真菌细胞色素P450在大肠杆菌中的表达

【背景】真菌细胞色素P450蛋白在大肠杆菌中表达水平低甚至不表达,近期研究发现通过对该类蛋白氨基端(N端)氨基酸序列的修饰可优化其表达水平。【目的】在大肠杆菌系统中表达预测功能为P450酶的焦曲霉094102菌株的Au8002蛋白,为真菌P450蛋白在大肠杆菌表达系统中的N端氨基酸序列修饰策略提供有效依据。【方法】对野生型P450蛋白Au8002的氨基酸序列进行分析,对其N端序列进行了3种序列修饰,并在诱导蛋白表达时添加P450生物合成前体5-氨基乙酰丙酸(5-ALA),研究N端氨基酸序列修饰策略及前体添加对真菌P450在大肠杆菌中蛋白表达的影响。【结果】SDS-PAGE和Westernblot检测结果显示,对目的蛋白进行的3种氨基酸序列修饰均使Au8002蛋白获得了表达,前体5-ALA的添加提高了目的蛋白表达量。其中对目的蛋白进行N端全长截短时可部分增加其可溶性,同时也验证了其特征性的CO结合能力。【结论】对预测为P450酶的菌株094102蛋白Au8002氨基端(N端)氨基酸序列的修饰有效解决了其在大肠杆菌内不表达的难题,实现了其可溶性表达;另一方面P450生物合成前体5-ALA的添加也能有效提高该类蛋白的表达水平,上述策略对改善其它该类蛋白在大肠杆菌内的表达水平具有借鉴意义。     

乳糖诱导木聚糖酶基因在大肠杆菌中的可溶性表达

以构建好的大肠杆菌工程菌BL21(DE3)/xylanase为研究对象,研究了以IPTG和乳糖作为诱导剂时重组蛋白的表达规律。在摇瓶发酵条件下研究了诱导剂浓度、诱导时机、诱导培养时间和诱导培养温度对目标蛋白表达的影响。实验结果表明,乳糖作为诱导剂时,重组菌产酶活力33.9 U/mg略高于IPTG作为诱导剂时重组菌产酶活力28.10 U/mg,这为乳糖作为诱导剂应用于重组大肠杆菌生产木聚糖酶提供了参考依据。     

乳糖诱导甜蛋白Monellin在大肠杆菌中的表达

根据已报道的单链Monellin甜蛋白的氨基酸序列,按大肠杆菌基因偏爱密码子,设计和人工合成了单链monellin基因。将单链monellin基因克隆到大肠杆菌表达载体pET-28a中,构建了重组表达载体pET28a-mon,转化大肠杆菌BL21(DE3),得到表达Monellin的大肠杆菌工程菌株。借助SDS-PAGE分析方法,研究了乳糖代替IPTG诱导大肠杆菌表达甜蛋白Monellin。通过对乳糖作为诱导剂表达条件进行优化,Monellin的表达量可占细胞总蛋白的33.09%,与IPTG诱导表达量接近。本研究结果为乳糖作为诱导剂应用于重组大肠杆菌生产甜蛋白Monellin提供了参考依据。     

乳糖诱导的重组人血管内皮抑素(rhEndostatin)蛋白的表达

研究用乳糖替代IPTG作为诱导剂进行重组蛋白的表达,观察乳糖对乳糖操纵子调控的基因工程菌发酵及重组血管内皮抑素表达的影响,从而选取最佳诱导表达条件。以重组人血管内皮抑素表达工程菌pETrhEN/BL21(DE3)作为研究对象,分别用IPTG和乳糖作为诱导剂,在摇瓶中进行表达实验。并对重组蛋白质表达量进行分析。然后在5 L发酵罐中进行验证。在摇瓶培养条件下,乳糖浓度大于0.5 g/L即可以诱导目的蛋白的表达。乳糖浓度1 g/L时诱导目的蛋白表达量与1 mmol/L的IPTG相当,当乳糖浓度为10 g/L,目的蛋白表达量达到最大。在发酵罐培养条件下,补料4 h后葡萄糖浓度基本耗尽,此时开始加入乳糖。诱导后1 h,即有重组蛋白表达,在诱导后4 h达到高峰(占菌体可溶性蛋白的56%),与此同时,诱导后5 h菌体浓度也达到最高值。在以乳糖操纵子为调控手段的工程菌表达系统中,可以使用乳糖作为诱导剂,诱导应在葡萄糖消耗完后进行。     

