固定化N-乙酰鸟氨酸脱酰基酶拆分消旋蛋氨酸

基因工程菌BL21/pET22b-argE可高效表达N-乙酰鸟氨酸脱酰基酶。将含酶细胞包埋于海藻酸钙凝胶中制成固定化细胞酶,用于消旋蛋氨酸的拆分,并将其拆分能力、拆分速度及操作稳定性与游离细胞酶相比较。结果表明:单批次转化固定化细胞酶的拆分能力和游离细胞酶相近,拆分速度较慢;但多批次转化的操作稳定性显著高于游离细胞酶。重复利用8次后的固定化细胞酶仍保有95%以上的酶活力,重复利用5次后的游离细胞酶活已降至20%左右。每克湿菌泥在游离和固定化条件下重复拆分产L-蛋氨酸的量分别为74.16mmol和241.93mmol。酶拆分液中的L-蛋氨酸经重结晶后光学纯度为98.3%。固定化细胞酶比游离细胞酶更具有工业化应用的潜质。     

表达条件对金属激活酶NAOase离子依赖的活性分析

N-乙酰鸟氨酸脱酰基酶为一种新型手性拆分酶制剂,酶活性依赖于Mg2＋、Mn2＋、Zn2＋及Co2＋ 中的某种金属离子。以高表达NAOase的重组菌DH10B/argE-pHsh为研究对象,考察不同培养条件下Mg2＋、Mn2＋、Zn2＋及Co2＋ 4种离子对重组菌的生长、酶的表达活性及表达量的影响。结果发现：同种离子在不同条件下对重组菌的生长影响不大,但对酶活性影响显著。4种离子在合适浓度时皆能提高酶活性,促进强度从高到低依次为Mn2＋、Mg2＋、Co2＋和Zn2＋。在TB培养基中,Mn2＋为15 mmol/L时：NAOase比酶活达到1 272.7 U/mL,是未添加时的4.67倍,激活作用显著高于其他离子。SDS-PAGE电泳实验表明4种离子的蛋白表达量基本相同。     

重组N-乙酰鸟氨酸脱乙酰基酶的高效表达与纯化

利用质粒pET-22b( )为表达载体,成功构建了高效表达N-乙酰鸟氨酸脱乙酰基酶的基因工程菌BL21-pET-22b( )-argE。研究了该菌的最适超声破壁条件及硫酸铵分级沉淀的最适范围,并以金属螯合亲和层析法纯化含有6-His-Tag的目的蛋白。结果表明:26℃诱导表达的菌体,最佳超声破碎条件为功率200 W,超声时间为15min;目的蛋白主要存在于40%~50%硫酸铵沉淀中。通过Ni-NTA亲和层析柱纯化目的蛋白,可以达到SDS-PAGE电泳纯,并显示单亚基相对分子质量为43 000。纯化倍数为139倍,回收率为11.1%。     

采用pHsh载体克隆与表达N-乙酰鸟氨酸脱乙酰基酶基因

利用质粒pHsh为表达载体,构建高效表达N-乙酰鸟氨酸脱乙酰基酶的基因工程菌E.coli DH10B/argE-pHsh.为提高酶活并降低生产成本,优化了诱导条件.结果表明:NAOase可在pHsh系统中高活性表达,诱导起始OD600为0.6,在空气摇床中42℃热激诱导5h重组菌比酶活达到152U/mL.     

重组N-乙酰鸟氨酸脱乙酰基酶包涵体的稀释复性

N-乙酰鸟氨酸脱乙酰基酶（N-Acetylornithine deacetylase,NAO）是一种重要的用于手性拆分的酶,具有广泛的底物选择性,常用于多种活性氨基酸的酶法拆分。采用稀释复性法研究了重组NAO包涵体的复性条件,如蛋白浓度、复性液中尿素浓度、pH、GSH浓度及c（GSH）/c（GSSG）比例,同时对稀释操作方式进行了考察,得到了较为适宜的复性条件。结果表明,尿素能有效抑制复性过程中蛋白质的聚集,随着蛋白质浓度的增加,复性效果变差。当复性缓冲液中尿素浓度为2 mol/L,GSH浓度为5 mmol/L,c（GSH）/c（GSSG）为2.5,pH为8.5,在4℃下进行分批稀释复性操作,复性后重组NAO的活性为1.077 U/mL,比酶活达到14.943 U/mg,与可溶性表达的NAO比较,复性率达到21.48%。     

