Production of recombinant protein in Escherichia coli cultured in extract from waste product alga,Ulva lactuca

This study examined the potential for waste product alga, Ulva lactuca, to serve as a media component for recombinant protein production in Escherichia coli. To facilitate this investigation, U. lactuca harvested from Jamaica Bay was dried, and nutrients acid extracted for use as a growth media. The E. coli cell line BL21(DE3) was used to assess the effects on growth and production of recombinant green fluorescent protein (GFP). This study showed that media composed of acid extracts without further nutrient addition maintained E. coli growth and recombinant protein production. Extracts made from dried algae lots less than six‐months‐old were able to produce two‐fold more GFP protein than traditional Lysogeny Broth media. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:784–789, 2014     

表达大肠杆菌谷氨酸-1-半醛氨基转移酶对红色荧光报告蛋白尿卟啉原Ⅲ甲基化酶的影响

谷氨酸-1-半醛氨基转移酶(Glutamate-1-semiadhyde aminotransferase,GSAT)是尿卟啉原Ⅲ生物合成上游途径的一个酶,尿卟啉原Ⅲ是红色荧光报告蛋白尿卟啉原Ⅲ甲基化酶(Uroporphyrinogen Ⅲ methyltransferase,UPMT)的底物。为了探明大肠杆菌共表达GSAT对UPMT荧光强度的影响,通过PCR扩增玉米upmt基因,将其插入pETDuet-1质粒中第2个顺反子中,构建的载体命名为pETU,表达UPMT的N端含有组氨酸标签;通过PCR扩增大肠杆菌编码GSAT的hemL基因,定点突变去除hemL基因中NcoⅠ序列,亚克隆至pET-51b质粒,再将获得的hemL基因插入pETU质粒的第一个顺反子中,构建pETeGU载体。表达GSAT的N端含有Strep标签。和单独表达upmt基因相比,表达2个基因后,蛋白印迹分析表明没有明显改变UPMT表达量,光谱扫描分析显示没有改变荧光物质的组成,但是增强了重组细胞的红色荧光物质三甲基咕啉的含量,该物质在354nm有特异吸收。用2mmol/L的GSAT抑制剂3-氨基-2,3二羟基苯甲酸处理后,表达两种酶的菌落荧光消失,表明重组GSAT可能增加内源尿卟啉原Ⅲ水平,从而增强重组UPMT催化产生的红色荧光。     

大肠杆菌染色体上严谨型启动子的构建

【背景】启动子的渗漏表达是代谢工程和合成生物学较为关注的问题,探索严谨型启动子使之能像开关一样控制基因的表达有助于解决这一问题。【目的】为避免在质粒上研究启动子带来的弊端,本研究将在染色体上对严谨型启动子进行构建和评价。【方法】基于4种调控元件四环素tetO、乳糖lacO、阿拉伯糖araC和鼠李糖rhaR的序列,以及2种来源的启动子PL和Plac序列,设计和组合构建了6个启动子PtetO2、PtetO3、PlacO2、PlacO3、PlacO+ara和PlacO+rha。应用CRISPR/Cas9系统将这6个启动子序列整合到大肠杆菌ATCC 8739染色体上,利用绿色荧光蛋白(Green fluorescent protein,GFP)的表达,分析这6个启动子的相对表达强度和严谨型控制情况。【结果】GFP表达分析显示,启动子PlacO+rha为最佳严谨型启动子,在无诱导剂时表达为0.02,有诱导剂时最大表达强度为lac Z基因启动子的12倍,相对控制范围为600倍。【结论】研究结果将为代谢工程和合成生物学中的精确调控基因表达奠定良好的应用基础。     

水稻黑条矮缩病毒第七号片段的cDNA克隆及其在大肠杆菌中的表达

运用RT-PCR技术,根据玉米粗缩病毒S6的末端序列设计引物,从玉米粗缩病感病材料中克隆得到一条2.2kb长的cDNA片段,序列分析表明,该片段全长2193bp,含两个开放阅读框,分别编码41.0kD和36.3kD的多肽。序列分析结果表明,该cDNA片段及其编码产物与水稻黑条矮缩病毒S7的cDNA序列及编码产物存在最高的相似性,推测该cDNA片段为水稻黑条矮缩病毒的S7片段,而不是玉米粗缩病毒的S6。分别将该片段的两个阅读框克隆到原核表达载体pET21d及pGEXKG中,经IPTG诱导后,两种蛋白均得到了高效的表达。表达产物回收后制备并得到了高效价的抗血清。     

Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein

In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.     

