Cytochrome b562 variants: a library for examining redox potential evolution

A general understanding of how cytochromes evolve within a fixed structure to optimize redox potential for specific bioenergetic processes does not exist. Toward this end, a library approach is used to investigate the range and distribution of redox potential which occurs when all sequence space available through mutation at two positions is examined within a fixed structural motif. Random mutation of Phe61 and Phe65 of cytochrome b562 (E. coli), and subsequent examination of a statistically significant sampling of this library, demonstrates that the redox potential can vary over 100 mV (>25% of the known accessible potential in native proteins with axial His-Met ligation) through mutation at these two positions. The redox potential of the wild-type protein occurs at an extremum of the distribution observed, indicating that Phe61 and Phe65 were most likely naturally selected to differentially stabilize the reduced state of the protein. At the other extremum, a compositionally conservative set of mutations (F61I, F65Y) leads to a 100 mV shift in the redox equilibrium toward the oxidized state. NMR analyses indicate that a charge-dipole interaction which results from mutation of phenylalanine to tyrosine at position 65 may be responsible.     

To the Final Goal: Can We Predict and Suggest Mutations for Protein to Develop Desired Phenotype?

Directed evolution of proteins is a good approach to develop desired phenotypes from existing proteins. Fully experimental protein evolution usually utilizes randomization of a given protein sequence by error-prone PCR or gene shuffling followed by high-throughput selection or timeconsuming screening method. However, these random methods create mutant library full of deleterious mutations. In addition, they need high-throughput screening or selection method to search for positive clones from an enormous size of mutant library. Construction of a mutant library while retaining the original function is important for efficient protein evolution because it greatly reduces time and effort for the identification of positive mutants. Therefore, researchers have tried to reduce the size of mutant library by minimizing the occurrence of deleterious mutants. Such efforts have led to the creation of a concept of ‘small but smart library’. For this goal, neutral drift theory has been applied. Although smart library greatly reduces the library size, it is still the beyond the capacity of low-throughput assay. In parallel, computational analysis of protein structure and efforts to discriminate mutatable residues from all residues of a given protein have been consistently pursued. Accumulated knowledge of protein evolution through random mutation and selection has improved our understanding of functions of amino acids in protein structure. Protein evolution by rational design is being developed based on such understanding. In this review, we describe how the use of semi-rationally designed library rather than completely random one has impacted the overall procedure of directed evolution. We also describe efforts made to evaluate the effect of single mutation. Such efforts will bring lazy boys to the final goal - computational mutation suggestion system.     

Identification of a single base change in a new human mutant glucose-6-phosphate dehydrogenase gene by polymerase-chain-reaction amplification of the entire coding region from genomic DNA.

We report the characterization at the molecular level of a mutant glucose-6-phosphate dehydrogenase (G6PD) gene in a Greek boy who presented with a chronic non-spherocytic haemolytic anaemia. In order to identify the mutation from a small amount of patient material, we adopted an approach which by-passes the need to construct a library by using the polymerase chain reaction. The entire coding region was amplified in eight sections, with genomic DNA as template. The DNA fragments were then cloned in an M13 vector and sequenced. The only difference from the sequence of normal G6PD was a T----G substitution at nucleotide position 648 in exon 7, which predicts a substitution of leucine for phenylalanine at amino acid position 216. This mutation creates a new recognition site for the restriction nuclease BalI. We confirmed the presence of the mutation in the DNA of the patient's mother, who was found to be heterozygous for the new BalI site. This is the first transversion among the point mutations thus far reported in the human G6PD gene.     

Construction of a California condor BAC library and first-generation chicken-condor comparative physical map as an endangered species conservation genomics resource

To support genomic analysis of the endangered California condor (Gymnogyps californianus), a BAC library (CHORI-262) was generated using DNA from the blood of a female. The library consists of 89,665 recombinant BAC clones providing approximately 14-fold coverage of the presumed approximately 1.48-Gb genome. Taking advantage of recent progress in chicken genomics, we developed a first-generation comparative chicken-condor physical map using an overgo hybridization approach. The overgos were derived from chicken (164 probes) and New World vulture (8 probes) sequences. Screening a 2.8x subset of the total library resulted in 236 BAC-gene assignments with 2.5 positive BAC clones per successful probe. A preliminary comparative chicken-condor BAC-based map included 93 genes. Comparison of selected condor BAC sequences with orthologous chicken sequences suggested a high degree of conserved synteny between the two avian genomes. This work will aid in identification and characterization of candidate loci for the chondrodystrophy mutation to advance genetic management of this disease.     

