Error Correction in Gene Synthesis

SURVEYOR digestion

Digest 12 μL of the PCR product with 1 μL Enhancer and 1 μL SURVEYOR.

Incubate at 42°C for 20 minutes.

Stopd by adding 1.5 μL of stop buffe

Expression and Purification of Aae-MutS

E. coli BL21(DE3) (Novagen, Madison, WI, USA) was transformed with plasmid containing mutS gene and cultured at 37 °C in 1.5 L of YT medium [0.8% (w/v) tryptone, 0.5% (w/v) yeast extract and 0.5% (w/v) NaCl] containing 50 μg/mL ampicillin. When the culture density reached 4 × 10 8 cells/mL, isopropyl β- D -thiogalactopyranoside was added to a final concentration of 100 μM to induce expression. The cells were grown at 37 °C for a further 4 h, harvested by centrifugation and lysed by sonication in buffer I (20 mM Tris-HCl and 50 mM NaCl, pH 7.8). The sonicate was heated to 70 °C for 10 min, centrifuged at 48,000× g for 20 min, and the supernatant was then loaded onto a Talon resin column (40 mL; Clontech, Palo Alto, CA, USA) equilibrated with buffer I. The column was washed with 300 mL of buffer I and then eluted with buffer I containing 150 mM imidazole. The fractions containing AaeMutS were detected by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and collected, and (NH 4 ) 2 SO 4 was added to the fraction to a final concentration of 1 M. The solution was loaded onto a Toyopearl-Phenyl column (Tosoh, Tokyo, Japan) equilibrated with buffer I containing 1 M (NH4)2SO4 . The column was washed with 100 mL of buffer I containing 1 M (NH 4 ) 2 SO 4 and then eluted with a 300 mL gradient of 1 to 0 M (NH 4 ) 2 SO 4 in buffer I. The fractions containing AaeMutS were detected by SDS-PAGE and concentrated with a Vivaspin concentrator (Vivascience, Göttingen, Germany). The protein solution was dialyzed against 20 mM Tris-HCl and 50 mM NaCl (pH 7.8).

Expression and Purification of Tth-RecA

E. coli BL21 (DE3) was transformed with pET-11a/Tth-RecA and then grown at 37 ∘ C in 1.5 liters of YT medium containing 50 μg/mL of ampicillin. When the density of cultures reached
2.9 × 10 8 cells/mL, isopropyl β-d-thiogalactopyranoside was added to 100 μM. The cells were grown at 37 ∘ C for 3 h after induction and harvested by centrifugation. The cells
were lysed by sonication in 50 mM Tris-HCl (pH 7.5) containing 100 mM NaCl and 1.5 M KCl and then heated to 60 ∘ C for 60 min. After centrifugation at 48,000 ×g for
20 min, the supernatant was isolated, and ammonium sulfate was added to a final concentration of 1.5 M. The solution was loaded onto a TOYOPEARL-Phenyl column (12 mL,
TOSOH, Tokyo, Japan) preequilibrated with 50 mM Tris-HCl (pH 8.0) containing 1.5 M ammonium sulfate. The column was washed with the same buffer and then eluted with 1.5–
0 M ammonium sulfate gradient in the same buffer. The fraction containing ttRecA was detected by SDS-PAGE and dialyzed against 50 mM Tris-HCl (pH 8.0). The solution was
loaded onto a TOYOPEARL-DEAE column (12 mL, TOSOH) preequilibrated with 50 mM Tris-HCl (pH 8.0). The column was washed with the same buffer and then eluted with 0–1.5 M
KCl gradient in 50 mM Tris-HCl (pH 8.0).

Gene synthesis using 0.3 μM Aae-Muts

add 1.5 ug Aae-Muts protein to 50 ul PCR reaction

combined use of MutS and RecA may allow even greater accuracy in PCR-based applications

examples:

1.   PCR was performed with 0.06 units/μL LA Taq Hot-Start Version (Takara, Shiga, Japan), 0.03 units/μL KOD polymerase (Toyobo, Tokyo, Japan) or 40 nM A. aeolicus DnaE, in 1× Takara LA PCR buffer II (Takara, Shiga, Japan) containing 20 nM template DNA and 0.3 uM Muts.

2.  1× Takara GC I buffer (Takara) containing 0.06 units/μL LA Taq (Takara); 5 ng/μL T. thermophilus  genomic DNA; 400 nM primers; 100 μM CoCl 2 ; 400 μM dATP, dTTP, dCTP and dGTP and various concentrations of TthMutS or AaeMutS.

Suppression of nonspecific amplification

use 0.2 uM Tth-RecA and 0.3 uM Tth-Muts

Error-removal using MutS mismatch-binding protein

The MutS protein is part of the bacterial MutHLS mismatch repair mechinary [38]. It detects and binds to a variety of mispaired bases and small single-strand loops in vivo. Methods have been developed that use MutS as an error-removal agent for gene synthesis. In one strategy, MutS protein from the thermophilic bacteria Thermus aquaticus was used to directly filter out full-length heteroduplexes [8]. After denaturation and reannealing, mismatch containing heteroduplexes are recognized and bound by the Taq MutS protein, which can be separated from the unbound homoduplexes (mostly error-free) by a gel-mobility shift assay (Fig. 2a). This method was reported to reduce error rate to 1 error per 3.8kb with one round of filtration, which represented several folds of improvement from raw synthesis products. Repeating the procedure one more time reportedly reduced the error rate to 1 error per 10 kb, which is good enough to ensure finding a correct sequence with a single round of cloning and sequencing. A major limitation with removing full-length heteroduplexes is that it requires a substantial fraction of the sequences to be error-free in order to survive the MutS binding filtration. Figure 2 Figure 2 Schematic of error removal strategies using MutS mismatch-binding protein. Assembled gene segments are denatured and re–annealed to form heteroduplexes where errors are revealed as mismatches between correct (blue) and mutant (red) sequences. ...

For error-rich sequences, an alternative MutS-based error-correction strategy is called “consensus shuffling” [39, 40] (Fig. 2b). Re-annealed polynucleotide products are first cleaved by a mixture of restriction endonucleases and the resulting overlapping short fragments are then subjected to MutS column filtration. Short, mismatch-containing pieces are captured by immobilized Taq MutS, while error-free fragments are eluted and re-assembled into full-length constructs by assembly PCR . Two iterations of consensus shuffling improve the error rate 3.5- to 4.3-fold to 1 error per 3.5kb [39].

Consensus shuffling is a mechanism that resembles DNA shuffling where several iterations can be made until the consensus sequences are concentrated enough to far outnumber other species. Consensus shuffling provides several advantages over direct MutS filtration of full-length sequences: i) it is more effective on longer DNA constructs where perfect hybridization after re-assortment is rare; ii) it tolerates more errors in the starting material; iii) the removal of the entire DNA heteroduplex is not necessary. Importantly, fragmentation is ideally performed with type IIS restriction endonucleases that cut outside of the recognition sites. In this way, distinct ends can be generated by the same restriction endonuclease so as to minimize nonspecific hybridization of cut fragments after error-filtration. However, despite the merit of the idea, consensus shuffling requires extraneous use of endonucleases and the reaction conditions may still need to be improved to achieve better outcome [39] Error-correction using mismatch-cleaving enzymes

Mismatch-cleaving enzymes here refer to a group of mismatch-specific endonucleases that recognize and cleave at or near mismatch sites in DNA heteroduplexes. The family embraces a variety of members including but not limited to: i) resolvases, such as phage T4 endonuclease VII [41], T7 endonuclease I [42] and E.coli endonuclease V [43]; ii) mismatch repair endonuclease MutH [38]; iii) single-strand specific nucleases, such as S1 nuclease from Aspergillus oryzae, P1 nuclease from Penicillium citrinum, mung bean nuclease, and CEL nuclease from celery [44, 45]. The ability of these endonucleases to cleave heteroduplex DNA at the mismatch sites has lead to their extensive applications as probes for mutation and polymorphism detection [44, 46–50]. The mismatch-cleaving activity also makes them very useful in error removal for gene synthesis and several different assays were developed utilizing this activity.

Resolvases

T7 endonuclase I was used in an early report:after assembly and amplification of a target DNA, the purified product was denatured, re-annealed and treated with T7 endonuclease I. Afterwards, the pool was run on a gel and the remaining intact band was excised and extracted, while the cleaved fragments were discarded [51]. This way of enriching error-free sequences seems to work for high-quality synthetic products where correct sequences out number mutants.

The efficacy of various resolvases was systematically explored to recognize and cleave single-base mismatches using a different error-removal assay, which is applicable for low-quality sequences. Three resolvases, T4 endonuclease VII, T7 endonuclease I and E.coli endonuclease V were tested respectively in the process of ligation-based gene synthesis and functional cloning of bacterial chloramphenicol-acetyltransferase (cat) gene. After treatment with T4 endo VII and E.coli endo V, the fraction of “functional clones” increased over 10-fold, and sequence analysis revealed an over 4-fold reduction of error-rate. T7 endonuclease I was not fully characterized in this setting [32]. Instead of using gel purification, it was demonstrated that following mismatch-cleavage, an additional 3’to 5’ exonuclease treatment to remove single-stranded overhangs was essential for error removal, either by adding E.coli exonuclease I or using the intrinsic 3’to 5’ exonuclease activity of proof-reading DNA polymerases (Fig. 3). The processed DNA fragments were reassembled into full-length products by assembly PCR. Figure 3 Figure 3 Schematic diagram showing the principal of error removal using mismatch-cleaving enzymes. The re-annealed heteroduplexes are cleaved in both strands by mismatch-specific enzymes 2–5bp downstream of the mismatches, generating stagger ends that ...

Another method, called circular assembly amplification, combines exonuclease and endonuclease treatments to improve gene synthesis quality [52]. In this method, the overlapping and complementary oligonucleotides are first ligated into circular molecules under stringent conditions. An exonuclease is added to degrade any un-circularized polynucleotides. The circular DNA constructs are then treated with E. coli endonuclease V, which linearizes and degrades the mismatch-containing circles in concert with the exonuclease. By using the above three tiers of selections, the method is able to reduce error-rate by at least 7-fold compared to the conventional PCA assembly protocol [52].

A problem with phage resolvase-based mismatch cleavage is preferential cleavage of certain types of mis-matches. For instance, T7 Endonuclease I has been reported to fail to achieve quantitative cleavage for selected mis-pairs [32]. In addition, the degree of specificity of T4-endonuclease VII has been found to be highly dependent on the length of the substrate and the sequence surrounding the mismatch sites [53, 54]. This enzymology limitation is intrinsic to these resolvases because they naturally function to recognized holiday junctions rather than mismatches in a heteroduplex [55]. Their potency and efficacy of mismatch cleavage are therefore compromised by both low sensitivity and high undesired background. Future work to optimize their substrate specificity or use enzyme combinations may potentially produce better results.

MutHLS

E. coli proteins MutH, MutL and MutS constitute a bacterial mismatch repair mechanism: MutS detects and binds to mispaired bases and small single-strand loops, MutL couples the MutH endonuclease to the MutS bound mutant sites, leading to incision of the unmethylated strand of a hemi-methylated dsDNA. The error containing staggered ends are trimmed by a helicase and an exonuclease, and the resulting gap is filled by a DNA polymerase and a ligase [5]. MutHLS can be used to remove polymerase-produced mutant sequences in PCR products [38]. Treating re-annealed PCR products with MutHLS allows MutS to bind to the mismatches and create incisions on both unmethylated strands at d(GATC) in the vicinity of the mismatch sites. The cleaved heteroduplexes are then removed by gel electrophoresis. Mismatch removal using E. coli MutHLS reduces the error rate by one order of magnitude and that is effective to G-T, A-C, G-G, A-A and small insertion/deletion mis-pairs, while less effective for other type of mismatches [38].

Single-strand specific nucleases

Single-strand specific nucleases are a family of multifunctional enzymes that are present ubiquitously and display a wide variety of characteristics. Although more than 30 single-strand nucleases have been isolated from various sources, only a few have been thoroughly characterized. The most widely used single-strand nucleases are almost exclusively extracellular glycoproteins, including S1 and P1 nucleases from fungi, the mung bean nuclease and the CEL nuclease from plant. These nucleases vary drastically in the degree of specificity towards different types of mismatches. For instance, S1 and P1 nucleases appear to be strongly specific to AT-rich regions [55]; S1 nuclease seems incapable of recognizing single base mismatches [56]; and quantitative single nucleotide mismatch cleavage by mung bean nuclease occurs only at pH 6–6.5 [57]. Moreover, contradictory results have been reported regarding the use of S1, P1 and mung bean nucleases in detecting different types of single nucleotide polymorphism (SNP) in heteroduplex DNA [57]. The incomplete capability of these single strand nucleases to identify all possible types of mismatches greatly discourages their general use in synthetic gene error correction.

CEL endonuclease is an ortholog of the S1 nucleases isolated from celery [45, 58]. The uniqueness of CEL endonuclease is that it is the first eukaryotic endonuclease known to cleave DNA with high specificity towards all types of base mismatches and DNA distortions at neutral pH. Following the discovery of CEL nuclease, the techniques for high-throughput mutation detection have significantly improved, and CEL has become the most commonly used enzyme in TILLING (targeting-induced local lesions in genomes) [59–62], polymorphism analysis [63, 64] and disease diagnosis [58, 63–68].

CEL endonuclease is a mannosyl glycoprotein that nicks a DNA strand at the 3’end of the base-substitution mismatch and DNA distortion. Prolonged incubation and a high enzyme to substrate ratio facilities double-stranded cleavage by CEL at opposite phosphodiester bonds 3’ to the mismatch, generating i) two single-base 3’ overhangs in the case of base-substitutions or ii) a short stagger end in the case of insertion/deletion [45, 58]. The commercialization of CEL endonuclease as Surveyor™ nuclease by Transgenomic, Inc (Omaha, NE, USA) has led to its extensive applications in mutation detection as a simple yet cost-effective method, resulting in the discovery of many novel mutations associated with human genetic disorders, including BRCA1 [45, 58, 69], EGFR [65], HCDC4 [66], p53 [67], mitochondrial [63, 64] and kidney-related [68] genes, etc. The broad substrate specificity and low undesired activity of CEL nucleases make them the most promising candidates for error-correction in synthetic gene assemblies.

Recent studies employing on-chip gene synthesis technology explored the effectiveness of Surveyor™ nuclease in error correction of microchip synthesized genes [25, 70] (Fig. 4). The incorporation of a 60-minute digestion step with Surveyor™ nuclease followed by PCR assembly with Phusion DNA polymerase was able to reduce the error rate of the synthetic gene products from 1 error per 526 bp to 1 error per 3,883 bp. Two iterations of enzymatic error cleavage reactions further reduced the error rate to 1 error per 8,701bp, representing an over 16-fold error reduction [70]. Selective sub-pool amplification of chip-synthesized oligonucleotides achieve an error-rate of 1 error per 7,017bp after enzymatic error correction using ErrASE kit, a commercially available CEL-based enzyme cocktail that corrects mismatches [71]in a similar fashion to [25]. Figure 4 Figure 4 Schematic diagram of error correction strategy using CEL mismatch-specific endonuclease. Multiple CEL error-correction cycles maybe integrated into a gene synthesis process. Each cycle consists of four steps: 1) Re-annealing of assembled gene constructs ...

The combined actions of mismatch cleavage by CEL nuclease and proof-reading by Phusion DNA polymerase makes the procedure particularly robust and suitable for correcting error-rich synthetic sequences. Re-annealing of low-quality synthetic sequences often yield little error-free duplexes that can survive the mismatch-binding filtrationor mismatch-cleavage. Rather than discarding all error-containing heteroduplexes, CEL nuclease makes double-stranded cuts next to the mismatches. The mismatch bases are then chewed away by the exonuclease activity of the proof-reading DNA polymerase. The resulting error-free fragments are reassembled into full-length gene constructs by assembly PCR (Fig. 4). This method enables error elimination at the single-base level, while salvaging a majority of the correct portions of the sequences. More importantly, the “inspection-correction-reassemble” cycle can be iterated multiple times until products with desired purity are obtained.