禽源性大肠杆菌O_1、O_2和O_(78)分离株外膜蛋白型的研究

对江苏省25个禽源性大肠杆菌O_1、O_2和O_(78)分离株的外膜蛋白型进行了测定。用改良的N-十二烷酰肌氨酸法提取其外膜蛋白,经SDS-PAGE电泳后,用考马斯亮蓝法进行染色。结果,8个O_1分离株,9个O_2分离株分属2个OMP型,其中的1个为二者所共有;而8个O_(78)分离株的OMP型也与该型相同。表明从江苏省分离到的禽源性大肠杆菌具有多样性的OMP型,而且这3个血清型的分离株中存在着共同的OMP型。     

Induction of {alpha}-Amylase is Repressed by Uniconazole, an Inhibitor of the Biosynthesis of Gibberellin, in a Dwarf Mutant of Rice, Waito-C

A system for induction of synthesis of     

Effect of Gibberellin Biosynthesis Inhibitors on Native Gibberellin Content, Growth and Floral Initiation in Sorghum bicolor

CCC, uniconazol, ancymidol, prohexadione-calcium (BX-112), and CGA 163′935, which represent three groups of gibberellin (GA) biosynthesis inhibitors, were applied as a soil drench to Sorghum bicolor cultivars 58M (phyB-1, phytochrome B-deficient mutant) and 90M (phyB-2, equivalent phenotypically to wild type, PHYB, except for small differences in flowering dates). The inhibitors that block steps before GA₁₂ (CCC, uniconazol, and ancymidol) lowered the concentrations of all endogenous early-C13α-hydroxylation pathway GAs found in sorghum: GA₁₂, GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, and GA₈. In contrast, the inhibitors that block the conversion of GA₂₀→ GA₁, (CGA 163′935 and BX-112) drastically reduced GA₁ and GA₈ levels, but they either did not change or caused accumulation of intermediates from GA₁₂ to GA₂₀. Combinations of pre-GA₁₂ inhibitors and GA₃ plus GA₁ strongly reduced GAs other than GA₁ and GA₃. Each of these compounds inhibited shoot growth in both cultivars and delayed floral initiation in 90M. Floral initiation of 58M was also delayed by CCC, uniconazol, and ancymidol but not by CGA 163`935 and BX-112. This separation of shoot elongation from floral initiation in sorghum is novel. Both inhibition of shoot growth and delayed floral initiation were almost completely relieved by a mixture of GA₃ and GA₁ in both 58M and 90M. This observation, plus the much lower levels of endogenous GA₃ than of GA₁ observed in these experiments, implies that GA₁ is the major endogenous GA active in shoot elongation. CGA 163′935 and BX-112 also failed to promote tillering in 58M, whereas inhibitors active before GA₁₂ did so. The possibility that the GA₂₀→ GA₁ inhibitors fail to block flowering and promote tillering in 58M because biosynthetic intermediates between GA₁₂ and GA₂₀ accumulate and/or because 58M is altered in GA metabolism in this same region of the biosynthetic pathway is discussed. Received April 7, 1998; accepted July 31, 1998     

抗菌肽GK1在大肠杆菌中的融合表达

为高效表达抗菌肽GK1并避免GK1的高抗菌活性对大肠杆菌宿主菌的致命影响, 将经改造后的人胰岛素原(mhPI)与GK1的融合基因(mhPI-GK1)克隆到表达载体pET28a中, 构建出表达质粒pET28a-mhPI-GK1, 转化至大肠杆菌BL21(DE3)中进行表达。融合蛋白在大肠杆菌中以包涵体形式表达, 表达量占菌体总蛋白的20%。经CNBr裂解、阳离子交换层析和RP-HPLC纯化后, 每升发酵液可获得5.7 mg纯度大于97%的重组GK1。质谱检测显示重组GK1的分子量为2794.0 D, 抑菌活性实验表明纯化后的重组GK1和化学合成GK1具有相同的抗菌活性。为利用基因工程方法大规模生产GK1奠定了基础。     

Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein

Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.     

Energy-dependent receptor activities of Escherichia coli K-12: mutated TonB proteins alter FhuA receptor activities to phages T5, T1, ⊘80 and to colicin M

Abstract The activity of the FhuA receptor in the outer membrane of Escherichia coli is dependent on the TonB, ExbB and ExbD proteins which are anchored to the cytoplasmic membrane. Only infection by phage T5 occurs independently of TonB, ExbB and ExbD. In this paper we describe mutated FhuA proteins which displayed either an increased or decreased FhuA activity to phage T5 when combined with mutated TonB proteins. These results suggest conformational changes in FhuA by TonB which are recognized by phage T5. Similar results were obtained with colicin M and the phages T1 and ⊘80. It is proposed that the FhuA mutant proteins assume conformations which are either improved or impaired by the TonB derivatives. For the direct interaction of FhuA with TonB regions which are located outside the TonB box of FhuA and the region around residue 160 of TonB are important.     

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

Abstract In an Escherichia coli strain, the levels of penicillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth ratw in chemostat cultures under different nutrient limitation conditions. A considerable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.     

Effect of different positively charged amino acids, C-terminally of the signal peptidase cleavage site, on the translocation kinetics of a precursor protein in Escherichia coli K-12

Abstract Introduction of positively charged amino acids immediately downstream of the signal sequence in prokaryotic precursor proteins is known to affect the export process. However, it is not clear whether different positively charged amino acids affect the export process similarly. To investigate this, the glutamate at position +2 of outer membrane protein PhoE was substituted by arginine, lysine of histidine. Pulse-chase experiments revealed that the Lys and Arg residues at position +2 caused a reduced processing rate, and that the effect was markedly more severe in the case of the Arg residue. Trypsin accessibility experiments revealed that the accumulated precursors were present in the cytoplasm. Since the degree of the inhibitory effect corresponded to the p K _{r a} of the different positively charged amino acids, this suggests that the positively charged residues must be deprotonated during the secretory process.     

Evidence for an internal entropy contribution to phosphoryl transfer: a study of domain closure, backbone flexibility, and the catalytic cycle of cAMP-dependent protein kinase.

While there is no question that ligands can induce large-scale domain movements that narrow (close) the active-site cleft of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK), the results from small-angle X-ray scattering, protein footprinting, and thermostability studies are inconsistent with regard to which ligands induce these movements. This inconsistency suggests a greater complexity of cAPK conformational dynamics than is generally recognized. As an initial step to study this issue in relation to the catalysis, a new method to measure cAPK domain closure was developed, and the state of domain closure and the local segmental flexibility at major steps of the cAPK catalytic cycle were examined with site-directed labeling and fluorescence spectroscopy. To achieve this, a C subunit mutant (F239C/C199A) was engineered that allowed for fluorescein 5-maleimide (donor) conjugation of F239C in the large lobe and tetramethylrhodamine (acceptor) conjugation of C343 in the small lobe. Domain closure was assessed as an increase in the efficiency of energy transfer between donor and acceptor. The anisotropy decay of fluoroscein 5-maleimide, conjugated to a site of cysteine substitution (K81C) in the small lobe of the C subunit was used to assess the local backbone flexibility around the B helix. The effects of substrate/pseudosubstrate (ATP and PKI(5-24)), a fragment of protein kinase inhibitor) and products (ADP and phosphorylated PKS) on domain closure and B helix flexibility were measured. The results show that domain closure is not tightly coupled to the flexibility around K81C. Moreover, although substrates/pseudosubstrate and products independently close the active-site cleft, only the substrates substantially decreased the backbone flexibility around the B helix. Because this order-to-disorder transition coincides with the phosphoryl transfer transition, the results suggest the existence of an internal entropy contribution to catalysis.     

