Anti-inflammatory effects of recombinant arginine deiminase originating from Lactococcus lactis ssp. lactis ATCC 7962

Arginine deiminase (ADI, E.C. 3.5.3.6), one of the arginine deprivation enzymes, exhibits anticarcinogenic activities. The present study investigated the anti-inflammatory activities of the purified recombinant ADI originating from Lactococcus lactis ssp. lactis ATCC7962 (LADI). LADI dose-dependently inhibited lipopolysaccharide (LPS)-induced upregulation of inducible nitric oxide synthase and the production of nitric oxide in RAW 264.7 murine macrophages. The induction of cyclooxygenase-2 expression and subsequent production of prostaglandin E2 by LPS was also attenuated by LADI treatment. Moreover, LADI inhibited the production of interleukin-6 in LPS-stimulated RAW 264.7 macrophages. These results indicate that LADI exerts anti-inflammatory effects, which may in part explain its chemopreventive potential.     

Expression, purification, and characterization of thymidylate synthase from Lactococcus lactis.

The thymidylate synthase (TS) gene from Lactococcus lactis has been highly expressed in Escherichia coli. The TS protein was purified by sequential chromatography on Q-Sepharose and phenyl-Sepharose. Six grams of cell pellet yielded 140 mg of homogeneous TS. TS is a highly conserved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L. lactis TS. By use of a 3-dimensional homology model, we have predicted covariant changes that might compensate for these differences. With the large amounts of L. lactis TS now available, studies can be pursued to understand the structure-function relationships of this enzyme compared to other TSs and to confirm the presumed roles of the compensatory changes predicted in the homology model.     

Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC 11105

The penicillin acylase gene (pac) from Escherichia coli ATCC 11105 was cloned into pUC 9 and the resulting vector (pUPA-9), when transformed into E. coli strain 5K, allowed the constitutive overproduction of mature penicillin acylase when grown at 28 degrees C. The enzyme was purified from the periplasmic fraction of E. coli pUPA-9 by hydrophobic interaction chromatography and anion exchange. Crystals of penicillin acylase were grown in batch using polyethylene glycol 8000 as a precipitant. The crystals (space group P1) diffracted to beyond 2.3 A.     

Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli.

Recombinant purine nucleoside phosphorylase (PNPase) from Escherichia coli was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate-liberating enzymes. The E. coli enzyme was overexpressed in E. coli by inserting the genomic fragment containing the deoD gene downstream of the isopropyl beta-d-thiogalactoside-inducible promotor of pSE380 expression vector. The recombinant protein was purified to approximately 90% homogeneity and with a yield of approximately 9000 units of activity/L of culture, using an efficient one-column procedure. A continuous spectrophotometric assay coupling P(i) release to the phosphorolysis of the nucleoside analogue 7-methylinosine (m(7)Ino) was recently described. Here, we report the steady-state kinetic parameters of the recombinant E. coli PNPase catalyzed reaction with m(7)Ino and P(i) as substrates and compare these parameters with those of a bacterial PNPase commercially available for use in coupled assays. Under the assay conditions described, the recombinant E. coli protein is active at higher pH values and is stable up to a temperature of approximately 55 degrees C and following multiple freeze-thaw cycles. It is activated by high ionic strength but loses some activity following dialysis or concentration under pressure. Finally, a new procedure for the synthesis of m(7)Ino from inosine is described which is safe and cost effective, making the use of this methylated nucleoside in PNPase-coupled P(i) assays more attractive.     

乳酸乳球菌启动子—信号肽活性片段在大肠杆菌中的克隆和表达

利用pGPB14为启动子-信号肽序列探测载体,在大肠杆菌中克隆乳酸乳球菌总DNA中具有启动子-信号肽功能的DNA片段。共得到42个具有红霉索、氨苄青霉素双重抗性的转化子。转化子的氨苄青霉素抗性水平在100～800μg/ml之间,β-内酰胺酶活力大多积累于周质空间,说明重组质粒中的外源插入片段确实具有发动转录和促进分泌的功能。Southern杂交结果表明,插入片段的确来源于乳酸乳球菌总DNA,其大小在80～400bp之间。DNA序列测定发现pSEQ8和pSEQ12中插入片段分属于pSEQ4和pSEQ17插入片段的一部分。在测序的4个DNA序列中都找到了启动子和起始密码子。其中2个含有典型的S.D.序列和非典型的信号肽序列,其他2个则没有发现典型的S.D.序列和信号肽序列。另外发现启动子上游序列对启动子转录起始的效率具有促进作用。     

Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, deaminated and lipid peroxidation-induced purine adducts. MPG from human and mouse has previously been cloned and expressed. However, due to the poor expression level in Escherichia coli (E. coli) and multi-step purification process of full-length MPG, most successful attempts have been limited by extremely poor yield and stability. Here, we have optimized the codons within the first five residues of human MPG (hMPG) to the best used codons for E. coli and expressed full-length hMPG in large amounts. This high expression level in conjunction with a strikingly high isoelectric point (9.65) of hMPG, in fact, helped purify the enzyme in a single step. A previously well-characterized monoclonal antibody having an epitope in the N-terminal tail could detect this codon-optimized hMPG protein. Surface plasmon resonance studies showed an equilibrium binding constant (K_D) of 0.25 nM. Steady-state enzyme kinetics showed an apparent K_m of 5.3 nM and k_cat of 0.2 min⁻¹ of MPG for the hypoxanthine (Hx) cleavage reaction. Moreover, hMPG had an optimal activity at pH 7.5 and 100 mM KCl. Unlike the previous reports by others, this newly purified full-length hMPG is appreciably stable at high temperature, such as 50 °C. Thus, this study indicates that this improved expression and purification system will facilitate large scale production and purification of a stable human MPG protein for further biochemical, biophysical and structure–function analysis.     

血小板第四因子在原核中的高效表达、分离纯化及活性测定

目的：构建重组表达质粒pET-32c/PF4,并在原核中进行表达,以探讨其对白血病细胞系（HEL）细胞增殖的作用。方法：通过PCR方法从含有PF4基因的PQE-60/PF4质粒中扩增PF4,用NcoⅠ/HindⅢ双酶切,克隆到原核表达质粒pET-32C中,使之在BL21表达,Ni-Chelating Sepharose亲和柱纯化,用细胞集落形成方法研究重组PF4对HEL的抑制作用。结果：菌株筛选后在大肠杆菌中获得可溶性高效表达,重组PF4占菌体总蛋白的22%,肠激酶酶切除去端啧合部分,获得了高纯度的重组人血小板第四因子（rh-PF4),纯度为95%以上,活性实验发现重组PF4可抑制HEL细胞集落的形成,抑制率为47%,结论：原核表达质粒pET-32C可高效可溶性表达PF4,重组PF4对HEL细胞的增殖有抑制作用。     

Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose

Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase. Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999     

Probiotic properties of Lactococcus lactis ssp. lactis HV219, isolated from human vaginal secretions

AIMS: To determine the resistance of Lactococcus lactis ssp. lactis HV219 to acids, bile, antibiotics, inflammatory drugs and spermicides, compare adsorption of the strain to bacteria and Caco-2 cells under stress, and evaluate the antimicrobial activity of bacteriocin HV219. METHODS AND RESULTS: Bacteriocin HV219 activity against Gram-positive and Gram-negative bacteria was confirmed by leakage of DNA and beta-galactosidase, and atomic force microscopy. Adsorption of bacteriocin HV219 to bacteria is influenced by pH, temperature, surfactants and salts. Initially, only 3% of HV219 cells adhered to Caco-2 cells. However, after 2 h, adherence increased to 7%. Strain HV219 and Listeria monocytogenes ScottA did not compete for colonization. Strain HV219 is sensitive to most antibiotics tested, but resistant to amikacin, ceftazidime, nalidixic acid, metronidazole, neomycin, oxacillin, streptomycin, sulphafurazole, sulphamethoxazole, sulphonamides, tetracycline and tobramycin. Ibuprofen, ciprofloxacin, diklofenak and nonoxylol-9 inhibited the growth of strain HV219. CONCLUSION: Strain HV219 is resistant to hostile conditions in the intestinal tract, including therapeutic levels of specific antibiotics and binds to Caco-2 cells, but not in competition with L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain HV219 will only be effective as probiotic if taken with specific antibiotics and not with anti-inflammatory drugs and spermicides.     

Expression, purification and characterization of soluble human thrombopoietin receptor from Escherichia coli

The extracellular domain (edMpl) of human thrombopoietin (TPO) receptor, c-Mpl was expressed in Escherichia coli by changing some nucleotides before and after the translation initiation codon. The mutations increased the expression by approx. 15-fold. The inclusion bodies were solubilized in 8 M guanidine-HCl under reducing conditions and refolded using a glutathione-redox system. The monomeric form of edMpl was purified to near homogeneity by two successive steps of ion-exchange chromatography using DEAE-Sephacel and Mono Q columns. The purified monomeric edMpl inhibited the TPO-dependent cell proliferation, suggesting that it was binding to TPO. Also, antisera raised against the edMpl bound specifically to the soluble receptor secreted by mammalian cells.     