海藻酸钙包埋氧化亚铁硫杆菌的固定化研究

采用非稳态法测定FeSO4在包埋和未包埋氧化亚铁硫杆菌的凝胶中的有效扩散系数。结果表明,FeSO4在凝胶中的有效扩散系数De随着海藻酸钠浓度的升高而降低,当海藻酸钠浓度为2%时最优;凝胶剂CaCl2的浓度对扩散系数的影响较小。包埋的氧化亚铁硫杆菌在10h达到增殖平衡,而FeSO4在包埋细菌的凝胶内扩散系数明显减少。     

重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

报道重组N-乙酰鸟氨酸脱乙酰基酶(NAOase)的研究进展。重组NAOase由大肠杆菌argE基因编码,在重组菌BL21(DE3)-pET22b-argE中的表达量为32.5%,大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶,比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时,约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后,获得了高纯度的NAOase。     

钝齿棒杆菌中异源表达N-乙酰鸟氨酸脱乙酰基酶合成L-鸟氨酸的研究

目的:对一株产鸟氨酸的钝齿棒杆菌Corynebacterium crenatum SYPA5-5/△proB/△argF(SYPO-1)进行代谢工程改造,筛选不同细菌来源的N-乙酰鸟氨酸脱乙酰基酶在大肠杆菌中克隆与表达,纯化后对其进行酶学性质的比较;将黏质沙雷氏菌Serratia marcescens Y213来源的Smarg E基因编码的N-乙酰鸟氨酸脱乙酰基酶在L-鸟氨酸生产菌株C.crenatum SYPO-1中过量表达,进一步提高L-鸟氨酸的产量。方法:通过利用pDXW10穿梭质粒对不同来源的N-乙酰鸟氨酸脱乙酰化酶进行克隆表达和酶学性质比较,选择性质最优来源的N-乙酰鸟氨酸脱乙酰基酶编码基因Smarg E在产L-鸟氨酸重组钝齿棒杆菌中表达,考察重组菌株发酵过程中参数的变化。结果:来源于S.marcescens Y213的N-乙酰鸟氨酸脱乙酰基酶比酶活最高为798.98U/mg,最适pH为7,最适温度为37℃,0.1mmol/L的Mg~(2+)、Li~+、Mn~(2+)促进酶的比酶活提高了50%;在钝齿棒杆菌中表达N-乙酰鸟氨酸脱乙酰基酶酶活达到128.4U/ml,显著提高了钝齿棒杆菌中胞内乙酰基循环水平;5L发酵罐发酵重组菌株96h,L-鸟氨酸的产量达到38.5g/L,比出发菌株,L-鸟氨酸的产量提高了33.2%,产率达0.401g/(L·h)。结论:筛选出最佳来源的N-乙酰鸟氨酸脱乙酰基酶,在鸟氨酸生产菌株C.crenatum(SYPO-1)中过量表达,可以促进鸟氨酸的前体物质N-乙酰鸟氨酸的快速消耗,实现鸟氨酸的积累。     

产乙酰鸟氨酸脱乙酰基酶基因工程菌的构建及遗传稳定性研究

利用质粒pET22b( )为表达载体,成功构建了产N-乙酰鸟氨酸脱乙酰基酶基因工程菌BL21 - pET22b( )-argE,并考察了重组质粒的稳定性.双酶切鉴定了质粒构建正确,SDS-PA GE电泳证实了该菌可高效表达目的蛋白.连续传代50次实验表明重组质粒具有结构稳定性.无选择压力连续传代时,质粒丢失严重;有选择压力时连续传代未发生质粒丢失现象,具有较好的分离稳定性.发酵过程中,用羧苄青霉素代替氨苄青霉素,质粒稳定率由77.78%提高到8 6.42%.羧苄青霉素浓度为200μg/mL时,质粒稳定率提高到98.33%.     

N-乙酰鸟氨酸脱酰基酶活性位点的构成研究

目的:用生物信息学软件预测出N-乙酰鸟氨酸脱酰基酶的活性中心的金属离子结合位点.方法:选用DEPC、WRK、PMSF、NBS、DTNB 5种化学试剂选择性修饰N-乙酰鸟氨酸脱酰基酶中组氨酸、天冬氨酸、谷氨酸、丝氨酸、色氨酸和半胱氨酸;同时考察Co2、Fe3 、Mg2 、Mn2 、ZN2 、Ni2 、Cu2 等金属离子对酶活性的影响.结果:在DEPC、WRK修饰后,酶的活力明显下降,而PMSF、NBS、DTNB对酶的活力影响不大;说明组氨酸和酸性氨基酸为酶活性中心的必需氨基酸,而丝氨酸残基、色氨酸残基、半胱氨酸残基不参与酶活性中心的组成;Co2 对酶反应有促进作用,验证了生物信息学的预测结果;底物N-乙酰-D,L-蛋氨酸对酶有较好的保护作用,保护作用随浓度增加而增加.结论:本研究为深入研究酶结构与功能的关系提供实验依据,为N-乙酰鸟氨酸脱酰基酶的工业应用提供理论参考.     

基因重组大肠杆菌表达HrpNEcc蛋白的发酵条件及诱导条件优化

对已构建好的表达HrpNEcc蛋白的工程菌BL21(DE3)/pET30a(+)hrpN Ecc的摇瓶发酵条件及乳糖诱导进行优化, 通过在7L发酵罐中放大发酵实验,以期提高蛋白产量并降低生产成本。在摇瓶中优化的发酵及诱导条件是：5% 的接种量,TB培养基,菌体培养至对数生长前期,添加3g/L外源诱导剂乳糖时,HrpNEcc蛋白产量可达417.60mg/L,比不添加乳糖时提高了36.73%,比用IPTG诱导时提高了16.85%。7L发酵罐中发酵,获得菌体湿重达到57.24g/L（WCW）,可溶性HrpNEcc蛋白产量占细胞总蛋白的50.2%,为3.29 g/L。     

His/GST-HDAC1在大肠杆菌中表达的研究

目的:对His/GST-HDAC1在大肠杆菌BL-21中的表达进行研究。方法:HDAC1的完整基因片断被克隆到pColdⅠ载体和pGEX载体上,并在其N末端分别联有His和GST;采用大肠杆菌BL21对HDAC1进行表达;采用亲和色谱对HDAC1进行纯化;用SDS-PAGE和蛋白质印迹来验证表达和纯化效果;对HDAC1活性进行测定。结果:多数HDAC1存在于大肠杆菌BL-21细胞裂解液的沉淀组分和纯化过程中的未结合组分中,小部分HDAC1可从细胞裂解液的上清液中得以纯化,但未显现出酶活性。用FPLC对HDAC1进行进一步分离,结果表明,HDAC1发生了分子聚集,使得它们的分子量大于正常分子量。结论:活性His/GST-HDAC1不能用大肠杆菌BL21成功表达。     

金属硫蛋白与单抗SZ51重组蛋白在大肠杆菌菌株BL21(DE3)pLysS中表达的研究

从基因工程水平构建了小鼠金属硫蛋白与抗人活化血小板单抗ＳＺ５１单链抗体的重组基因产物,并在大肠杆菌菌株ＢＬ２１（ＤＥ３）ｐＬｙｓＳ中成功地进行了表达．该重组蛋白以包涵体形式存在于菌体蛋白中,分子量为３８ｋＤ,ＥＬＩＳＡ实验证明该重组蛋白既具有血栓部位活化血小板的单抗活性,又具有小鼠ＭＴ单抗的活性     

Production of Recombinant Proteins in the lon-Deficient BL21(DE3) Strain of Escherichia coli in the Absence of the DnaK Chaperone

