Insertional re-activation of a chloramphenicol acetyltransferase misfolding mutant protein

The deletion of nine residues from the C-terminus of the bacterialchloramphenicol acetyltransferase (CAT) results in depositionof the mutant protein in cytoplasmic inclusion bodies and lossof chloramphenicol resistance in Escherichia coli. This foldingdefect is relieved by C-terminal fusion of the polypeptide withas few as two residues. Based on these observations, efficientpositive selection for the cloning of DNA fragments has beendemonstrated. The cloning vector encodes a C-terminally truncatedCAT protein. Restriction sites in front of the stop codon allowthe insertion of target DNA, resulting in the production ofproperly folded CAT fusion proteins and regained chloramphenicolresistance. The positive selection of recombinants is accomplishedby growth of transformants on chloramphenicol-containing agarplates. The method appears particularly convenient for the cloningof DNA fragments amplified by the PCR because minimal informationto restore CAT folding can be included in the primers. The cloningof random sequences shows that the folding defect can be relievedby fusion to a wide variety of peptides, providing great flexibilityto the positive selection system. This vector may also contributeto the determination of the role of the C-terminus in CAT folding.     

历史中心区复兴中的地域性问题与对策

本文以上海思南路47～48街坊改造为例,探讨城市形态更新与居民外迁问题,并期待相关地域性社会对策的出现。     

RNA结合蛋白RB47、RB60在叶绿体翻译调节中的作用

光调节的叶绿体mRNA的翻译需要mRNA5′,非翻译区(5′ UTR)相互作用的中间作用因子.叶绿体多聚腺苷酸结合蛋白(cPABP)RB47特异性地与PsbAmRNA的5′ UTR结合,并对于该mRNA的翻译是很重要的.定位于叶绿体中与cPABP伴随纯化出来的一种蛋白质二硫化物异钩酶RB60表现出调节cPABP与PsbAmRNA的5′ UTR结合的特性.这种调节是通过可逆地改变有氧化还原特征的cPABP的氧化还原状态来实现,或通过ADP依赖的磷酸化来实现.这种机制就在调节叶绿体的基因表达中提供了一个简单可逆的开关机制.     

The effects of induction conditions on production of a soluble antitumor sFv in Escherichia coli

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 (0.02–0.03 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.     

Structure-activity studies of human tumour necrosis factors

The mechanism by which tumour necrosis factors (TNF and lymphotoxin,also called TNFß and TNFß respectively) exerttheir cytotoxic activity on many malignant cells, remains largelyunknown. Furthermore, the broad array of differentiation (geneinduction) and mitogenic activities towards many primary cellsis still a subject of intensive investigation. TNF is an importantmediator in inflammation, immune responses and infection-relatedphenomena and these activities contribute to the severe toxidtyseen when TNF is used as an anticancer agent. The first stepin the mechanism of action is the specific binding of the ligandto its receptors and dissection of the molecular mechanism involvedin this interaction is the subject of this review. The reasonsfor the interest in this aspect are obvious: first, the developmentof strong antagonistic TNF analogues can be useful in dampeningthe potentially lethal or debilitating effects of an overproductionof the cytokine (as in septic shock or rheumatoid arthritis).Secondly, since two distinct TNF receptors exist, constructionof TNF muteins that distinguish between both types may leadto derivatives of this plekrtropic agent with a more restrictedbiological activity pattern. Ideally, one would like to developa TNF mutant that has retained its cytotoxic action on tumourcells without inducing the deleterious systemic toxteity. Suchan optimized TNF molecule could become a potent anticancer agent     

A new family of sugar-inducible expression vectors for Escherichia coli

A set of 11 expression vectors was constructed, each of themharbouring a cloning cassette under the control of the promoter.Some of these vectors enable expression of foreign proteinsin the cytoplasm, while others include a synthetic sequencecoding for a very efficient secretion signal sequence. Otherfeatures are an fl origin of replication (in plus or minus orientation)and a promoter^up mutation that enhances the already very highlevel of expression from these vectors. With such a versatilevector family, cloning, sequencing and sitedirected mutagenesiscan be performed on the same vector, and the level of expressioncan be defined according to the specific constraints of a givenprotein.     