argE基因在大肠杆菌中的表达优化

N-乙酰鸟氨酸脱酰基酶可在重组菌BL21（DE3）-pET22b-argE中表达。首先确定了该酶的细胞表达定位,再研究了诱导温度、诱导剂种类及浓度、诱导起始菌体密度、诱导时间等因素对重组菌生长及目的蛋白表达活性的影响。结果表明,IPTG和乳糖皆可诱导目的蛋白表达,乳糖的诱导效果优于IPTG。在诱导起始0D600为0．46时加入15g／L乳糖,20℃诱导18h最适于目的蛋白的活性表达。表达条件优化后,酶活从1．68U／mL提高至282．99U／mL,约为原来的168倍。     

pEASY-TAZ-5原核表达载体的构建及在大肠杆菌中表达

TAZ基因在心磷脂代谢异常导致线粒体功能障碍中起重要作用。本研究旨在构建人TAZ-5(h TAZ-5,Δ5)的基因工程菌,用乳糖诱导TAZ-5蛋白的表达,为后续h TAZ-5纯化、抗血清的制备及生理功能检测奠定基础。以h TAZ全长为材料,通过PCR方法扩增人h TAZ-5基因,将其插入到原核表达载体p EASY中。经菌落PCR筛选阳性克隆,并通过DNA测序确定原核表达载体p EASY-TAZ-5构建成功。将p EASY-TAZ-5转入大肠杆菌(E.coli)感受态细胞Transetta(DE3)中,用乳糖诱导蛋白表达,分别对诱导剂浓度、时间、时机及温度等条件进行优化,确定最优的乳糖诱导条件,并对表达产物进行可溶性分析。结果表明PCR扩增出约786 bp的目的产物,与预期的片段长度一致;h TAZ-5蛋白最优表达条件为终浓度2.0 g/L乳糖、A600=0.8、37℃下诱导4 h,可以达到良好的诱导效果;收集菌体中的上清和沉淀,进行可溶性分析,目的蛋白以包涵体的形式存在。Western Blot分析表明重组蛋白TAZ表达成功。成功构建p EASY-TAZ-5原核表达载体,乳糖可诱导h TAZ-5(Δ5)基因表达,且效果很好。     

乳糖替代IPTG诱导脱色酶TpmD基因在大肠杆菌中的高效表达

本文考察了乳糖代替IPTG诱导三苯基甲烷类染料脱色酶TpmD在大肠杆菌BL21(DE3)中表达的可行性, 分别对用乳糖作为诱导剂时的诱导时机、乳糖浓度、诱导持续时间和添加方式进行优化并与IPTG诱导的差异等方面进行了比较分析, 确定了乳糖诱导的最佳条件。结果表明, 在工程菌对数生长中期(OD600约为0.8)添加终浓度为0.4 mmol/L的乳糖诱导6 h的条件下能获得最大量的目的蛋白和菌体量。由于乳糖可以作为碳源被菌体利用, 分批添加乳糖效果优于一次性添加。乳糖诱导条件下目的蛋白表达量占总蛋白的35.62%, 与IPTG诱导条件下的35.03%无明显差异。乳糖诱导后外源蛋白的表达时间有所滞后, 但收获的菌体量高于IPTG诱导, 显示出了乳糖同样是一种T7启动子的廉价高效诱导剂, 可以代替昂贵的IPTG用于脱色酶TpmD的规模化发酵, 同时也为其他重组蛋白的生产提供了有益的参考和借鉴。     