To eliminate unavoidable contamination of purified recombinant proteins by DnaK, we present a unique approach employing a BL21(DE3) ΔdnaK strain of Escherichia coli. Selected representative purified proteins remained soluble, correctly assembled, and active. This finding establishes DnaK dispensability for protein production in BL21(DE3), which is void of Lon protease, key to eliminating unfolded proteins.Obtaining substantial amounts of pure protein is essential in innumerable biological studies and indispensable to the biochemical characterization of proteins. The ease of growth, well-characterized genetics, and the large number of tools for gene expression have long made Escherichia coli the organism of choice for protein overproduction. The BL21(DE3) strain is widely used for recombinant protein production because of its engineered capacity to produce T7 polymerase and its deficiency in Lon and OmpT proteases.DnaK is an abundant protein (about 1% of the total protein of E. coli) (17) that interacts with a wide range of newly synthesized polypeptides (28) and assists their proper folding and assembly into oligomers by preventing protein aggregation. DnaK, together with ClpB ATPase, is also required to disaggregate preformed protein aggregates (12, 20), and it participates in the degradation of damaged proteins by Lon and ClpP (26, 27).The inactivation of dnaK has been shown previously to increase the insoluble fractions of certain aggregation-prone recombinant proteins (7), and DnaK alleviates the aggregation of certain heterologous proteins when coproduced with the protein of interest (8). However, fruitful coproduction of recombinant proteins with chaperones has been challenged by recent findings demonstrating that chaperones increase the solubility but not necessarily the quality of proteins (13, 15).The DnaK binding site, a five-residue hydrophobic core flanked by two basic residue-enriched regions, occurs on average every 36 residues in protein sequences (25). One consequence is unwanted DnaK contamination of recombinant proteins during purification in E. coli, even after several chromatographic steps (1, 3, 11, 14, 16, 21, 23).One challenge in protein purification is to obtain the highest level of purity in the fewest steps. Biologically active impurities can jeopardize research or therapeutic applications even if present in trace amounts. One approach developed to circumvent DnaK contamination is extensive washing of columns with ATP since DnaK in its ATP-bound state has low affinity for protein (3). However, this strategy lengthens the purification procedure, is expensive, and is of inconsistent effectiveness (1, 14).In an attempt to eliminate DnaK contamination, we have investigated whether recombinant proteins could be produced in the absence of DnaK. Toward that end, we constructed a ΔdnaK derivative of the extensively employed E. coli B host strain BL21(DE3). The consequences of the absence of DnaK for the production, solubilities, correct assembly, and activities of several recombinant proteins in BL21(DE3) have been studied. Obtaining a BL21(DE3) ΔdnaK strain has allowed us to elucidate to what extent such a major E. coli chaperone is indispensable to protein overproduction in the particular genetic context of an E. coli strain that lacks Lon, an ATP-dependent protease responsible for degrading unfolded proteins (10).dnaK in BL21(DE3) was inactivated by the introduction of a null allele, ΔdnaK::Kan, from the E. coli PopC4617 strain by P1 transduction (see Table S1 in the supplemental material). Transductants were selected at 30°C in Luria-Bertani medium complemented with kanamycin. The absence of dnaK was verified by colony PCR using the specific primers dnaK-Nter (5′-GGTAAAATAATTGGTATCGACCTGG-3′) and dnaK-Cter (5′-GTCTTTGACTTCTTCAAATTCAGCG-3′) (see Fig. S1 in the supplemental material). Immunoblotting using an anti-DnaK antibody showed that the obtained transductant (EN2) did not produce DnaK (see Fig. S1 in the supplemental material). The EN2 strain has been deposited at the Collection Nationale de Culture de Microorganismes at the Institut Pasteur (with identification number CNCM I-3863).E. coli K-12 dnaK mutants usually have a narrow range of permissive temperatures for growth (around 30°C) and exhibit multiple cellular defects, such as impaired cell division and the inhibition of DNA and RNA synthesis (4, 6, 18, 22). Inactivating dnaK in the genetic background of