地衣芽孢杆菌普鲁兰酶编码基因的鉴定

对地衣芽孢杆菌基因组序列分析显示。其中标注为amyX的基因可能编码普鲁兰酶。以PCR方法,从地衣芽孢杆菌染色体DNA中扩增出amyX基因蛋白编码区,插入大肠杆菌表达载体pET28aT7启动予下游。含重组质粒的大肠杆菌BL21（DE3）在IPTG诱导下表达出有活性的普鲁兰酶。酶学性质初步分析表明,重组普鲁兰酶最适反应温度为40℃,最适pH值为6．0。     

Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.     

Effect of lacY expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers

The role of the Escherichia coli lactose permease (LacY) in the homogeneous induction of the lactose-inducible promoters P(tac) and P(trc) by the natural inducer lactose and the synthetic inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) was investigated. Lactose requires active transport by LacY, whereas IPTG can freely penetrate the cell wall. In E. coli strains lacking a functional LacY, IPTG is required for induction of P(tac) and P(trc). In E. coli strains carrying a functional LacY, induction of P(trc) and P(tac) with intermediate concentrations of lactose gave rise to two subpopulations, one fully induced and one uninduced, whereas a single, fully induced population resulted when high inducer concentrations were used. In contrast, induction with IPTG gave rise to a single population of cells at all inducer concentrations in both lacY and lacY(+) strains.     

Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells

An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro.     

Non-toxic expression in Escherichia coli of a plasmid-encoded gene for phage T4 lysozyme

The phage T4 gene coding for lysozyme has been cloned into a plasmid under control of the (trp/lac) hybrid tac promoter and expressed in Escherichia coli with no significant toxic effect to actively growing cells. E. coli D1210 (lacIq) transformed with this plasmid produced active T4 lysozyme at levels up to 2% of the cellular protein after induction with isopropyl-beta-D-thiogalactoside. A strain producing active lysozyme was shown to be under a selective disadvantage when co-cultured with a similar strain producing inactive lysozyme. Purified strains, however, are reasonably stable in culture and under normal storage conditions.     

植原体免疫主导膜蛋白Imp基因原核表达载体构建及表达

旨在构建植原体免疫主导膜蛋白Imp基因原核表达载体,并进行初步表达。以重组克隆质粒pMD18-T-Imp为模板,PCR扩增Imp基因片段。构建表达载体pET-28a(+)-Imp,转化宿主菌E.coliBL21(DE3)。筛选阳性克隆,提取重组质粒作PCR鉴定、酶切鉴定及IPTG诱导表达鉴定。PCR及双酶切结果显示,重组质粒pET-28a(+)-Imp构建成功。经IPTG诱导BL21(pET-28a(+)-Imp)表达约20 kD的蛋白,与预期的携带6×His-Tag的目的蛋白(19.5 kD)大小相符,主要以包涵体形式存在。结果显示,构建的表达载体pET-28a(+)-Imp在E.coliBL21(DE3)中能够达一定量表达,为进一步纯化Imp蛋白奠定基础。     

Flow cytometric analysis of metabolic stress effects due to recombinant plasmids and proteins in Escherichia coli production strains

Overexpression of recombinant proteins in Escherichia coli often leads to a severe growth retardation of the host cells. Using flow cytometry, we analyzed the temporal development of the cellular content of DNA, total protein, and the recombinant product (human superoxide dismutase) in different strains. In cells carrying plasmids utilizing the phage T7 promoter 10 (pET vectors), induction with IPTG leads to an increase in protein content and size, an increase and a wide spreading of DNA content distribution, and a termination of cell division. These effects occurred with pET plasmids with or without an insert, but not with another plasmid which utilizes the tac promoter.     

Improved penicillin amidase production using a genetically engineered mutant of Escherichia coli ATCC 11105

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene. (c) 1993 John Wiley & Sons, Inc.     

Pseudomonas putida S1海藻糖合成酶表达质粒的构建及诱导条件优化

采用PCR方法从Pseudomonas putida S1中克隆出编码海藻糖合成酶的基因treS,并与质粒pQE30T相连,构建了表达质粒pQE—TS2。将此重组质粒转化宿主菌E．coliM15进行诱导表达。十二烷基磺酸钠-聚丙烯酰胺凝胶SDS—PAGE电泳结果表明,treS基因在大肠杆菌中获得了高效表达。通过对诱导温度、诱导剂浓度、加诱导剂时间和诱导时间的优化研究,在菌液生长至OD600值为0．6时,加入诱导剂IPTG至终浓度0．01mmol／L,20℃诱导20h,蛋白的表达量达到每克干细胞89mg的蛋白,粗酶液酶活达到19U／mL。     

Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.     

在大肠杆菌中反义RNA抑制乙型肝炎病毒核心抗原的表达

将乙型肝炎病毒核心抗原(HBcAg)基因的DNA序列分别按正、反方向置tac启动子的控制下,并克隆进入同一个质粒中,转化大肠杆菌JM103后,观察到HBcAg和HBeAg的合成均明显低于仅含有HBcAg基因的细菌,最大抑制率可达70％～80％,分子杂交可见反义RNA存在。     

Release of periplasmic enzymes and other physiological effects of beta-lactamase overproduction in Escherichia coli

When Escherichia coli containing the plasmid ptac11 is induced with 10(-4) M isopropyl-beta-thiogalactopyranoside (IPTG), 90% of the beta-lactamase activity of an overnight culture is present in the medium. The high extracellular activity of beta-lactamase does not result from cell lysis but from an increase in the permeability of the outer membrane. The excreting cells release several other periplasmic enzymes into the extracellular fluid and are more sensitive to lysis by detergents. It was also shown that in these cells the level of two membrane proteins, OmpA and OmpC, is decreased. None of these phenomena were observed with the plasmid pDW17, which has a mutation in the tac promoter that reduces its activity to one fourth of the tac promoter.     

Functional differences between manganese and iron superoxide dismutases in Escherichia coli K-12.

Superoxide dismutases are enzymes that defend against oxidative stress through decomposition of superoxide radical. Escherichia coli contains two highly homologous superoxide dismutases, one containing manganese (MnSOD) and the other iron (FeSOD). Although E. coli Mn and FeSOD catalyze the dismutation of superoxide with comparable rate constants, it is not known if they are physiologically equivalent in their protection of cellular targets from oxyradical damage. To address this issue, isogenic strains of E. coli containing either Mn or FeSOD encoded on a plasmid and under the control of tac promoter were constructed. SOD specific activity in the Mn and FeSOD strains could be controlled by the concentration of isopropyl beta-thiogalactoside in the medium. The tolerance of these strains to oxidative stress was compared at equal Mn and FeSOD specific activities. Our results indicate that E. coli Mn and FeSOD are not functionally equivalent. The MnSOD is more effective than FeSOD in preventing damage to DNA, while the FeSOD appears to be more effective in protecting a cytoplasmic superoxide-sensitive enzyme. These data are the first demonstration that Mn and FeSOD are adapted to different antioxidant roles in E. coli.     

一株高效抗砷喜温硫杆菌工程菌的构建

利用DNA体外重组技术,将大肠杆菌质粒载体pUM3上的抗砷基因簇片段亚克隆到含有强启动子（tac启动子）并具有广泛寄主范围特性的IncQ族质粒pMMB24上,删除调节基因片段,构建了含有强启动子、可在tra基因诱动下转移的组成型表达的抗砷质粒pSDRA4。通过接合转移的方式将其导入专性自养极端嗜酸性喜温硫杆菌Acidithiobacillus caldus中,构建了冶金工程菌Acidithiobacillus caldus (pSDRA4),接合转移频率为（1.444±0.797）×10-4。表明在大肠杆菌和喜温硫杆菌之间成功地建立了一个遗传转移系统。经检测,重组质粒在喜温硫杆菌中具有较好的稳定性,在无选择压力条件下传代50次基本保持稳定（重组质粒保留76% 以上）。经抗砷性能检测,与野生菌相比,构建的喜温硫杆菌工程菌抗砷能力明显提高,从10mmol/L提高到45mmol/L。