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1.
Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket 总被引:1,自引:0,他引:1
Muller BH Lamoure C Le Du MH Cattolico L Lajeunesse E Lemaître F Pearson A Ducancel F Ménez A Boulain JC 《Chembiochem : a European journal of chemical biology》2001,2(7-8):517-523
We describe a strategy that allowed us to confer on a bacterial (E. coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability. First, we identified mutations, at positions other than those occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation. We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being outside. Second, the gene encoding the inactive mutant was submitted to random mutagenesis. Enzyme activity was restored upon the single mutation D330N, at a position that is 12 A away from the center of the catalytic pocket. Third, this mutation was combined with other mutations previously reported to increase AP activity slightly in the presence of magnesium. As a result, at pH 10.0 the phosphatase activity of both mutants D330N/D153H and D330N/D153G was 17-fold higher than that of the wild-type AP. Strikingly, although the two individual mutations D153H and D153G destabilize the enzyme, the double mutant D330N/D153G remained highly stable (T(m)=87 degrees C). Moreover, when combining the phosphatase and transferase activities, the catalytic activity of the mutant D330N/D153G increased 40-fold (k(cat)=3200 s-1) relative to that of the wild-type enzyme (k(cat)=80 s-1). Due to the simultaneous increase in K(m), the resulting k(cat)/K(m) value was only increased by a factor of two. Therefore, a single mutation occurring outside a catalytic pocket can dramatically control not only the activity of an enzyme, but also its thermostability. Preliminary crystallographic data of a covalent D330N/D153G enzyme-phosphate complex show that the phosphate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant previously reported. 相似文献
2.
Belin P Dassa J Drevet P Lajeunesse E Savatier A Boulain JC Ménez A 《Protein engineering, design & selection : PEDS》2004,17(5):491-500
We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding. For an efficient selection, we found that time of toxicity induction must be precisely determined and recombinant protein must be expressed as a fusion with a protein whose activity is easily detectable on plates, thus allowing elimination of non-productive mutants. Choosing the expression to the periplasmic space of an scFv fragment fused to the N-terminus of alkaline phosphatase as a model, we selected chromosomal mutations that reduce aggregation-induced toxicity and showed that they concomitantly improve production of a functional recombinant hybrid. The effects of the mutations isolated could then be cumulated with those of other strategies used for recombinant scFv production. Thus, we could ensure a 6- to 16-fold increase in production of a functional scFv-PhoA hybrid. This is the first report demonstrating the possibility of directly selecting on agar plates E.coli strains improved for functional recombinant protein production from a large bacterial mutant library. 相似文献
3.
Browner Michelle F.; Rasor Peter; Tugendreich Stuart; Fletterick Robert J. 《Protein engineering, design & selection : PEDS》1991,4(3):351-357
In order to understand how allosteric switches regulate boththe catalytic activity and molecular interactions of glycogenphosphorylase, it is necessary to design and analyze variantproteins that test hypotheses about the structural details ofthe allosteric mechanism. Essential to such an investigationis the ability to obtain large amounts of variant proteins.We developed a system for obtaining milligram amounts (>20 mg/1) of rabbit muscle phosphorylase from bacteria. Phosphorylaseaggregates as inactive protein when a strong bacterial promoteris used under full inducing conditions and normal growth conditions.However, when the growth temperature of bacteria expressingphosphorylase is reduced to 22°C we obtain active musclephosphorylase. The degree to which the induced expression ofphosphorylase protein is temperature sensitive depends on thestrain of bacteria used. New assay and purification methodswere developed to allow rapid purification of engineered phosphorylaseproteins from bacterial cultures. The rabbit muscle phosphorylaseobtained from the bacterial expression system is enzymaticallyidentical to the enzyme purified from rabbit muscle. The expressedprotein crystallizes in the same conditions used for growingcrystals of protein from rabbit muscle and the crystal formis isomorphous. Rabbit muscle phosphorylase is one of the largestoligomeric mammalian enzymes successfully expressed in Escherichiacoli. Our results indicate that optimization of a combinationof growth and induction conditions will be important in theexpression of other heterologous proteins in bacteria. 相似文献
4.