Cloning of a chromosomal fragment from Lactococcus lactis subsp. lactis partially complementing Escherichia coli recA functions

A recA-like gene was isolated from a gene library of Lactococcus lactis subsp. lactis by intergeneric complementation of an E. coli recA mutant. A plasmid was obtained which fully complemented the RecA response to DNA damaging agents and UV inducibility of prophage, but not P1 plating efficiency in an E. coli recA mutant. The cloned DNA fragment also partially complemented the rec mutation in Lc. lactis MMS36. Hybridization studies showed that there was no detectable sequence homology between the recA gene of E. coli and Lc. lactis subsp. lactis chromosomal DNA.     

灵杆菌非特异性核酸酶的原核表达、纯化及活性分析

以灵杆菌基因组DNA为模板,PCR扩增非特异性核酸酶 (Non-specific nuclease,NU) 基因,并克隆到pMAL-c4X载体上构建重组表达载体pMAL-c4X-NU。经测序及 BLASTN发现其与灵杆菌Serratia marcescens核酸酶基因的同源性为97％。将构建的表达载体pMAL-c4X-NU转入大肠杆菌BL21,经IPTG诱导实现了胞内表达78 kDa的麦芽糖结合蛋白-NU融合蛋白 (Maltose-binding protein-NU,MBP-NU),其最佳诱导表达条件为37 ℃,0.75 mmol/L IPTG诱导1.5 h。用Amylose resin纯化得到了目的蛋白。活性检测表明MBP-NU具有同时降解DNA和RNA的活性,在37 ℃、pH 8.0时活性最高,比活力为1.11×106 U/mg,目标蛋白的纯化效率可达10.875 mg/L。纯化的目标蛋白中无蛋白酶活性存在。0.5 mmol/L乙二胺四乙酸 (Ethylene diamine tetraacetic acid,EDTA)、1 mmol/L苯甲基磺酰氟 (Phenylmethanesulfonyl fluoride,PMSF) 以及150 mmol/L KCl对MBP-NU的活性几乎无影响,因此MBP-NU可作为蛋白质纯化过程中核酸的高效降解酶。     

Protein characterization of lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages isolated from cheese wheys

Proteins of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris bacteriophages were studied using antibody inhibition assay and immunoblotting. Antisera were prepared against four representative L. lactis ssp. lactis and L. lactis ssp. cremoris phages (D59-1, F4-1, G72-1, and I37-1), which were selected from 17 isolates, derived from commercial cheese wheys. The reactivities of the four antisera with 13 other phage isolates were tested. Among these isolates, two phage groups having distinct serological properties were found. Group I reacted with the antisera against phages D59-1/F4-1 and Group II reacted with the antisera against phages G72-1/I37-1. Strongly lytic phages, capable of lysing phage-resistant host strains, were found to share protein similarities with the phage protein group I, and phages isolated from phage-sensitive host strains belonged to the phage protein group II. Furthermore, group I was composed of all prolate and some isometric phages, whereas group II was composed solely of the isometric phages. Thus, the two serologically distinct phage groups were not correlated with the two morphological groups, prolate and isometric. Proteins of the four phages were further characterized by immunoblotting and silver staining. A 22.5-kDa antigenic polypeptide of phage I37-1, and three polypeptides of 65, 37, 21 kDa in phage F4-1 were responsible for the cross-reactivities in group II and group I, respectively. Correspondence to: R. A. Ledford     

K5裂解酶在大肠杆菌中的表达、纯化及酶学性质分析

K5多糖裂解酶(Elma)能够裂解半合成肝素的底物-K5多糖,裂解产物是半合成法生产低分子量肝素的底物。利用PCR方法扩增elma,构建表达载体pET-28a-Elma,将构建好的质粒转化至大肠杆菌BL21中,以0.2 mmol/L的IPTG在16℃诱导5 h实现了高效表达,SDS-PAGE分析表明Elma表达量可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法和G-75分子筛层析纯化目的蛋白,其纯度大于95%。通过PAGE多糖电泳发现裂解前后的K5多糖分子量有明显的减小。根据Elma裂解产物产生双键从而在232 nm处有吸光度的变化来测Elma的酶活。其最适反应温度为37℃,反应的最适pH值为7.0。底物特异性分析发现Elma除K5多糖外对肝素和透明质酸也有降解作用。     

Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363

This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.     

Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli

A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ‘‘natural” cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential application in the production of fruit juices product industry.     

Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39?kDa. The effects of induction temperature, concentration of isopropyl-β-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23?U/mL (8.64?U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .