Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture

The arabinose-inducible promoter P(BAD) is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible P(tac) and P(taclac) promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P(BAD)) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}     

Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library

A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.     

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.     

Characterization of the depletion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis

The ispF gene product in Escherichia coli has been shown to catalyze the formation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) in the deoxyxylulose (DOXP) pathway for isoprenoid biosynthesis. In this work, the E. coli gene ispF and its Bacillus subtilis orthologue, yacN, were deleted and conditionally complemented by expression of these genes from distant loci in the respective organisms. In E. coli, complementation was achieved through integration of ispF at the araBAD locus with control from the arabinose-inducible araBAD promoter, while in B. subtilis, yacN was placed at amyE under control of the xylose-inducible xylA promoter. In both cases, growth was severely retarded in the absence of inducer, consistent with these genes being essential for survival. E. coli cells depleted of MEC synthase revealed a filamentous phenotype. This was in contrast to the depletion of MEC synthase in B. subtilis, which resulted in a loss of rod shape, irregular septation, multicompartmentalized cells, and thickened cell walls. To probe the nature of the predominant deficiency of MEC synthase-depleted cells, we investigated the sensitivity of these conditionally complemented mutants, grown with various concentrations of inducer, to a wide variety antibiotics. Synthetic lethal behavior in MEC synthase-depleted cells was prevalent for cell wall-active antibiotics.     

天然兔源噬菌体单链抗体库的构建与鉴定

目的:构建天然兔源噬菌体单链抗体库。方法:采用RT-PCR法从未免疫的兔子脾脏中克隆得到抗体重链可变区(VH)与轻链可变区(VL)基因,重叠PCR将VH和VL拼接成scFv片段,将scFv连接到噬菌粒pComb3XSS上,电转入XL1-Blue菌中,得到单链抗体库,并用此抗体库筛选抗肌酸激酶抗体。结果:构建了容量为4×108,基因重组率95%的单链抗体库,DNA指纹图谱显示抗体库多样性良好。以肌酸激酶为抗原,从该库中筛到3株抗肌酸激酶的抗体。结论:分析表明构建的天然兔源单链抗体库质量良好,可用于快速筛选、制备多种单链抗体。     

一种核糖体展示单链抗体文库的构建与鉴定

用柑桔溃疡病致病菌Xanthomonas axonopodis pv. citri（Xac）全菌免疫小鼠,提取小鼠脾细胞mRNA,RT-PCR扩增小鼠抗体重链可变区（VH）和轻链可变区（VL）,采用linker (Gly4Ser)3连接VH和VL,构建用于核糖体展示方法筛选阳性单链抗体（scFvs）的文库。通过将scFv文库DNA转化大肠杆菌JM109,随机挑取克隆子测序以分析单链抗体文库的多样性。结果显示,用柑桔溃疡病菌免疫后的小鼠抗血清效价为2500倍左右,扩增的VH大小为350bp左右,VL的大小为650bp左右,linker连接后的单链抗体文库DNA大小为1200bp左右。测序结果显示,单链抗体文库多样性好。以Xac为靶,筛选到了抗Xac的特异性抗体,说明该抗体库可用于柑桔溃疡病菌单链抗体的筛选。     

Independent regulation of two genes in Escherichia coli by tetracyclines and Tet repressor variants

We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities. One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline. The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline. We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.     

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.     

Genetic and physical analyses of Caulobacter crescentus trp genes.

Caulobacter crescentus trp mutants were identified from a collection of auxotrophs. Precursor feeding experiments, accumulation studies, and complementation experiments resulted in the identification of six genes corresponding to trpA, trpB, trpC, trpD, trpE, and trpF. Genetic mapping experiments demonstrated that the trp genes were in two clusters, trpCDE and trpFBA, and a 5.4-kilobase restriction fragment from the C. crescentus chromosome was isolated that contained the trpFBA gene cluster. Complementation experiments with clones containing the 5.4-kilobase fragment indicated that trpF was expressed in Escherichia coli and that all three genes were expressed in Pseudomonas putida. This expression was lost in both organisms when the pBR322 tet gene promoter was inactivated, indicating that all three genes were transcribed in the same orientation from the tet promoter. Thus, the C. crescentus promoters do not seem to be expressed in E. coli or P. putida. Complementation of the C. crescentus trp mutants indicated that the tet promoter was not necessary for expression in C. crescentus and suggested that at least two native promoters were present for expression of the trpF, trpB, and trpA genes. Taken together, these results indicate that C. crescentus promoters may have structures that are significantly different from the promoters of other gram-negative species.     

Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity

Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Antibody clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a β-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5′-end of the β-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of 7.6 × 10⁹, greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.     

抗TNF-α单链抗体噬菌体展示文库的建立和鉴定

应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.     

Multicopy Tn10 tet plasmids confer sensitivity to induction of tet gene expression.

We inserted the Tn10 tetracycline resistance determinant (tet) into the multicopy plasmid pACYC177, and we examined the phenotype of Escherichia coli K-12 strains harboring these plasmids. In agreement with others, we find that Tn10 tet exhibits a negative gene dosage effect. Strains carrying multicopy Tn10 tet plasmids are 4- to 12-fold less resistant to tetracycline than are strains with a single copy of Tn10 in the bacterial chromosome. In addition, we find that multicopy tet strains are 30- to 100-fold less resistant to the tetracycline derivative 5a,6-anhydrotetracycline than are single-copy tet strains. Multicopy tet strains are, in fact, 10- to 25-fold more sensitive to anhydrotetracycline than are strains that lack tet altogether. The hypersensitivity of multi-copy strains to anhydrotetracycline is correlated with the effectiveness of anhydrotetracycline as an inducer of tet gene expression, rather than its effectiveness as an inhibitor of protein synthesis. Anhydrotetracycline is 50- to 100-fold more effective than tetracycline as an inducer of tetracycline resistance and as an inducer of beta-galactosidase in strains that harbor tet-lac gene fusions. In contrast, anhydrotetracycline appears to be two- to fourfold less effective than tetracycline as an inhibitor of protein synthesis. Both anhydrotetracycline and tetracycline induce synthesis of tet polypeptides in minicells harboring multicopy tet plasmids. Differences between E. coli K-12 backgrounds influence the tetracycline and anhydrotetracycline sensitivity of multicopy strains; ZnCl2 enhances the tetracycline and anhydrotetracycline sensitivity of these strains two- to threefold. We propose that the overexpression of one or more Tn10 tet gene products inhibits the growth of multicopy tet strains and accounts for their relative sensitivity to inducers of tet gene expression.     

Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.