Interactions of chemically exfoliated graphene oxide (GO) nanosheets and Escherichia coli bacteria living in mixed-acid fermentation with an anaerobic condition were investigated for different exposure times. X-ray photoelectron spectroscopy showed that as the exposure time increased (from 0 to 48 h), the oxygen-containing functional groups of the GO decreased by ~60%, indicating a relative chemical reduction of the sheets by interaction with the bacteria. Raman spectroscopy and current–voltage measurement confirmed the reduction of the GO exposed to the bacteria. The reduction was believed to be due to the metabolic activity of the surviving bacteria through their glycolysis process. It was found that the GO sheets could act as biocompatible sites for adsorption and proliferation of the bacteria on their surfaces, while the bacterially-reduced GO (BRGO) sheets showed an inhibition for proliferation of the bacteria on their surfaces. It was shown that the slight antibacterial property of the BRGO sheets and the detaching of the already proliferated bacteria from the surface of these sheets contributed to the growth inhibition of the bacteria on the surface of the reduced sheets. 相似文献
5.
In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from VH manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation. 相似文献
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Fujita Izumi; Komeiji Yuto; Yamato Ichiro 《Protein engineering, design & selection : PEDS》1995,8(9):935-938
A mutational study was performed on the corepressor (Ltryptophan)binding site of the trp repressor of Escherichia coli. Threonine81, one of the residues forming the hydrophobic pocket of thebinding site, was replaced with Ser, Cys and Met by cassettemutagenesis. Biochemical characterization showed that all thesemutations caused a moderate decrease in tryptophan binding activity(free energy change 1 kcal/mol). The results suggested thatthe binding pocket is rather flexible in the vicinity of Thr81.On the other hand, the mutations produced a discernible decreasein the repressor activity in vivo, apparently by weakening oreliminating the hydrogen bond between Thr81 and the operatorDNA, as well as by introducing possible side-chain rearrangement. 相似文献
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采用聚乙烯亚胺(PEI)和戊二醛(GA)对棉纤维进行化学修饰,考察了载体改性后的性能和对固定化大肠杆菌产丁二酸的影响。改性后的载体菌体负载量提高了63.3%。培养基中葡萄糖浓度为43 g/L,添加改性棉纤维120g/L,以MgCO3为缓冲盐,进行批式发酵,丁二酸浓度达到29.6 g/L,比未改性棉纤维提高了11.3%;丁二酸收率达到70.5%,比改性前提高了7.5%;丁二酸生产速率达到0.66 g/(L?h), 比改性前提高了37.5% 。对该材料固载的细胞进行7次重复批式发酵,丁二酸产量、转化率和产率没有下降趋势,具有一定的重复稳定性。 相似文献
13.
Li Zhou Wen‐jing Cui Zhong‐mei Liu Zhe‐min Zhou 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2016,91(3):576-584
D‐lactate is an important chiral intermediate and a substrate for polylactic acid (PLA) production. Escherichia coli accumulate D‐lactate as the primary fermentative product, and synthesis is directly controlled by the activity of lactate dehydrogenase, the efficiency of the carbon resource utilization, and the redox state. D‐lactate accumulation is complicated by the flux through the competing metabolic routes and the indirect regulation of energy metabolism, as well as by the unexpected interconnectivities of the cellular components in the remote metabolic routes. These effects have been extensively studied, and a number of metabolic engineering strategies have been applied to overproduce D‐lactate with high titers, yields and productivities in E. coli that have been able to reduce the production costs and precisely control the fermentation process. This review summarizes the related strategies and suggests directions for further study; these directions provide guidelines for the development of other metabolic products using E. coli as an industrial platform. © 2015 Society of Chemical Industry 相似文献
14.