Study on the relationship between structure and enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1

To enhance the enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1 (APE1547), a directed evolution approach is employed to generate mutant library from the native enzyme. A mutation (TBC26) is identified after one round of epPCR. The enantioselectivity of TBC26 is increased up to 2.6-fold compared to that of wild type enzyme. TBC26 contains five amino acid substitutions (R11G, L36P, V223A, I551L, A564T). The five mutation sites are spatially distant to the catalytic center. According to the published crystal structure of WT and considering the changes of secondary and tertiary structure, here we try to explain the change of enantioselectivity of the TBC26. The results suggest that the change of enantioselectivity of enzyme has a close relationship to the configuration of the enzyme.     

TRAB: testing whether mutation frequencies are above an unknown background

To rigorously determine whether a gene or a set of genes have alterations that are involved in carcinogenesis requires a comparison of the prevalence of identified changes to a control mutation frequency present in tumor DNA. To facilitate this task, we develop a testing approach and the associated R library, called TRAB, that evaluates whether the frequency of somatic mutation in a given gene is higher than that observed in a control group of genes. Specifically, we test the null hypothesis that the frequency belongs to a control population of frequencies, against the alternative hypothesis that the frequency is higher. Mutation frequencies in the control group are themselves allowed to be variable. TRAB computes the a posteriori probability and the Bayes factor for the hypothesis using a hierarchical Bayesian approach.     

Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9^th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.     

Method to protect a targeted amino acid residue during random mutagenesis

To generate a random mutant library that is free from mutation at a particular amino acid residue, we replace the codon of interest with a detachable, short DNA sequence containing a BsaXI recognition site. After PCR mutagenesis, this sequence is removed and intramolecular ligation of the sequences flanking the insert regenerates the gene. The three-base cohesive ends for ligation correspond to the codon for the targeted residue and any sequences with mutations at this site will fail to ligate. As a result, only the variants that are free from mutation at this site are in the proper reading frame. In a random library of C₃₀ carotenoid synthase CrtM, this method was used to exclude readily accessible mutations at position F26, which confer C₄₀ synthase function. This enabled us to identify two additional mutations, W38C and E180G, which confer the same phenotype but are present in the random library at much lower frequencies.     

噬菌体展示HIV-1 Tat38-61碱性区51和55位随机突变体文库的构建

为了构建噬菌体展示Tat38-61(51N/55N) 碱性区突变体文库,进一步研究HIV-1 Tat38-61表位的分子进化筛选,采用含随机核苷酸序列的引物,通过Overlap PCR的方法获得51和55位氨基酸随机突变的全长Tat编码序列,再以此为模板PCR扩增出两端含有Xba I识别序列的Tat38-61突变体片段HIV-1 Tat38-61(51N/55N),克隆至噬菌体展示载体pCANTAB5S上,转化大肠杆菌TG1,经M13K07辅助噬菌体拯救,构建噬菌体展示Tat38-61(51N/55N) 碱性区突变体文库。结果显示文库的库容量为5.0×106,滴度为2.65×1012 TU/mL,阳性克隆率为56.50％;序列分析显示文库中51、55位核苷酸与氨基酸均呈随机性分布,达到了对文库进行分子进化筛选的要求,为获得可用作疫苗候选物的新型Tat突变体奠定基础。     

Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening

Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed Fc_CH3A) with mutations in one CH3 domain (CH3A) on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted Fc_CH3B) with mutations in the other CH3 domain (CH3B). In the mated cells, secreted Fc_CH3B is displayed on the cell surface through heterodimerization with the displayed Fc_CH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80–90%) with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.     

A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries

A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 10⁹ sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an alpha-synuclein ep-PCR library and NNS synthetic oligonucleotide libraries are given as examples.     

Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map

Identification of all human protein–protein interactions will lead to a global human protein linkage map that will provide important information for functional genomics studies. The yeast two-hybrid system is a powerful molecular genetic approach for studying protein–protein interactions. To apply this technology to generate a human protein linkage map, the first step is to construct two-hybrid cDNA libraries that cover the entire human genome. With a homologous recombination-mediated approach, we have constructed a modular human EST-derived yeast two-hybrid library in the Gal4 activation domain-based vector, pACT2. Quality analysis of this library indicated that the approach of constructing two-hybrid cDNA libraries from individually arrayed human EST clones is feasible, and such a two-hybrid library is suitable for detecting protein–protein interactions. This is also the first time that a comprehensive two-hybrid system cDNA library has been constructed from a collection of individually arrayed EST clones.     

Flagellar basal body flg operon as a virulence determinant of Vibrio vulnificus

Vibrio vulnificus, a halophilic estuarine bacterium causing a rapidly progressing fatal septicemia, is highly cytotoxic to eukaryotic cells. To identify new virulence factors associated with cytotoxicity, we constructed a mariner-based transposon (Tn Himar1) library of the highly virulent clinical isolate MO6-24/O having a double mutation in the hemolysin and protease genes. The Himar1 mutant library was extensively screened for the mutants showing decreased cytotoxicity to HeLa cells. We selected a cytotoxicity defective mutant having a Himar1 insertion in an open reading frame showing 96% identity to Vibrio parahaemolyticus FlgC, a flagella basal body rod protein. The Tn Himar1 insertion mutation also resulted in a significant decrease in motility, adhesion, cytotoxicity, and lethality to mice. This is the first report showing that flg genes, which are components of the flagellum biogenesis gene cluster, might play an important role in the virulence of V. vulnificus.     

重组人淋巴毒素随机点突变组合文库的构建

构建重组人淋巴毒素（rhLT）随机点突变组合文库以进行体外分子进化及结构和功能的研究。应用含随机核苷酸序列的引物,通过Overlap PCR的方法分别对rhLT 的46、106和130位氨基酸进行定点随机突变,获得各单点随机突变体库。通过基因操作将这三个单点随机突变体库拼接并克隆于Pmd_18T载体建立三点组合突变体文库,DNA测序鉴定突变位点的随机性和多样性,原核表达该变异体库,体外测定生物学活性。成功获得rhLT三点随机点突变组合文库,其转化克隆数达到1.5×10⁵,是多样性理论值的4.5倍。50个样品的序列分析显示各个位点的核苷酸和氨基酸序列的突变都呈随机性分布。对原核表达的30个样品进行生物学活性测定,结果70%(21个)的样品无活性、23.3%(7个)的样品活性低于rhLT、6.7%(2个)的样品活性高于rhLT。成功构建了rhLT随机点突变组合文库,该库不仅在一级结构上具有良好的随机性和多样性,而且具有生物学活性的多样性,为应用噬菌体展示等高通量筛选策略对淋巴毒素进行体外分子进化和结构与功能的深入研究打下了基础。     

Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.     

A DNA barcode library for Korean Chironomidae (Insecta: Diptera) and indexes for defining barcode gap

Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified larvae. The interspecies and intraspecies COI sequence variations were analyzed. Sophisticated indexes were developed in order to properly evaluate indistinct barcode gaps that are created by insufficient sampling on both the interspecies and intraspecies levels and by variable mutation rates across taxa. In a variety of insect datasets, these indexes were useful for re-evaluating large barcode datasets and for defining COI barcode gaps. The COI-based DNA barcode library will provide a rapid and reliable tool for the molecular identification of Korean chironomid species. Furthermore, this reverse-taxonomic approach will be improved by the continuous addition of other speceis’ sequences to the library.     