BL21(DE3), an E. coli B strain which is already deficient in OmpT and Lon proteases, did not lead to a dramatic difference in the exponential growth rate at 30°C, but at stationary phase, EN2 cells exhibited slightly reduced ability to form colonies on plates (data not shown). As expected for dnaK mutants, EN2 cells demonstrated impaired growth at 42°C (data not shown). Inactivating dnaK in BL21(DE3) did not induce major morphological defects, and EN2 cells were never found to form long filaments, as dnaK mutants with other genetic backgrounds have previously been reported to do (5) (data not shown). Therefore, the EN2 strain can easily be cultivated at 30°C.We next investigated whether inactivating dnaK in BL21(DE3) would impair the production and solubilities of different recombinant proteins (whose features are summarized in Table S2 in the supplemental material). These proteins belong to organisms of different kingdoms, and their molecular masses range from 19 to 51 kDa; therefore, they potentially correspond to DnaK substrates since the masses of polypeptides interacting with DnaK range from 14 to 90 kDa (28). Many of them exist as oligomers and may require the assistance of DnaK for proper assembly. These proteins were also chosen for their different levels of production and solubility in E. coli. Four of them (CpxP, ClpP1, PA28α, and proteasome-activating nucleotidase [PAN]) are totally soluble, and two of them (ClpP2 and green fluorescent protein [GFP]) are aggregation prone and may require the presence of DnaK to prevent their aggregation. Importantly, all these proteins were contaminated by DnaK when purified from E. coli (see Fig. ​Fig.33).Open in a separate windowFIG. 3.Purification of recombinant proteins in the absence of DnaK. Aliquots of 10 μg of CpxP, ClpP1, ClpP2, GFP, and PAN purified from BL21(DE3) cells (lanes 1, 3, 5, 7, and 9) or EN2 cells (lanes 2, 4, 6, 8, and 10) were loaded onto an SDS-12% PAGE gel. (A) Proteins were revealed by Coomassie blue staining. (B and C) DnaK (B) and GroEL (C) were detected by Western blotting. Sizes of molecular mass markers (lanes MW) are given in kilodaltons and indicated to the left of the gel. The asterisk indicates the position of DnaK on the gel. The two major bands in the purified PAN sample correspond to full-length 50-kDa His-PAN and the 40-kDa PAN fragment resulting from the internal initiation of translation, which copurify as oligomeric complexes (30). The gels shown are representative of results from at least three independent experiments.The production of recombinant proteins in exponentially growing BL21(DE3) and EN2 cells in Luria-Bertani medium at 30°C was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 h. The same biomasses of BL21(DE3) and EN2 cells were sonicated in 1 ml of lysis buffer (50 mM Tris, pH 7.5, 100 mM KCl, 1 mM dithiothreitol). Soluble proteins were separated from aggregated proteins and cellular debris by 30 min of centrifugation at 14,000 × g and 4°C. Pellets containing protein aggregates were resuspended in 1 ml of Tris, pH 7.5, containing 1% sodium dodecyl sulfate (SDS). Total extracts and soluble and insoluble fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Levels of production and solubility of recombinant proteins in the absence of DnaK. Aliquots of 10 μg of total extracts (T-un and T) and soluble (S) and insoluble (P) fractions from uninduced (T-un) and IPTG-induced (T, S, and P) BL21(DE3) and EN2 cells overexpressing CpxP (A), ClpP1 (B), PA28α (C), ClpP2 (D), GFP (E), or PAN (F) were analyzed by SDS-12% PAGE on gels stained by Coomassie blue. Sizes of molecular mass markers (lanes MW) are given in kilodaltons and indicated to the left of each gel. Arrowheads indicate the positions of recombinant proteins. The gels shown are representative of results from at least three independent experiments.The levels of production of all tested proteins in EN2 and BL21(DE3) cells were similar, as demonstrated by the protein amounts in total extracts (Fig. ​(Fig.1,1, lanes T). Moreover, dnaK inactivation did not affect the solubilities of recombinant proteins, even those such as CpxP (Fig. ​(Fig.1A),1A), ClpP1 (Fig. ​(Fig.1B),1B), and PA28α (Fig. ​(Fig.1C)1C) produced in high amounts or those such as ClpP2 (Fig. ​(Fig.1D)1D) and GFP (Fig. ​(Fig.1E)1E) prone to aggregation. These findings were surprising since the function of the DnaK chaperone is to prevent protein aggregation during synthesis