Homology modelling of annexin I: implicit solvation improves side-chain prediction and combination of evaluation criteria allows recognition of different types of conformational error

Annexin I homology models were built from the annexin V crystalstructure. Three methods for side-chain prediction were testedbased on molecular mechanics conformational search, the useof a rotamer database, or a combination of these two methods.We showed that rotamer-based methods were more efficient andthat molecular mechanics energy minimizations, prior to rotamerselection, did not afford clearly improved predictions. Modelsbuilt in vacuo and with an implicit solvation term were comparedwith the annexin I crystal structure which became availableduring the course of this study. The analysis of solvation energies,root mean square deviations, Xi angles and hydrogen bonds showedthat models built with implicit solvation were of better quality.In annexin V, repeat III displays A-B and D-E loop conformationsquite different from other repeats. Since the sequence differencessuggest that repeat III in annexin I might present a conformationsimilar to other repeats, two annexin I models with differentrepeat III conformations were built and compared to determinewhether the correct conformation could have been predicted.We show that using a combination of evaluation criteria, itis possible to discriminate unequivocally between the nativeand the incorrect fold, stressing that only one criterion shouldnot be used to evaluate protein structures.     

Predictive modelling of the 3-D structure of interleukin-13

Several atomic structures are now available for the family ofhelical cytokines, which includes growth hormone as well asmany of the interleukins. Using structural information fromfive members of this family, two alternative models of interleukin(IL)-13 are proposed. IL-13 has biological properties similarto those of IL-4 and, like the other interleukins, is a potentiallyimportant pharmaceutical target. The model of IL-13 is discussedand compared with the known interleukin structures.     

6-Phospho-{beta}-galactosidases of Gram-positive and 6-phospho-{beta}-glucosidase B of Gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of staphylococcus aureus

The 6-phospho-ß-galactosidase of Staphylococcus aureus,Lactococcus lactis and Lactobacillus casei and 6-phospho-ßglucosidaseB of Escherichia coli build a subfamily inside a greater enzymefamily, named the glycosal hydrolase family 1, which, hi addition,contains nine ß-glycosidases of different origins.Kinetic and immunological evidence is provided in this reportwhich strengthens the relationship of the four 6-phospho-ß-glycosidases.It is shown that the 6-phospho-ß-galactosidases and6-phospho-ß-glucosidase B are able to split aromaticß-galactoside phosphates and ß-glucosidephosphates. The turnover numbers of hydrolysis of substrateswith different epimerization at C-4 of the glycon vary up to15-fold only. Two polydonal antisera, one derived against thenative 6-phospho-ß-galactosidase from S.aureus andthe other derived against the 6-phospho-ß-glucosidaseB, cross-reacted with both enzymes. Peptides of the proteinswere separated by reverse phase HPLC. The cross-reacting peptideswere sequenced and shown to be localized at almost the sameposition in the aligned primary structures of both enzymes.An insertion of nine amino adds near these antigenic domainsis unique for the 6-phospho-ß-glycosidases and missingwithin the sequences of the ß-glycoside-specific membersof the family. The lacG gene of a 6-phospho-ß-galactosidasenegative S.aureus mutant was doned into E.coli and sequenced.In the totally inactive mutant protein only the glycine at position332 was changed to an arginine. This amino acid is part of thesequence insertion near the antigenic domain reacting with bothantisera. These data support the assumption that the regionis of great importance for the function of the enzymes and thatit is possible it determines the specificity of the phosphorylatedform of the substrates. In addition, the 6-phospho-ß-galactosidaseof S.aureus was modified by sitedirected mutagenesis of thecorresponding lacG gene hi order to replace residues Glul60and Glu375, which were suspected of being involved hi the generalacid catalysis of substrate hydrolysis, with glutamine residues.The mutant protein 160EQ retained some catalytic activity whilethe protein 375EQ was totally inactive. Glu375 is the activesite nudeophile of the 6-phospho-ß-galactosidase ofS.aureus. It is located in the sequence motif ENG where Glu358was identified as the catalytkally active nudeophile hi theß-glucosidase of Agrobacterium.     

Identifying a putative common binding site shared by substance P receptor and an anti-substance P monoclonal antibody

Substance P G-protein coupled receptor and the antigen recognitionsite of a monoclonal antibody raised against substance P sharea stretch of five contiguous identical amino acids. This observationprompted us to build an atomic model of both the receptor andthe antibody and to analyse their common features. In particular,we report here that a pocket of similar size and compositionis present in both proteins, strongly suggesting a similarityin the mode of binding of both macromolecules to substance P.From the analysis of our models, the available data on the modeof binding of the antibody to substance P and recent data onsubstance P receptor mutants, we concluded that the pocket isvery likely to be involved in binding of the C-terminal 'messagesequence' of the tachykinin. This allowed us to suggest specificsite-directed mutants of the receptor which should shed somelight on the mechanism of peptide recognition by G-protein coupledreceptors.