Purification of the MglC/E membrane proteins of the binding protein-dependent galactose transport system of Salmonella typhimurium.

The high affinity galactose transport system of Salmonella typhimurium consists of four proteins, a periplasmic galactose binding protein (the MglB protein), and three inner membrane-associated proteins, the MglA, MglC and MglE proteins. We purified the MglC/E proteins from an MglC/E hyperproducing strain after solubilisation of inclusion bodies in guanidine hydrochloride followed by renaturation in a detergent-containing buffer and affinity chromatography on a MglB-Sepharose column. The MglC/E proteins are devoid of ATPase activity and they complement an extract from a strain carrying a plasmid with the mglA gene for reconstitution of the MglB-dependent galactose transport in proteoliposomes.     

Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting.

The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retroviral RNA to its three DNA forms. Fine-structure analysis of the linear double-stranded viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the 3' recessed termini that normally precedes integration.     

大肠杆菌异源重组蛋白质的变性与复性

描述了大肠杆菌异源重组蛋白质的形成、制备、变性和复性,综述了国内外变性、复性的有效方法。     

Bacteriophage T4 gene 32 protein shares antigen determinants with reconstituted HeLa hnRNP proteins in western blots.

A polyclonal antiserum against purified bacteriophage T4 gene 32 protein was raised in rabbits. In Western blots it detected a number of SDS-PAGE separated nuclear and ribosomal proteins of HeLa cells. Using a renaturing blotting system, however, larger hnRNP proteins ranging between 66.000 and 82.000 Da preferentially reacted with this antiserum. In addition hnRNP group A core proteins were detected to a minor extent. Nucleic acid binding proteins like histones or ribosomal proteins were not stained by this antibody after renaturation.     

Renaturation of DNA: a novel reaction of histones.

Histones isolated from several sources, either singly or in combination promote the renaturation of complementary single strands of DNA, as measured by the acquisition of resistance to S1 nuclease. The reaction is rapid (T1/2 less than 1 min), and is stoichiometric rather than catalytic. Renaturation is stimulated by Mg2+, Mn2+, and Ca2+, but is strongly inhibited by Zn2+. Crude extracts of early embryos of Drosophila melanogaster possess renaturation activity which is protease sensitive, heat-stable, and acid-soluble, suggesting that most or all of it can be attributed to histones. This observation thus provides a functional assay for histones that should prove useful in studies of chromatin and histone-DNA interactions, as well as for the identification and isolation of histones and histone-like proteins in crude extracts.     

Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the beta subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.     

包涵体复性研究进展(英文)

用基因工程技术在大肠杆菌高水平表达重组蛋白时,通常形成无生物活性的包涵体。包涵体在体外经分离、溶解与重折叠后可实现复性,表现为具有生物活性的蛋白。总结了包涵体的相关复性技术,重点介绍重折叠的最新进展情况 。     

Identification of sequence elements in the human cytomegalovirus DNA polymerase gene promoter required for activation by viral gene products.

To determine the mechanisms involved in the regulation of human cytomegalovirus early gene expression, we have examined the gene that encodes the viral DNA polymerase (UL54, pol). Our previous studies demonstrated that sequences required for activation of the pol promoter by immediate-early proteins are contained within a region from -128 to +20 and that cellular proteins can bind to this activation domain. In this study, we demonstrate by competition analysis that binding of cellular proteins to pol is associated with an 18-bp region containing a single copy of a novel inverted repeat, IR1. Time course analysis indicated that viral infection increased the level of protein binding to IR1, concurrent with the activation of the pol promoter. Mutation of the IR1 element abrogated binding of cellular factors to the pol promoter and reduced by threefold the activation by immediate-early proteins. Similarly, mutation of IR1 rendered the promoter poorly responsive to activation by viral infection. Mutation of additional sequence elements in the pol promoter had little effect, indicating that IR1 plays the major role in pol promoter regulation. These studies demonstrate that the interaction between cellular factors and IR1 is important for the regulation of expression of the polymerase gene by viral proteins.     

应用RNA干扰沉默猪内源性反转录病毒

目的:尝试应用RNA干扰(RNAi)沉默猪源PK-15细胞中的猪内源性反转录病毒(PERV),并通过反转录酶活性及pol基因相对荧光定量PCR检测沉默效果。方法:依据GenBank公布的PERV pol基因序列,采用Invitro-gen公司的BLOCK-iT RNAi Designer软件设计Stealth小干扰RNA(siRNA)序列;将合成的siRNA转染PK-15细胞,72 h后检测细胞上清PERV反转录酶活性及细胞内pol基因拷贝数并评价沉默效果。结果:反转录酶活性及pol基因拷贝数检测结果表明,设计的3条Stealth siRNA序列中,位于pol基因3272～3296 bp的序列能有效沉默PERV。结论:RNAi方法可有效使猪源PK-15细胞中的PERV沉默,为进一步研究天然抗病毒分子与PERV的相互作用提供了实验基础,同时也为猪源异种移植研究中去除PERV提供了一种可供尝试的方法。     

Oxidative renaturation of lysozyme at high concentrations

Newly synthesized cloned gene proteins expressed in bacteria frequently accumulate in insoluble aggregates or inclusion bodies. Active protein can be recovered by solubilization of inclusion bodies followed by renaturation of the solubilized (unfolded) protein. The recovery of active protein is highly dependent on the renaturation conditions chosen. The renaturation process is generally conducted at low protein concentrations (0.01-0.2 mg/mL) to avoid aggregation. We have investigated the potential of successfully refolding reduced and denatured hen egg white lysozyme at high concentrations (1 and 5 mg/mL). By varying the composition of the renaturation media, optimum conditions which kinetically favor proper folding over inactivation were found. Solubilizing agents such as guanidinium chloride (GdmCl) and folding aids such as L-arginine present in low concentrations during refolding effectively enhanced renaturation yields by suppressing aggregation resulting in reactivation yields as high as 95%. Quantitatively the kinetic competition between lysozyme folding and aggregation can be described using first-order kinetics for the renaturation reaction and third-order kinetics for the overall aggregation pathway. The rate constants for both reactions have been found to be strongly dependent on denaturant and thiol concentration. This strategy supercedes the necessity to reactivate proteins at low concentrations using large renaturation volumes. The marked increase in volumetric productivity makes this a viable option for recovering biologically active protein efficiently and in high yield in vitro from proteins produced as inclusion bodies within microbial cells. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 221-230, 1997.     

Renaturation of recombinant proteins produced as inclusion bodies

Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble inclusion bodies. Within the last few years specific methods and strategies have been developed to prepare active proteins from these inclusion bodies. These methods include (i) isolation of inclusion bodies after disintegration of cells by mechanical forces and purification by washing with detergent solutions or low concentrations of denaturant, (ii) solubilization of inclusion bodies with high concentrations of urea or guanidine-hydrochloride in combination with reducing reagents, and (iii) renaturation of the proteins including formation of native disulphide bonds. Renatured and native disulphide bond formation are accomplished by (a) either air oxidation, (b) glutathione reoxidation starting from reduced material, or (c) disulphide interchange starting from mixed disulphides containing peptides. The final yield of renatured proteins can be increased by adding low concentrations of denaturant during renaturation.     

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.