3,4-Dihydroxy-2-butanone 4-phosphate (DHBP) and GTP are the precursors for riboflavin biosynthesis. In this research, improving the precursor supply for riboflavin production was attempted by overexpressing ribB and engineering purine pathway in a riboflavin-producing Escherichia coli strain. Initially, ribB gene was overexpressed to increase the flux from ribulose 5-phosphate (Ru-5-P) to DHBP. Then ndk and gmk genes were overexpressed to enhance GTP supply. Subsequently, a R419L mutation was introduced into purA to reduce the flux from IMP to AMP. Finally, co-overexpression of mutant purF and prs genes further increased riboflavin production. The final strain RF18S produced 387.6 mg riboflavin · L?1 with a yield of 44.8 mg riboflavin per gram glucose in shake-flask fermentations. The final titer and yield were 72.2%and 55.6%higher than those of RF01S, respectively. It was concluded that simultaneously engineering the DHBP synthase and GTP biosynthetic pathway by rational metabolic engineering can efficiently boost riboflavin production in E. coli. 相似文献
15.
The effects of induction conditions on production of a soluble antitumor sFv in Escherichia coli 总被引:2,自引:0,他引:2
Jaymie R.Sawyer; Schlom Jeffrey; Kashmiri S. V. S. 《Protein engineering, design & selection : PEDS》1994,7(11):1401-1406
CC49 is a second generation monoclonal antibody(mAb) with highaffinity to a pancarcinoma antigen, TAG-72. A single-chain Fv(sFv)ofCC49 may have a role in managing human carcinomas. Most reportedsFvs have been expressed as insoluble products that must besolubilized and renatured. Soluble sFv expression is advantageousas activity can be assayed directly from the periplasmic fraction.Also, gene-level immunoconjugates may not be amenable to refoldingprotocols. Using a vector that carries the tac promoter andomp A signal, we have examined the effects of four variableson the expression and accumulation of soluble CC49 sFv: (i)linker sequence joining VL and VH, (ii) isopropylthio-ß-D-galactosideconcentration for induction, (iii) temprature, and (iv) theaddition of nonmetabolizable sugars to the medium. We have beenable to demonstrate, using rapidly prepared periplasmic extracts,that the yield of soluble sFv improves by the addition of 0.4Msucrose to the medium and by inducing expression with a verylow concentration of IPTG (0.020.03 mM). Under theseinduction conditions periplasmic extracts demonstrate increasedexpression of the sFv, as shown by the larger amount of a 27kDa band on SDS-polyacrylamide gel, and an increased abilityto inhibit binding of the mAb CC49 to immobilized tumor extracts. 相似文献
16.
在重组大肠杆菌发酵生产类人胶原蛋白(HLC)过程中,快速确定不同反应器中的补料速率,抑制乙酸产生,高水平表达HLC.采用溶氧探测补料技术,在不同规模反应器中,分批-补料培养重组大肠杆菌生产HLC,根据脉冲补料方式时溶氧信号的响应特征来辩识是否产生乙酸并确定补料速率.在实验室规模获得的最终细胞密度和HLC质量浓度分别为6... 相似文献
17.
de Lamotte-Guery F; Pruvost C; Minard P; Delsuc MA; Miginiac-Maslow M; Schmitter JM; Stein M; Decottignies P 《Protein engineering, design & selection : PEDS》1997,10(12):1425-1432
Proline 40 in Escherichia coli thioredoxin is located close to the redox
active site (Cys32-Cys35) within the alpha2 helix. The conservation of this
residue among most of the thioredoxins suggests that it could play an
important role in the structure and/or function of this protein. We have
substituted Pro40 for Ala by using site- directed mutagenesis and expressed
the mutant P40A in E.coli. The effects of the mutation on the biophysical
and biological properties of thioredoxin have been analyzed and compared
with molecular dynamics simulations. Modeling predicted that the
replacement of Pro40 by Ala induced a displacement of the active site which
exposes Trp31 to the solvent and opens a cleft located between helices
alpha2 and alpha3. The solvation free energy (SFE) calculation also
indicated that P40A became more hydrophobic as W31 became more accessible.