乳糖作为诱导剂对重组目的蛋白表达的影响

将重组粒细胞-巨噬细胞集落刺激因子/白细胞介素3(GM-CSF/IL-3)融合蛋白表达菌BL21(DE3)(pFu)作为研究对象,对于以乳糖作为诱导剂时重组目的产物的诱导表达规律进行了深入的研究。分析比较了不同培养基中,不同生长阶段进行诱导对于产物表达的影响。对诱导所需的乳糖浓度、诱导持续时间长短等因素亦进行了研究。实验结果表明,在对诱导条件进行优化控制的前提下,利用乳糖作为诱导剂可以达到与IPTG类似的诱导效果。随后的研究中,将乳糖作为诱导剂应用于高密度发酵过程。这些研究结果为乳糖作为诱导剂最终应用于重组基因工程药物的工业化生产提供了有益的参考和借鉴。     

Trx-NAP 5融合蛋白在大肠杆菌中的表达及其活性检测

目的:用大肠杆菌表达获得重组线虫抗凝血肽5(rNAP 5),为研究开发NAP5的功能与应用提供原料来源。方法:将扩增的NAP5基因经BamHⅠ和HindⅢ双酶切后与表达载体pET-32a连接。构建好的重组表达质粒转化至大肠杆菌BL21(DE3)后,分别经IPTG和乳糖诱导表达,并探讨诱导表达条件,分析表达产物的可溶性情况。表达产物经镍亲和纯化后,用凝血酶原时间(PT)和活化部分凝血活酶时间(aPTT)检测体外抗凝活性。结果:成功构建了pET-32a/NAP5表达载体,IPTG和乳糖均能诱导目的蛋白在大肠杆菌BL21(DE3)中高效地可溶性表达。优化条件下每升LB培养基可获可溶性目的融合蛋白量达65.3mg。纯化的蛋白能明显延长PT及aPTT,7.0mg/L的蛋白平均约延长5.09倍aPTT,2.55倍PT。结论:在大肠杆菌中成功表达了具有很好生物活性的Trx-NAP5融合蛋白,为研究开发NAP5的功能与应用奠定了基础。     

乳糖诱导重组多价人精子表位肽在大肠杆菌中的表达

旨在研究用乳糖替代IPTG作为诱导剂进行重组多价人精子抗原表位肽的表达及诱导表达的优化条件.在摇瓶发酵条件下,通过改变培养基组成、诱导时机、诱导温度、诱导剂浓度、诱导时间和诱导方式等条件,利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统,研究以上条件改变对GST-重组多价人精子抗原表位肽融合蛋白表达量的影响,并与IPTG诱导结果相比较.结果显示,在摇瓶试验中,最优表达条件为选用TB培养基,在菌体对数生长的中期进行诱导,诱导温度37℃、乳糖诱导终浓度3 mmol/L、诱导时间6 h.GST-重组多价人精子抗原表位肽融合蛋白的表达量占菌体总蛋白的30.5％,且主要以可溶形式表达,与IPTG的诱导结果相同.分批流加乳糖和一次性加入乳糖诱导,效果一样.乳糖可以替代IPTG作为诱导剂诱导GST-重组多价人精子抗原表位肽融合蛋白的表达,优化条件下可获得与IPTG相同的诱导表达效果.     

Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts

In a previous publication (Narhi, L. O., and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome that is strongly induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this single polypeptide can catalyze the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase (cytochrome P-450BM-3) has now been cloned by an immunochemical screening technique. The Escherichia coli clone harboring the recombinant plasmid produces a 119,000-dalton protein that appears to be electrophoretically and immunochemically identical to the B. megaterium enzyme and contains the same N-terminal amino acid sequence. The recombinant DNA product also exhibits the characteristic cytochrome P-450 spectrum and is fully functional as a fatty acid monooxygenase. In E. coli, the synthesis of P-450BM-3 is directed by its own promoter included in the DNA insert and proceeds constitutively at a very high rate but is not stimulated by pentobarbital. However, when the cloned P-450BM-3 gene, either intact or in a truncated form, is introduced back into B. megaterium via an E. coli/Bacillus subtilis shuttle vector, its expression is constitutively repressed but is induced by pentobarbital. This finding demonstrates that the regulatory region of the P-450BM-3 gene that responds to barbiturates is included in the cloned DNA. The evidence also indicates that pentobarbital cannot directly act on the gene to cause induction but presumably interacts with another component such as a repressor molecule that is present in B. megaterium but is absent in the E. coli clone.     