and to cooperate with DnaJ, GrpE, and ClpB in the disaggregation of aggregates. It seems that, even for aggregation-prone recombinant proteins, solubility may not necessarily be dependent on endogenous DnaK. This finding may reflect the different folding requirements of specific proteins. Another explanation may be the presence of another chaperone with an overlapping conjoint function. In fact, a consequence of the absence of DnaK in cells is higher levels of production of heat shock proteins such as GroEL/GroES (29). Consistent with these data, EN2 cells produced higher amounts of GroEL than BL21(DE3) cells (data not shown), and these higher amounts may compensate for the absence of DnaK in preventing protein aggregation, as was shown previously for endogenous E. coli proteins and other recombinant proteins (8, 28). An abundance of different chaperones playing nonspecialized roles in recombinant protein folding in E. coli cells may permit toleration of the loss of DnaK, without impairing cell capacity as a protein production factory.Since the examined proteins could fold and assemble independently of DnaK, we next tested whether a protein known to interact with DnaK could be produced in the absence of this chaperone. Nemo, the IκB kinase complex regulatory component of the NF-κB signaling pathway in eukaryotes, was shown previously to tightly bind and be contaminated by DnaK when produced in E. coli (1). When recombinant His-tagged Nemo was produced in BL21(DE3) under our conditions, it was barely detectable on electrophoresis gel (Fig. 2A and B). However, immunodetection using an anti-His₆ antibody (Roche) at a 1:2,000 dilution showed that the absence of DnaK resulted in an increase in Nemo production (Fig. ​(Fig.2D).2D). When Nemo was produced in higher amounts, most of the protein was found in the soluble fraction, indicating that it could be produced as a soluble species in the absence of DnaK (Fig. ​(Fig.2D,2D, lane 5). Increased production of Nemo in the absence of DnaK could be explained by a role of this chaperone in Nemo degradation. Producing Nemo in a BL21(DE3) strain that is deficient in the protease ClpP did not increase its cellular amount (data not shown), indicating that if Nemo was degraded in a DnaK-dependent manner in BL21(DE3) (which already lacks Lon protease), ClpP was not responsible for this proteolysis or the absence of ClpP was compensated for by another protease.Open in a separate windowFIG. 2.Levels of production and solubility of recombinant Nemo in the absence of DnaK. Aliquots of 10 μg (A and C) or 20 μg (B and D) of total extracts (T) and soluble (S) and insoluble (P) fractions from uninduced and IPTG-induced BL21(DE3) and EN2 cells overexpressing Nemo were loaded onto an SDS-10% PAGE gel. Proteins were detected by Coomassie blue staining (A and C), and His-tagged Nemo was detected by Western blotting (B and D). Sizes of molecular mass markers (lanes MW) are given in kilodaltons and indicated to the left of each gel. The gels shown are representative of results from at least three independent experiments.We next tested whether recombinant proteins produced in the absence of DnaK would remain soluble and active during their purification. Samples of 200 ml of cells overproducing CpxP, ClpP1, ClpP2, or GFP or 500 ml of PAN-overproducing cells were sonicated in 2 ml of lysis buffer (50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl, 10 mM imidazole). The soluble fraction obtained after 30 min of centrifugation at 38,000 × g and 4°C was loaded onto 400 μl of nickel-nitrilotriacetic acid resin, and His-tagged proteins were purified according to the recommendations of the resin manufacturer (Qiagen). After elution, His-tagged proteins were dialyzed against 50 mM Tris, pH 7.5, concentrated, and analyzed by electrophoresis.By this procedure, recombinant proteins were purified to the levels of homogeneity indicated in Fig. ​Fig.3A.3A. Samples of 10 μg of purified proteins were used for the immunodetection of contamination by DnaK (using an anti-DnaK antibody from Stressgen at a 1:2,000 dilution). We found that DnaK in BL21(DE3) cells contaminated all preparations of purified recombinant proteins, albeit to different extents (Fig. ​(Fig.3B,3B, lanes 1, 3, 5, 7, and 9). As expected, dnaK inactivation prevented such contamination (Fig. ​(Fig.3B,3B, lanes 2, 4, 6, 8, and 10). It is noteworthy that most of the proteins purified from BL21(DE3) were also contaminated