PreCR Repair Mix components

Taq DNA Ligase

Endonuclease IV

Bst DNA Polymerase

Fpg Uracil-DNA Glycosylase (UDG)

T4 PDG (T4 Endonuclease V)

Endonuclease VIII 

Reaction Conditions: DNA template, 100 µM dNTPs, 1X ThermoPol Reaction Buffer, 1X NAD+ and 1 µl PreCR Repair Mix in a total reaction volume of 50 µl. Incubate at 37°C.

1X ThermoPol Reaction Buffer: 10 mM KCl 10 mM (NH4 )2 SO4 20 mM Tris-HCl 2 mM MgSO4 0.1%Triton X-100 pH 8.8 @ 25°C 1

http://www.google.sr/patents/CN101538561B?cl=zh&hl=nl

0008] (1)称取芹菜，每Ikg芹菜加入80〜120ml缓冲液A，捣成勻浆，压榨出液体，过滤， 用IM的Tris溶液调节pH至8. 0左右，得粗酶液；

[0009] (2)将粗酶液在45〜50°C下保温15〜20min，然后3000 X g〜5000 X g离心20〜 40min，取上清液；

[0010] (3)加入硫酸铵粉末至饱和度为25%，3000Xg〜5000Xg离心20〜40min，收 集上清液；所得上清液中加入硫酸铵粉末至饱和度为80%，3000Xg〜5000Xg离心20〜 40min,收集沉淀物；

[0011] (4)将所得沉淀物用缓冲液B溶解，透析脱盐；

[0012] (5)加入刀豆凝集素，混勻，4〜10°C下搅拌6〜16小时使CEL I核酸酶与刀豆凝 集素充分结合，然后静置20〜60min ；

[0013] (6)抽滤去清液，在滤渣中加入缓冲液C溶解出CEL I核酸酶，超滤浓缩。在4〜10°c条件下，上DEAE-S印harose FF,然后依次用洗脱液D和洗脱液E各500ml以0. 5〜 lml/min的流速进行梯度洗脱；

[0014] (7)收集洗脱液，用RF-I Nicking方法检测各收集管中的CEL I核酸酶活力，合并 酶活力在3 X 107U/mg蛋白的洗脱液；

[0015] 上述步骤中，所述缓冲液A由以下方法制备得到：在pH8. 0的0. IM Tris-HCl缓 冲液中添加N2SO3和苯甲基磺酰氟（Phenylmethanesulfonyl fluoride, PMSF)至浓度分别 为0. OlM和ImM ；所述缓冲液B由以下方法制备得到：在pH8. 0的0. IM Tris-HCl缓冲液 中添加苯甲基磺酰氟至浓度为ImM;所述缓冲液C由以下方法制备得到：在pH8.0的50mM Tris-HCl缓冲液中添加甲基甘露糖苷和Triton X-100至浓度分别为30mM和0. 01%;所述 洗脱液D由以下方法制备得到：在pH8. 0的Tris-HCl缓冲液中添加甲基甘露糖苷、Triton X-100和KCl至浓度分别为10mM、0. 01%和IOmM ；所述洗脱液E由以下方法制备得到：在 pH8. 0的Tris-HCl缓冲液中添加甲基甘露糖苷、Triton X-100和KCl至浓度分别为10mM、 0. 01% 和 0. 5M。

[0016] 本发明方法在抽提液中采用添加少量亚硫酸钠减少和清除提取液中有色物质；采 用热变性的物理方法，快速清除大量耐温性差的杂蛋白；利用CEL I是糖蛋白能与活化的 刀豆凝集素结合的特点，使CEL I核酸酶与刀豆凝集素结合除去其他杂蛋白，使CEL I得到 进一步的的纯化；最后选择DEAE-S印harose FF作为适宜介质进行一次层析纯化出CEL I。 综上，本发明方法抛弃了现有方法中的多次高速或超速离心以及多次凝胶过滤和离子交换 层析的手段，采用合适的物理化学方法达到快速将CEL I核酸酶从芹菜中提取出来并纯化 的目的。