Enhanced expression of cytochrome P450s from lac-based plasmids using lactose as the inducer

The cytochrome P450 expression systems used in Escherichia coli are highly regulated and involve the use of the lac repressor to control expression. Induction in these systems utilizes the nonmetabolizable analog of lactose, isopropyl-beta-D-thiogalactopyranoside (IPTG), which is the most expensive compound required for an E. coli expression system. To determine if the natural inducer lactose could be used to induce cytochrome P450 expression we examined the expression of three P450 enzymes in E. coli using two different expression systems, pTrc99A and the T7-based PET22b vector. For both systems lactose was found to induce expression of active P450 to concentrations that exceeded the levels achieved with IPTG. A 20-liter fermentation of a P450 expression system in the pTrc plasmid in which lactose was used as the inducer resulted in 2.4 micromol P450/liter, with a total yield of 2 g of cytochrome P450. The use of lactose for protein expression in E. coli should be broadly useful for the inexpensive, large-scale production of heterologous proteins in E. coli.     

乳糖代替IPTG诱导己糖激酶在大肠杆菌中的表达

构建了己糖激酶GLK高效表达的工程菌株BL21(DE3)/pET-glk,考察了乳糖代替IPTC诱导己糖激酶GLK在大肠杆菌BL21(DE3)中表达的可行性.实验结果表明,工程菌对数生长中期(OD<,600>约为1.0)添加终浓度为10 g/L的乳糖于25℃的条件下诱导6 h能获得最大量的目的蛋白和菌体量,目的蛋白表达...     

Interactions of substrates at the surface of P450s can greatly enhance substrate potency

Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.     

Active site topologies of bacterial cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3). In situ rearrangement of their phenyl-iron complexes.

The reactions of cytochromes P450101 (P450cam), P450108 (P450terp), and P450102 (P450BM-3) with phenyldiazene result in the formation of phenyl-iron complexes with absorption maxima at 474-478 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 decreases the 474-478 nm absorbance and shifts the phenyl group from the iron to the porphyrin nitrogens. Acidification and extraction of the prosthetic group from each of the ferricyanide-treated enzymes yields a different mixture of the four possible N-phenylprotoporphyrin IX regioisomers. The ratios of the regioisomers with the phenyl ring on pyrrole rings B, A, C, and D (in order of elution from the high performance liquid chromatography column) are, respectively: cytochrome P450cam, 0:0:1:4; P450terp, 0:0:0:1; and P450BM-3, 2:10:2:1. The isomer ratio for recombinant cytochrome P450BM-3 without the cytochrome P450 reductase domain (2:9:2:1) shows that the reductase domain does not detectably perturb the active site topology of cytochrome P450BM-3. Potassium ions modulate the intensity of the spectrum of the phenyl-iron complex of cytochrome P450cam, but do not alter the N-phenyl isomer ratio. Computer graphics analysis of the crystal structure of the cytochrome P450cam phenyl-iron complex indicates that the active site of cytochrome P450cam is open above pyrrole ring D and, to a small extent, pyrrole ring C, in complete agreement with the observed N-phenylprotoporphyrin IX regioisomer pattern. The regioisomer ratios indicate that the active site of cytochrome P450terp is only open above pyrrole ring D, whereas that of cytochrome P450BM-3 is open to some extent above all the pyrrole rings but particularly above pyrrole ring A. The bacterial enzymes thus have topologies distinct from each other and from those of the mammalian enzymes so far investigated, which have active sites that are open to a comparable extent above pyrrole rings A and D.     

Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents

Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.     

A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A

Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the positions omega-1, omega-2, and omega-3. A rapid and continuous spectrophotometric activity assay for cytochrome P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was developed. In contrast to the commonly used activity assays for this enzyme, relying on the consumption of oxygen or NADPH or the use of 14C-labeled carboxylic acids, the pNCA assay can even be used with crude extracts of the recombinant enzyme from lysed Escherichia coli cells. The kinetics of p-nitrophenolate formation are directly measured at a wavelength of 410 nm using a spectrophotometer or microtiter plate reader. Sensitivity of the assay is greatly enhanced if p-nitrophenoxydodecanoic or p-nitrophenoxypentadecanoic acid are used with the F87A mutant instead of the wild-type P450 BM-3 enzyme.