by GroEL, although this contamination was minor. In EN2 cells, where GroEL expression is increased, we did not systematically observe greater contamination by GroEL (Fig. ​(Fig.3C).3C). Moreover, CpxP and GFP, the proteins that exhibited the greatest DnaK contamination, were not the most contaminated by GroEL, and GroEL did not copurify with PAN in the absence of DnaK. Thus, the absence of DnaK did not necessarily lead to a higher level of contamination by GroEL.Despite the absence of DnaK, all purified recombinant proteins remained soluble even after being concentrated. Since some aggregates are soluble and solubility does not always guarrantee a native active conformation (15, 19), the activities (when readily measurable) or native conformations of some of the purified proteins were examined. One microgram of purified PAN was used to measure ATP hydrolysis at 55°C as described earlier (2). PAN proteins purified from BL21(DE3) and EN2 cells had comparable ATPase activities, with means ± standard errors of 762.33 ± 145.51 and 968.32 ± 198.85 nmol mg⁻¹ h⁻¹ (n = 3), respectively. The fluorescence emission spectrum (at an excitation wavelength of 400 nm) of GFP purified from EN2 cells was indistinguishable from that of GFP purified from BL21(DE3) cells (Fig. ​(Fig.4A),4A), indicating that GFP remained correctly folded when produced in the absence of DnaK. CpxP, a component of the Cpx signal transduction pathway, was the protein that exhibited the greatest DnaK contamination (11). It self-associates into dimers (M. Miot and J.-M. Betton, unpublished data), and to test its correct assembly, 100 μl of purified CpxP at 1 mg/ml in a buffer of 25 mM Tris, pH 7.5, and 150 mM NaCl was loaded onto a size exclusion chromatography column (Superdex 200 HR10/30; GE Healthcare) and eluted with the same buffer at a flow rate of 0.5 ml/min. Recombinant CpxP purified from BL21(DE3) eluted at a volume of 14.98 ml (Fig. ​(Fig.4B),4B), corresponding to a species with an apparent molecular mass of 39.85 kDa (a dimer of Cpx). Recombinant CpxP purified from EN2 eluted at a nearly identical volume of 14.97 ml (Fig. ​(Fig.4B).4B). Thus, the absence of DnaK did not alter CpxP dimeric assembly and did not produce any soluble higher-molecular-mass aggregate species.Open in a separate windowFIG. 4.Folding and assembly of proteins in the absence of DnaK. (A) Fluorescence emission spectra of 8-μg/ml GFP preparations purified from BL21(DE3) and EN2 cells, recorded with an FP-6200 spectrofluorimeter (Jasco) at a scan rate of 250 nm min⁻¹ using a bandwidth of 5 nm for both excitation and emission beams. The spectra shown are representative of results from at least two independent experiments. (B) Size exclusion chromatograms for CpxP proteins purified from BL21(DE3) and EN2 cells. Arrowheads indicate the elution volumes of the standards, and their masses are given in kilodaltons. The chromatograms shown are representative of results from at least three independent experiments.Altogether, these findings indicate that high levels of correctly folded, assembled, and active soluble recombinant proteins can be produced in the absence of endogenous DnaK chaperone in BL21(DE3). Surprisingly, our study showed that the inactivation of dnaK in BL21(DE3), which does not contain Lon, did not result in an increase in the aggregation of recombinant proteins, as was seen previously in E. coli K-12 (24). It seems that in BL21(DE3) cells, and in E. coli B cells in general, factors other than DnaK and Lon may be fundamental in managing the accumulation of aggregated proteins. Through the detailed characterization of a BL21(DE3) ΔdnaK strain and testing of the production of proteins of different natures, origins, and sizes, including aggregation-prone proteins, our study demonstrates that this EN2 strain offers a strategy that can be generally and extensively used to avoid unwanted contamination by DnaK. In addition, since DnaK has ATPase activity, the EN2 strain is particularly well suited for the production and purification of recombinant ATPases, eliminating the undifferentiable ATPase contamination. Given that GroEL, another major chaperone in E. coli, has also been found to contaminate purified recombinant proteins (9), it would be of additional interest to find conditions under which both dnaK and groEL could be eliminated in the BL21(DE3) strain without impairing its survival and its remarkable protein factory capacities.     

