plasmids can sequentially improve the solubility of EH-IscS protein, and the pCold-SUMOa plasmid can improve the yield of target protein and the proportion of the target protein in the total protein of the whole cell compared with pET28a-SUMO, pET-SUMO and pBAD-SUMO plasmids, and the whole cell after induction of protein expression can be directly used for nuclear magnetic resonance analysis. In conclusion, the escherichia coli cold shock solubilizing expression vector has good application potential in the aspect of promoting the soluble expression of heterologous polypeptides.

Example 4 isolation and purification of recombinant cysteine desulfurization enzyme

4.1 separation and purification of recombinant cysteine desulfurization enzyme by nickel column affinity chromatography

The IscS, EH-IscS, SUMO-EH-IscS, GST-EH-IscS, TF-EH-IscS and SUMO-NFS1(55-457) TF-NFS1(55-457) proteins are separated and purified by nickel column affinity chromatography. The specific experimental procedures are as follows:

4.1.1 preparation of buffer for separating and purifying protein:

solution A: also known as DB (desaling Buffer) or Binding Buffer, pH 8.0, used for protein preservation, purification combined with column Buffer, 4 degrees C storage.

And B, liquid B: also known as Ellution Buffer, is used for eluting the protein bound on the chromatographic column and is stored at 4 ℃ in the dark.

4.1.24 ℃, centrifuging at 6,000rpm for 6min, and collecting thalli after culturing at 15 ℃ for 24 hours;

4.1.3 resuspending the cells with a desaling Buffer and centrifuging, washing twice repeatedly, and then resuspending the cells with 30ml of precooled desaling Buffer;

4.1.4 cell disruption: crushing the cells for 4 times by using a high-pressure cell crusher at the temperature of 4 ℃ and under the conditions of 1000-;

4.1.5 after disruption, the suspension was centrifuged at 24,000rpm for 45min at 4 ℃ and the supernatant was purified after filtration through a 0.45. mu.M filter.

4.1.6 starting the computer, the protein purifier, starting the running program and waiting for the connection of the instrument and the computer;

4.1.7 ddH₂o cleaning the A1 and B1 pipelines flowing through the A pump and the B pump, pumping wash three times, cleaning the A1 and B1 pipelines by using the A liquid and the B liquid,

the pump wash is carried out twice;

4.1.8 connection of Ni column for purification, washing the Ni column with 100% Elution Buffer at a flow rate of 3ml/min for 3min, replacing 20 volumes of A solution on the Ni column to balance the Ni column at a flow rate of 3ml/min for about 10-15min to stabilize the baseline, ddH₂O, cleaning the sample loading column;

4.1.9 injecting the filtered protein supernatant into the sample loading column, starting the nickel column affinity chromatography purification program, and waiting for the instrument to automatically complete the protein purification

Procedures, i.e., equilibration, loading, elution, and collection.

The specific parameters of the nickel column affinity chromatography purification procedure are as follows:

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

And (3) elution: eluting the hybrid protein 10cv 3ml/min, 6% Elution Buffer

Eluting the target protein: 15cv 3ml/min, 100% Elution Buffer

Collecting: collecting 2ml of target protein according to a sample tube corresponding to a protein absorption peak displayed on a program;

4.1.10 protein desalting: after the protein purification is finished, the Desalting column desaling tube is replaced, and after the column is balanced, the nickel column is used for purifying the protein sample

The product is sequentially desalted.

Collecting: 2ml of the target protein was collected in a sample tube corresponding to the protein absorption peak shown in the program and placed on ice.

4.1.11 determination of protein concentration

The protein was scanned at a full wavelength of 250-700nm using an ultraviolet-visible spectrophotometer (U3900). Since the protein has a specific absorption peak at 280nm, the concentration of the corresponding protein is calculated by the following formula by its specific extinction coefficient at 280nm (the extinction coefficient of each protein is calculated from the contained amino acids).