[0017] 本发明方法提取CEL I核酸酶的效率较高，每100千克芹菜经步骤1后可得总粗 酶活力1.81X107，比活9.5X102U/mg蛋白的粗酶液，继续经步骤⑵〜（7)分离纯化后可 得纯的CEL I核酸酶1.545 X IO6〜1.895 X IO6U,酶活回收率达到8. 54〜10. 47% ；所得酶 的纯度达电泳纯，酶比活可达3X 107U/mg蛋白。本发明方法与经典的Yang等人的方法相 比，所得酶的纯度及比活没明显差异，但回收率提高了 5倍以上。

[0018] 附图说明

[0019] 图1是本发明方法提取核酸酶CEL I的SDS-PAGE图。

[0020] 图2是本发明方法提取核酸酶CEL I的酶切鉴定图。

具体实施方式

[0021] 例 1

[0022] 1、制备

[0023] 称取市场上购买的西芹（即西洋芹菜）100千克，去掉叶片，加入缓冲液A 8L，捣 成勻浆，榨出滤液74L。用IM Tris水溶液液调整滤液的pH至8. 0(Tris液用量为7. 5L)， 然后在50°C条件下保温20分钟后置于冰水中冷却；5000 Xg离心40分钟，去掉残渣，取上 清液；一边搅动一边缓慢加入硫酸铵粉末（每升约加入144g)至25%的饱和度，5000Xg离 心40min，去掉沉淀，收集上清液，一边搅拌一边加入硫酸铵粉末至80 %的饱和度（每升约 加入390g)，在5000 X g条件下离心40min，去掉上清液，保留沉淀。沉淀溶于8L缓冲液B 中，用同样的Tris缓冲液透析脱盐。脱盐后溶液加入刀豆凝集素200ml (已活化，且用缓冲液B平衡），4°C下搅拌过夜，使酶与刀豆凝集素充分结合。静置1小时，用抽滤漏斗去掉清 液，加入缓冲液C 300ml (分5次进行），充分混勻，使酶溶于缓冲液中，过滤；滤液使用超滤 膜浓缩（分子量截留10000道尔顿)至30ml，上DEAE-S印harose FF柱（30mmX 400mm)，以 缓冲溶液D和E各500ml作洗脱液，采用TH-500梯度混合仪（上海沪西分析仪器厂生产） 进行梯度洗脱，洗脱流速为0. 5ml/min,每Smin收集一管洗脱液；用RF-I Nicking方法检 测CELI活力高峰管，再用SDS-聚丙烯凝胶电泳法检测其蛋白纯度，之后合并蛋白为单一条 带的活力高峰管酶液，此洗脱液即为CEL I核酸酶蛋白洗脱液，共获得酶液20ml。

[0024] 2、鉴定

[0025] (1)所得酶液用SDS-PAGE检测，在4.3kDa处可见一蛋白条带，说明所得酶液应为 CEL I核酸酶，纯度达电泳纯（见图1)。

[0026] (2)蛋白质含量分析按考马斯亮蓝法测定。其原理是考马斯亮蓝G-250在游离 态下呈红色，当它与蛋白质的疏水区结合后变为青色，前者最大光吸收在465nm，后者在 595nm。在一定蛋白质浓度范围内（0〜100 μ g/ml)，蛋白质-色素结合物在595nm波长下 的光吸收与蛋白质含量成正比。

[0027] (3)所得酶液采用RF-I Nicking方法测定酶活。该方法以pUC 19为底物，用不同 浓度的酶液切割，切割产物经电泳后用Gel-PRO Analyzer凝胶定量分析软件分析RF I,RF II和RF III各区带荧光强度响应值。如图2所示，栏1为Lambda DNA/Hind IIIDNA分子 量标样，栏2为RF I形式的pUC19质粒对照，用2 μ 1水代替酶液；栏3〜7分别为反应体 系中加入2μ1不同浓度的CEL I提取酶液。由于在一定酶浓度范围内区带的荧光强度与 酶活性线性相关，利用公式I计算得出CEL I核酸酶的比活。

[0028] 酶活力

结果计算结果，所提取酶液每毫升含蛋白3. 158yg、酶活力为94750U，酶的比活 为3. 032 X 107U/mg蛋白；20ml提取液中酶的总活力1. 895 X IO6U, CEL I核酸酶回收率达到 10. 47%。