Construction and characterization of a normalized yeast two-hybrid library derived from a human protein-coding clone collection

The nuclear yeast two-hybrid (Y2H) system is the most widely used technology for detecting interactions between proteins. A common approach is to screen specific test proteins (baits) against large compilations of randomly cloned proteins (prey libraries). For eukaryotic organisms, libraries have traditionally been generated using messenger RNA (mRNA) extracted from various tissues and cells. Here we present a library construction strategy made possible by ongoing public efforts to establish collections of full-length protein encoding clones. Our approach generates libraries that are essentially normalized and contain both randomly fragmented as well as full-length inserts. We refer to this type of protein-coding clone-derived library as random and full-length (RAFL) Y2H library. The library described here is based on clones from the Mammalian Gene Collection, but our strategy is compatible with the use of any protein-coding clone collection from any organism in any vector and does not require inserts to be devoid of untranslated regions. We tested our prototype human RAFL library against a set of baits that had previously been searched against multiple cDNA libraries. These Y2H searches yielded a combination of novel as well as expected interactions, indicating that the RAFL library constitutes a valuable complement to Y2H cDNA libraries.     

Protein structure prediction using mutually orthogonal Latin squares and a genetic algorithm

We combine a new, extremely fast technique to generate a library of low energy structures of an oligopeptide (by using mutually orthogonal Latin squares to sample its conformational space) with a genetic algorithm to predict protein structures. The protein sequence is divided into oligopeptides, and a structure library is generated for each. These libraries are used in a newly defined mutation operator that, together with variation, crossover, and diversity operators, is used in a modified genetic algorithm to make the prediction. Application to five small proteins has yielded near native structures.     

Generation of genome-wide CD8 T cell responses in HLA-A*0201 transgenic mice by an HIV-1 ubiquitin expression library immunization vaccine.

HIV-1 is a fundamentally difficult target for vaccines due to its high mutation rate and its repertoire of immunoevasive strategies. To address these difficulties, a multivalent, proteasome-targeted, live genetic vaccine was recently developed against HIV-1 using the expression library immunization approach. In this HIV-1 vaccine all open reading frames of HIV-1 are expressed from 32 plasmids as Ag fragments fused to the ubiquitin protein to increase Ag targeting to the proteasome to enhance CTL responses. In this work we demonstrate the ability of the HIV-1 library vaccine to simultaneously provoke robust HLA-A*0201-restricted T cell responses against all 32 HIV-1 library vaccine Ags after single immunization by gene gun. These CD8 T cell responses included HLA-A*0201-restricted CTL activity, CD8/IFN-gamma T cell responses, and HLA tetramer binding against defined immunodominant epitopes in gag, pol, env, and nef as well as potent CD8/IFN-gamma responses against undefined HLA-A*0201-restricted epitopes in all remaining Ags of the library. CD8 responses mediated by single gag, pol, env, and nef plasmids from the vaccine demonstrated little reduction in specific T cell responses when these plasmids were diluted into the context of the full 32-plasmid library, suggesting that Ag dominance or immune interference is not an overt problem to limit the efficacy of this complex vaccine. Therefore, this work demonstrates the ability of the HIV-1 library vaccine to generate robust multivalent genome-wide T cell responses as one approach to control the highly mutable and immunoevasive HIV-1 virus.     

Highly efficient selection of epitope specific antibody through competitive yeast display library sorting

Combinatory antibody library display technologies have been invented and successfully implemented for the selection and engineering of therapeutic antibodies. Precise targeting of important epitopes on the protein of interest is essential for such isolated antibodies to serve as effective modulators of molecular interactions. We developed a strategy to efficiently isolate antibodies against a specific epitope on a target protein from a yeast display antibody library using dengue virus envelope protein domain III as a model target. A domain III mutant protein with a key mutation inside a cross-reactive neutralizing epitope was designed, expressed, and used in the competitive panning of a yeast display naïve antibody library. All the yeast display antibodies that bound to the wild type domain III but not to the mutant were selectively sorted and characterized. Two unique clones were identified and showed cross-reactive binding to envelope protein domain IIIs from different serotypes. Epitope mapping of one of the antibodies confirmed that its epitope overlapped with the intended neutralizing epitope. This novel approach has implications for many areas of research where the isolation of epitope-specific antibodies is desired, such as selecting antibodies against conserved epitope(s) of viral envelope proteins from a library containing high titer, high affinity non-neutralizing antibodies, and targeting unique epitopes on cancer-related proteins.