产邻苯二酚工程菌的构建及发酵条件的优化

从实验室保存的一株能高效降解苯胺的不动杆菌中克隆到完整的苯胺双加氧酶基因簇,序列分析表明该基因簇包含6个完整的ORF,全序列与已报道的不动杆菌YAA的苯胺双加氧酶基因簇在氨基酸水平上有较高的同源性。将该基因簇连接于pLAFR6载体,电转化至E.coli,构建了该基因簇的工程菌。发酵条件优化表明,在苯胺浓度0.5mg/mL,采用pH7.0的LB培养基,E. Coli DH5α为宿主菌,于37℃培养,接种量为3％的条件下,邻苯二酚产量在20h达到0.546mg/mL,底物分子水平转化率可达92.4％。     

谷氨酸菌体破碎条件的优化研究

以释放胞内物质为主要目的的细胞破碎条件,采用均质破碎法、超声波法、加酶法和碱性自溶法等对谷氨酸菌作进行破碎试验,并对其相应的工艺参数进行了优化研究。结果表明,使用碱性自溶法在pH10.0,温度70℃,干菌浓度为10％时,自溶40min后蛋白质的释放率接近80％,表明该法对释放胞内物质的作用效果较理想。     

褐藻胶降解菌株S10鉴定及产酶条件优化

探讨了褐藻胶降解菌株S10的生长条件及其对产褐藻胶降解酶活力的影响。以分离自海参肠道的褐藻胶降解菌株S10为研究对象，采用形态学观察结合16S rDNA序列分析，对菌株S10进行菌种鉴定并对其生理生化特性进行测定。以降解酶活力为指标，利用单因素、Plackett-Burman(PB)和响应面法对培养基成分和培养条件进行优化；最后对优化前后的菌株生长量、产酶活力和粗酶液稳定性进行分析。结果表明，菌株S10属于溶藻孤菌(Vibrio algindyticus);当pH 7、接种量2%(体积分数)、装液量150 mL、温度26℃、转速150 r/min、NaCl 3%(质量分数，下同)、海藻酸钠含量1.12%、硫酸铵含量0.44%、培养时间35.95 h条件下，褐藻胶降解酶活力最大(188.18 U/min)。优化后产酶活力提高30%;4℃低温更有利于该酶保存。综上，优化后的菌株S10产褐藻胶降解酶活力较高，能更好地用于降解褐藻胶，可为提高褐藻胶的利用率和进一步发掘褐藻胶寡糖的利用价值提供参考。     

反应分离耦合技术生产L-苹果酸工艺过程的优化研究

运用生物反应分离耦合原理,以富马酸钙为底物,采用游离延胡索酸酶,直接转化生产苹果酸钙。该法相对目前广泛采用的包埋式固定化方法具有工艺流程短、操作简便、转化率、收率高等特点,研究结果表明,转化温度为40℃,PH为7．0－7．5时,每升延胡索酸酶液能在20－28h间将3．2kg的富马酸钙转化生产成苹果酸钙,转化率高达99．9％,富马酸在产品中的残留在0．1％左右,产品符合美国药典标准,成本与化学合成法生产的DL－苹果酸相当。     

基因工程菌Pseudomonas sp.B4的构建及其产邻苯二酚发酵条件的初步研究

利用PCR技术以Pseudomonas sp. B3-1基因组DNA为模板,扩增出2.9kb编码苯甲酸双加氧酶基因簇benABC。将该基因簇连接于pLAFRJ载体,电转化至E．coli DH5α,再通过三亲本结合法导入野生菌株Pseudomonas sp. B3-1中,得到了一株邻苯二酚产量提高的基因工程菌,命名为Pseudomonas sp.B4。发酵条件优化表明,当苯甲酸钠浓度为6.0 g/L,聚蛋白胨浓度为2.0 g/L,温度为32℃以及pH值为6.0时,工程菌在200rpm旋转摇床发酵36小时后,邻苯二酚产量达到0.7 mg/ml,比优化前提高了20％。     

脂肪酶抑制剂产生菌Bacillus sp.LF深层发酵条件研究

随着生活水平的不断提高 ,肥胖在中国也开始成为影响人们健康的主要危险之一。目前市场上常见的减肥产品主要是作用于大脑 ,通过抑制食欲减少食物的摄入 ,由于这类药可能产生大脑、心血管及神经系统的副作用 ,不宜长期使用。瘦素 (Leptin)是一种肥胖基因表达的多肽激素 ,也是通过食欲中枢发挥作用 ,可以抑制食欲 ,提高代谢率 ,达到减肥的目的。因而另一类减肥药品脂肪酶抑制剂近年来受到国内外重视。脂肪酶抑制剂可直接阻断人体对脂肪的吸收 ,它不需要通过影响中枢神经系统来抑制人体的食欲 ,而是阻止脂肪在胃肠道的吸收。文献报道脂肪酶抑…