[0030] 例 2

[0031] 称取市场上购买的本芹（即中国芹菜）100千克，去掉叶片，加入缓冲液A 12L，捣 成勻浆，榨出滤液77L。用IM Tris水溶液液调整滤液的pH至8. 0(Tris液用量为7. 3L)， 然后在50°C条件下保温15分钟后置于冰水中冷却；3000 Xg离心20分钟，去掉残渣，取上 清液；一边搅动一边缓慢加入硫酸铵粉末（每升约加入144g)至25%的饱和度，3000Xg离 心20min，去掉沉淀，收集上清液，一边搅拌一边加入硫酸铵粉末至80 %的饱和度（每升约 加入390g)，在3000 X g条件下离心20min，去掉上清液，保留沉淀。沉淀溶于8. 2L缓冲液 B中，用缓冲液B透析脱盐。脱盐后溶液加入刀豆凝集素200ml (所用刀豆凝集素已活化， 且用缓冲液B平衡），4°C下搅拌过夜，使酶与刀豆凝集素充分结合。静置20分钟，用抽滤 漏斗去掉清液，加入缓冲液C 280ml (分7次进行），充分混勻，使酶溶于缓冲液中，过滤；滤 液使用超滤膜浓缩（分子量截留10000道尔顿）至30ml，上DEAE-S印harose FF柱【柱场 为30mm (直径）X 400mm (高）】，采用TH-500梯度混合仪（上海沪西分析仪器厂生产）进行 梯度洗脱，洗脱流速为lml/min，每Smin收集一管洗脱液；用RF-I Nicking方法检测CEL I活力高峰管，再用SDS-聚丙烯凝胶电泳法检测其蛋白纯度，之后合并蛋白为单一条带的活 力高峰管酶液，此洗脱液即为CELI核酸酶蛋白洗脱液，共获得酶液24ml。按例1方法检测， 每毫升酶液含蛋白2. 138 μ g、酶活力为64400U，酶的比活力为3. OlXlO7 ；24ml提取液酶的 总活力为1. 545X IO6U, CEL I核酸酶回收率为8. 54%。用SDS-PAGE检测，达电泳纯核酸酶 CEL I。

[0032] 例 3

[0033] 称取市场上购买的西芹100千克，去掉叶片，加入缓冲液A 10L，捣成勻浆，榨出滤 液。用IM Tris水溶液调整滤液到pH至8. 0，然后在50°C下保温18分钟后置于冰水中冷 却；4000Xg离心30分钟，去掉残渣，取上清液；一边搅动一边缓慢加入硫酸铵粉末至25% 的饱和度，4000 X g离心30min，收集上清液，一边搅拌一边加入硫酸铵粉末至80 %的饱和 度，在4000 X g条件下离心30min，去掉上清液，保留沉淀。沉淀溶于7L缓冲液B中，用同样 的Tris缓冲液透析脱盐。脱盐后溶液加入刀豆凝集素200mL(已活化，且用缓冲液B平衡）， 4°C下搅拌过夜，使酶与刀豆凝集素充分结合。静置40分钟，抽滤去掉清液部份，加入缓冲 液C 300ml (分5次进行），充分混勻，使CEL I核酸酶溶于缓冲液中，过滤；滤液使用超滤 膜浓缩（分子量截留10000道尔顿)至100ml，上DEAE-S印harose FF柱【柱场为30mm (直 径)X400mm(高）】，采用TH-500梯度混合仪（上海沪西分析仪器厂生产)进行梯度洗脱， 洗脱流速为0. 6ml/min,每8min收集一管洗脱液；用RF-I Nicking方法检测CEL I活力高 峰管，再用SDS-聚丙烯凝胶电泳法检测其蛋白纯度，之后合并蛋白为单一条带的活力高峰 管酶液，此洗脱液即为CEL I核酸酶蛋白洗脱液，获得酶液19. 2ml。按例1方法检测，每ml 酶液含蛋白3. 091^、酶活力为92687仏测得酶比活为3\10力/11^蛋白；19. 2ml酶提取液酶 的总活为1. 78X IO6U, CEL I核酸酶回收率达9. 83%。用SDS-PAGE检测，达电泳纯核酸酶 CELI。