These predictions were totally in agreement with the experimental results.
The mutant P40A exhibited chromatographic behavior and fluorescence
properties very different from those of the wild-type (WT) protein, in
relationship with the displacement of W31. The determination of the free
energy of unfolding of P40A showed that the mutant was globally
destabilized by 2.9 kcal/mol. However, the effect of the mutation on the
transition curve was highly unusual as the midpoint of the unfolding
transition increased, indicating that some local structures were actually
stabilized by the mutation. Despite these structural modifications, neither
the ability of the protein to reduce a chloroplastic enzyme nor its
reactivity with the bacterial reductase decreased. The only functional
difference was the higher stability of P40A in light activation of
NADP-malate dehydrogenase under air, which suggests that the mutant was
less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40
is not essential for maintaining the redox function of thioredoxin but
rather is required for the stability of the protein.
相似文献
18.
Jianghua Li Long Liu Jing Wu Jun Sun Guocheng Du Jian Chen Wenyi Tao 《Korean Journal of Chemical Engineering》2010,27(4):1233-1238
Statistics-based experiment designs were used to optimize the culture medium (glucose, yeast extract, IPTG, tween-60, and CaCl2) for cutinase production by recombinant Escherichia coli. A 25-1 fractional factorial design augmented with center points revealed that glucose, yeast extract, and IPTG were the most significant factors, whereas the other factors were not important within the levels tested. The method of steepest ascent was used to approach the proximity of optimum, followed by a central composite design to develop a response surface for culture condition optimization. The optimum culture medium for cutinase production was found to be: glucose 33. 92 g/L, yeast extract 30.92 g/L, and IPTG 0.76 g/L. A cutinase production of 145.27±1.5 U/mL, which was in agreement with the prediction, was observed in triplicate verification experiments. The results obtained here verified the effectiveness of the applied methodology and may be helpful for cutinase production on an industrial scale. 相似文献
19.
《中国生物制品学杂志》2017,(4)
目的检测恶性疟原虫表面蛋白25(Plasmodium falciparum surface protein 25,Pfs25)与鼠伤寒沙门菌1相鞭毛蛋白(phase 1 flagellin of Salmonella enterica serovar Typhimurium,FliC)的融合蛋白FliC-Pfs25,在硫氧还蛋白还原酶基因(thioredoxin reductase gene,trxB)和谷胱甘肽还原酶基因(glutathione reductase gene,gor)双突变大肠埃希菌Origami2(DE3)中的表达。方法将含有Pfs25和FliC基因的重组质粒pET28a(+)-fliC-pfs25转化Origami2(DE3),IPTG诱导表达融合蛋白FliC-Pfs25,破菌上清经Ni-Sepharose纯化,纯化产物进行SDS-PAGE及Western blot鉴定。将纯化的融合蛋白配制成不同的疫苗制剂免疫BALB/c小鼠,ELISA法检测小鼠免疫血清中抗Pfs25抗体水平。结果融合蛋白FliC-Pfs25在Origami2(DE3)中以可溶性形式表达;纯化的融合蛋白可被两个抗Pfs25的单抗mAb 1A6和mAb 4D10识别;融合蛋白与铝佐剂结合在小鼠中激发出抗Pfs25的抗体应答,但Origami2(DE3)表达的FliCPfs25与抗Pfs25单抗的反应以及在小鼠中激发抗Pfs25抗体的能力,均弱于大肠埃希菌BL21(DE3)表达的FliCPfs25。结论融合蛋白FliC-Pfs25在大肠埃希菌Origami2(DE3)中成功获得表达,但trxB和gor两个基因的突变未显示出能够提高该融合蛋白在大肠埃希菌中的表达量。 相似文献