The kinetics of RCC1 inclusion body formation in Escherichia Coli

The Regulator of Chromosome Condensation protein (RCC1) is located in both the soluble and inclusion body (IB) fractions of the whole cell lysate when expressed in Escherichia coli BL21 (pLysS) at temperatures below 30 degrees C. When bacterial growth was carried out at 20 degrees C, the majority of the RCC1 remained soluble up to 5.5 h postinduction, When the temperature was raised to 25 degrees C, RCC1 IB was dominant by 1.5 h postinduction. The shift in RCC1 IB formation with temperature suggests that in addition to increased translation rates, folding and aggregation processes may contribute to RCC1 IB formation at higher temperatures. (c) 1995 John Wiley & Sons, Inc.     

Baculoviral polyhedrin as a novel fusion partner for formation of inclusion body in Escherichia coli

Baculoviral polyhedrin, which originated from Autographa californica nuclear polyhedrosis virus (AcNPV), was employed for the first time as a novel fusion partner for expression of foreign proteins in an Escherichia coli system. We characterized the expression of recombinant polyhedrin protein fused to green fluorescent protein (GFP). The polyhedrin fusion protein ( approximately 58 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 30% of the total cellular protein. The E. coli expressing polyhedrin-GFP fusion protein showed higher cell growth ( approximately 1.8-fold) and higher GFP yield ( approximately 3.5-fold) than the strain expressing soluble single GFP. Interestingly, the polyhedrin fusion portion showed almost the same characteristics as the native baculoviral polyhedrin; it was rapidly solubilized under alkaline conditions, similar to the conditions found in the insect midgut. In addition, the polyhedrin fusion portion was rapidly digested by alkaline proteases in insect Plutella xylostella midgut as well as by alpha-chymotrypsin, a protease that has similar properties to insect midgut polyhedra-associated alkaline proteases. These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for production of foreign proteins, especially harmful proteins, in E. coli expression systems.     

Communication between eukaryotic translation initiation factors 1 and 1A on the yeast small ribosomal subunit

We have used expressed protein ligation to site-specifically label eukaryotic translation initiation factors (eIFs) 1 and 1A at their C termini with tetramethyl rhodamine. These fluorescent proteins were used in steady-state anisotropy-based binding experiments to measure the dissociation constants of the factors and the yeast small (40S) ribosomal subunit for the first time. These studies demonstrate that both eIF1 and eIF1A are capable of binding to the 40S subunit in the absence of any other initiation factors or mRNA, arguing against previous suggestions that eIF3 is required for recruitment of eIF1 to the small ribosomal subunit. Strikingly, the data also demonstrate that there is approximately ninefold thermodynamic coupling in the binding of the two factors to the 40S subunit. This indicates that eIF1 and eIF1A communicate with one another when bound to the 40S subunit. Communication between these two factors is likely to be important for coordinating their functions during the initiation process. The data presented here provide a foundation on which to build a quantitative understanding of the network of interactions between these essential factors and the rest of the initiation machinery.     

Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein

A green fluorescent protein (gfp) gene was ligated to the Lactobacillus reuteri-specific nisin-inducible expression-secretion vector pNIES, generating a pNIES-GFP vector capable of secreting the cloned gene as a GFP-fusion protein with fluorescent activity. To develop this system as a live vehicle carrying the heat-stable enterotoxin (ST) and heat-labile enterotoxin B (LT(B)) of the enterotoxigenic Escherichia coli (ETEC), a recombinant 5'-ST-LT(B)-3' DNA fragment was cloned into pNIES-GFP. The resulting L. reuteri/pNIES-GFP:STLT(B) system was found to possess the capability of adhering to the mice gut, secreting GFP:STLT(B) product at 0.14 and 0.026 pgcell(-1) under induced and noninduced conditions, respectively. Further analysis of the GFP:STLT(B) product confirmed its ganglioside-binding ability, LT(B) antigenicity and relative freedom from the ST-associated toxicity, making it suitable for use as an oral vaccine in mice. Oral inoculation of the L. reuteri/pNIES-GFP:STLT(B) culture in mice elicited significant (P<0.01) serum IgG and mucosal IgA antibodies against the STLT(B) antigen. These immunized mice were subsequently challenged with ETEC and showed full protection against the fluid influx response in the gut. This is the first report of using L. reuteri as a vaccine carrier to induce complete immunologic protection against ETEC.