Protein concentration calculation formula: c_pro＝(OD₂₈₀-OD₇₀₀) 1000/extinction coefficient (unit: mu M)

4.2 Experimental results: (1) the protein purity of the recombinant cysteine desulfhydrase obtained by SDS-PAGE and Coomassie brilliant blue staining identification and purification is higher than 90%, and the recombinant cysteine desulfhydrase can be directly used for subsequent enzymology experiment analysis (as shown in figure 2); (2) the purified protein is subjected to full-wavelength scanning analysis, and the chimeric EH-IscS, GST-EH-IscS and SUMO-EH-IscS are consistent with the Escherichia coli IscS, a characteristic absorption peak of cysteine desulfurization enzyme prosthetic group pyridoxal phosphate (PLP) appears at 395nm, and the protein is light yellow or yellow. The human cysteine desulfhydrase NFS1(55-457) and SUMO-NFS1(55-457) show PLP characteristic absorption peaks at 420nm, and the protein is also light yellow or yellow. The purified TF-EH-IscS is light yellow, but its cysteine desulfhydrase activity is very weak. The purified TF-NFS1(55-457) protein is colorless and has a very weak characteristic absorption peak at 420nm, indicating that the target protein is not bound to PLP, because the Trigger factor tag is too large (52kDa) and may affect the spatial structure of the target protein, while the SUMO tag is relatively small (16kDa), which is used as a molecular chaperone to facilitate the correct folding of the target protein and does not affect the spatial structure of the target protein (as shown in FIG. 3).

Example 5 determination of cysteine desulfurization enzyme Activity

5.1 determination of iron and Sulfur content in Nth protein sample

5.1.1 determination of iron content in Nth protein samples

The iron content in the protein sample is determined by adopting a feloxazine method, and the basic principle is as follows: under the action of L-cysteine, iron in the protein sample is released and combined with the phenazine (Ferrozine) to form Fe-Ferrozine complex. The complex has a specific absorption peak at 564nm and an extinction coefficient of 27.9mM^-1cm^-1And measuring the value of the absorption peak, so that the iron content in the protein can be calculated by using a formula.

After the system was added as above, the mixture was heated at 85 ℃ for 20min, left at room temperature for 30min, centrifuged at 12,000rpm for 5min, the supernatant was scanned at a full wavelength of 450-700nm, and a Desalting Buffer was used as a negative control.

Calculating the iron content according to the formula: c_Fe＝(OD₅₆₄-OD₇₀₀) 1000/27.9 (unit: mu M)

5.1.2 determination of Sulfur content in Nth protein samples

Determination of sulfur content in protein samplesThe DPD method is adopted, and the basic principle is as follows: sulfide and FeCl₃And (3) interaction, wherein DPD is used as a color developing agent, a characteristic absorption peak can be formed at 669nm, and the sulfur content in the protein can be calculated by using a formula and the extinction coefficient of sulfur.

After the system was added as above, the mixture was left at room temperature for 30min, centrifuged at 12,000rpm for 5min, the supernatant was subjected to 450-fold 800nm full-wavelength scanning, a desaling Buffer was used as a negative control, and the extinction coefficient of sulfur was calculated by Nth.

Calculating the sulfur content according to the formula: c_S＝(OD₆₆₉-OD₈₀₀) 1000/S extinction coefficient (unit: mu M)

Extinction coefficient of S ═ OD₆₆₉-OD₈₀₀)*1000/Nth C_S

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

5.2 determination of cysteine Thiosylase Activity in protein samples IscS, EH-IscS, GST-EH-IscS, SUMO-EH-IscS, TF-EH-IscS, SUMO-NFS1(55-457) and TF-NFS1(55-457)

1ml of reaction system was prepared before activity determination: taking protein with known concentration, diluting with solution A (DB) to final concentration of 10 μ M, adding 1M DTT 3 μ L (final concentration of 3mM), mixing, incubating at 37 deg.C for 5min, immediately adding 20 μ L of 0.1M L-cysteine (final concentration of 2mM), reversing mixing, immediately taking 160 μ L to determine sulfur content, defining the time as 0min, taking sample (160 μ L) every 5min to determine sulfur content, terminating at 15min, and repeating for 4 times.

The catalytic reaction system is as follows:

the above system was mixed and incubated at 37 deg.C for 5min, 20. mu.l of 0.1M L-cysteine was added, 160. mu.L of the mixture was taken to measure the sulfur content at that time, and the measurement was performed every 10min thereafter.

Sulfur content determination system:

the sulfur measurement system is stood still on a dry thermostat at 37 ℃ and heated for 20min, after centrifugation at 12,000rpm for 5min, a full-wavelength scan of 500nm-800nm is carried out with U3900, and a characteristic absorption peak at 669nm can be seen.

According to formula C_S＝(OD₆₆₉-OD₈₀₀) Extinction coefficient of/S1000 (unit: μ M) and how much of the sulfur content directly reflects the magnitude of the IscS protease activity. DB as a negative control, protein Nth (a very stable [4Fe-4S ]]) As a positive control, the extinction coefficient of sulfur was calculated from the content of iron in the positive control Nth. (Note: the method for measuring the Fe and S contents in Nth is as described in 5.1)

And (3) drawing a time-sulfur content curve chart after the measurement is finished: and (4) drawing a time-sulfur content curve chart according to the calculated sulfur content, and analyzing the enzyme activity of each protein.

5.3 Experimental results: (1) as shown in fig. 3C, SUMO-EH-IscS protein cysteine desulfurase activity is the highest and higher than that of escherichia coli IscS protein, and EH-IscS protein cysteine desulfurase activity is lower than that of IscS protein, which indicates that SUMO chaperones can improve EH-IscS protein solubility on one hand and can significantly improve EH-IscS protein activity on the other hand, as the reason may be related to SUMO chaperones that can enhance EH-IscS protein stability and prevent self-aggregation, and the specific molecular mechanism thereof is to be further studied; (2) the TF-EH-IscS protein has weak cysteine desulfurase activity, because the Trigger factor label is too large (52kDa), which may affect the spatial structure of the target protein and is consistent with the analysis result; (3) human cysteine desulfhydrase NFS1 is active by binding to ISD11, and thus, it has no cysteine desulfhydrase activity when SUMO-NFS1(55-457) and TF-NFS1(55-457) proteins are tested alone.

Example 6 Effect of different hydrotropic tags on the stability of recombinant cysteine desulfhydrase

The purified IscS, EH-IscS, GST-EH-IscS, SUMO-EH-IscS and TF-EH-IscS protein concentrations were diluted to 1. mu.M, respectively, using DB, and then incubated at 37 ℃ for 30min, and absorbance at 320nm of the protein samples was measured every 5 min. If the protein sample undergoes self-aggregation, the absorbance at 320nm increases with time. The horizontal axis represents time, and the vertical axis represents absorbance at 320 nm. As shown in fig. 4: (1) although the chimeric cysteine desulfurization enzyme EH-IscS has better activity of cysteine desulfurization enzyme, the protein has poor stability, and the self-aggregation phenomenon is more obvious when the protein concentration is higher; (2) the recombinant protein expressed after the EH-IscS is fused with the SUMO and the TF tag has good stability. In addition, the document reports that NFS1 is a protein which is easy to self-agglutinate, but in the experiment, SUMO-NFS1(55-457) and TF-NFS1(55-457) proteins have good stability, which indicates that the solubility-promoting tag can enhance the solubility of the protein and also can prevent the self-agglutination of the protein.

Example 7 construction of recombinant plasmid expressing Ulp1 protease

7.1 expression and purification of Ulp1 protease

The cDNA of Saccharomyces cerevisiae BY4743 is used as a template, the PCR technology is adopted to amplify the active fragment at the position 404-621 of the small-molecule ubiquitin-like specific protease 1 of the Saccharomyces cerevisiae, and the active fragment is cloned into the Nde I restriction enzyme cutting site of the pET28b plasmid BY utilizing the seamless cloning technology to construct the Ulp1-pET28b recombinant plasmid.

The recombinant plasmid of Ulp1-pET28b is transformed into BL21(DE3) competent cells by a calcium chloride method, and E.coli Ulp1-pET28b/BL21 genetic engineering strains are constructed.

Example 8 preparation of Ulp1 protease

8.1 expression and purification of Ulp1 protease

Mu.l of E.coli Ulp1-pET28b/BL21 strain stored at-30 ℃ was inoculated into LB medium containing 50. mu.g/ml kanamycin by using a pipette gun, and cultured overnight at 37 ℃ with shaking at 250 rpm. Expanding culture to OD₆₀₀Approximately 1.0, IPTG was added to a final concentration of 200. mu.M, and the cells were harvested after shaking culture at 37 ℃ and 250rpm for 3 hours.

The isolation and purification of the Ulp1 protease was as described in example 4 of the present invention.

8.2 definition of the enzymatic Activity of Ulp1 protease

100. mu.g of control protein was reacted at 25 ℃ for 1 hour in 50mM Tris-HCl, pH 7.5,150 mM NaCl, and the amount of enzyme Ulp1 required for cleavage of more than 95% was defined as 1U.

Reagent formula of 8.3 Ulp1 protease

3.5mg/ml Ulp1 protease was stored in bioreagent containing 50mM Tris-HCl pH 7.5,150 mM NaCl, 20% glycerol, 1mM benzamidine, 0.2mM phenylmethylsulfonyl fluoride, 0.1mM ethylene glycol bistetraacetic acid, 0.1% 2-mercaptoethanol, 0.03% dodecyl polyglycol ether.

8.4 storage and stability of Ulp1 protease

The Ulp1 protease preserved by the biological reagent of the invention 8.3 can be preserved at-70 ℃ for 1 year from the production date.

Example 9 removal of SUMO tag from recombinant proteins

9.1 preparation of recombinant proteins with native N-terminus Using Ulp1 protease

According to the proportion of 1U: 500-. And selecting the enzyme cutting proportion with the cutting efficiency of more than 90 percent to prepare the recombinant protein with the natural N end on a large scale. Wherein the SUMO protein and the Ulp1 protease both have 6 XHis tags, and are purified and removed by nickel column affinity chromatography after enzyme digestion reaction. The cleavage efficiency of Ulp1 protease is shown in FIG. 5.

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

<110> Wenzhou university of medical science

<120> construction method and application of escherichia coli cold shock solubilizing expression plasmid

<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 (2)

1. A method for soluble expression of foreign protein in Escherichia coli is characterized in that: by adopting a cross PCR technology, fusing a nucleic acid coding sequence of 1-263 th amino acid at the N end of the escherichia coli cysteine desulfurization enzyme IscS protein with a nucleic acid coding sequence of 316-457 th amino acid at the C end of the humanized cysteine desulfurization enzyme NFS1 protein to construct a chimeric cysteine desulfurization enzyme expression gene with a sequence shown as SEQ ID NO. 25; connecting the chimeric cysteine desulfhydrase expression gene to pCold-SUMOa plasmid by seamless cloning technology to obtain recombinant plasmid and expressing; the construction method of the pCold-SUMOa plasmid is as follows: pE-SUMOpro Kan plasmid is used as a template, a primer SUMO-F with a sequence shown as SEQ ID N0.3 and a primer SUMO-R with a sequence shown as SEQ ID N0.4 are used for amplification to obtain a Smt3 fusion protein sequence with a sequence shown as SEQ ID N0.19, pCold TF plasmid after single enzyme digestion by Sal I is used as a template, a primer pCold-F with a sequence shown as SEQ ID N0.1 and pCold-R with a sequence shown as SEQ ID N0.2 are used for amplification to obtain a pCold plasmid skeleton fragment, and then the two fragments are connected by a seamless cloning technology to obtain a recombinant plasmid which is named as pCold-SUMOa.

2. The chimeric cysteine desulfurization enzyme constructed according to the method of claim 1, wherein: the stability of the chimeric cysteine desulfhydrase is higher than that of the humanized cysteine desulfhydrase, and the activity of the chimeric cysteine desulfhydrase is slightly lower than that of the cysteine desulfhydrase of escherichia coli.

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