Overlap PCR Research Articles

Neurotrophin-dependent Tyrosine Phosphorylation of Ras Guanine-releasing Factor 1 and Associated Neurite Outgrowth Is Dependent on the HIKE Domain of TrkA

Ras guanine-releasing factor 1 (RasGrf1), a guanine nucleotide exchange factor for members of the Ras and Rho family of GTPases, is highly expressed in the brain. It is regulated by two separate mechanisms, calcium regulation through interaction with its calcium/calmodulin-binding IQ domain and serine and tyrosine phosphorylation. RasGrf1 is activated downstream of G-protein-coupled receptors and the non-receptor tyrosine kinases, Src and Ack1. Previously, we demonstrated a novel interaction between the intracellular domain of the nerve growth factor-regulated TrkA receptor tyrosine kinase and an N-terminal fragment of RasGrf1. We now show that RasGrf1 is phosphorylated and interacts with TrkA, -B, and -C in co-transfection studies. This interaction and phosphorylation of RasGrf1 is dependent on the HIKE domain of TrkA (a region shown to interact with pleckstrin homology domains) but not on any of the phosphotyrosine residues that act as docking sites for intracellular signaling molecules such as Shc and FRS-2. The PH1 domain alone of RasGrf1 is sufficient for phosphorylation by the TrkA receptor. A potential role for Trk activation of RasGrf1 is suggested through transfection studies in PC12 cells in which RasGrf1 significantly increases neurite outgrowth at low doses of neurotrophin stimulation. Notably, this neurite outgrowth is dependent on an intact HIKE domain, as nnr5-S10 cells expressing a TrkA HIKE domain mutant do not exhibit potentiated neurite outgrowth in the presence of RasGrf1. These studies identify RasGrf1 as a novel target of neurotrophin activation and suggest an additional pathway whereby neurotrophin-stimulated neurite outgrowth may be regulated.

Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL-37, in Escherichia coli

The human cationic antimicrobial peptide hCAP-18/LL-37 is the unique cathelicidin identified in human to date. It has broad spectrum of antimicrobial activities and LPS-neutralizing activity and is involved in angiogenesis. Both purified and synthetic LL-37 or its derivatives were used in the study on LL-37. However, production of LL-37 in Escherichia coli has not been established. In this study, its precursor instead of the mature peptide was adopted for expression to avoid the lethal effect of recombinant LL-37 on host cells. A thrombin recognition site was introduced between the cathelin-like domain and LL-37 domain by overlap PCR to construct fragment encoding modified precursor (mhCAP-18) to facilitate the final release of the recombinant peptide. Then mhCAP-18 was fused in-frame to thioredoxin gene under the control of inducible T7 promoter to construct expression vector pET-mhCAP-18. The soluble form fusion protein was expressed in E. coli and purified by Chelating Sepharose column chromatography. Thrombin digestion of the fusion protein yielded recombinant GSLL-39, which was then purified by cation-exchange chromatography. Recombinant GSLL-39, which has two extra residues on its N-terminus when compared with its native counterpart, showed similar antimicrobial activities against both Gram-negative and Gram-positive bacteria.

Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-beta-d-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a approximately 200-fold improvement over the wild type in reactivity (k(cat)/K(m)) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.

Versatile PCR-mediated insertion or deletion mutagenesis

based procedure that can be used to generate precisely located mutants with any length of insertion or deletion. The strategy for insertion mutagenesis employs an established overlap extension PCR protocol, but involves three templates including an insert fragment of any length. The protocol for insertion requires four chimeric primers that are derived partly from the sequence to be inserted and partly from the template, in addition to two outermost flanking primers. Three PCR products are first prepared separately, including one insert fragment and two adjacent fragments using appropriate primer pairs. The insert fragment thus generated contains terminal complementarities at both ends to two adjacent fragments. In the second step, an overlap extension PCR is carried out, using the outermost primer

Cloning and analysis of mono-specific scFv fragments from chicken to allergenic proteins of Periplaneta americana (American cockroach)

Rationale Although many cockroach proteins bind IgE, only 5 allergenic proteins of Blattella germanica and 3 of Periplaneta americana have been identified and characterized. Phage display of scFvs (single chain fragment variable antibodies) panned against a lysate of cockroach allergens allows us to quickly identify novel cockroach allergenic proteins, and will facilitate the standardization of cockroach allergen extracts. Methods A 6 month old female white leghorn chicken was immunized with American cockroach extract and antibody production was verified through analysis of IgY. RNA was isolated from the spleen and bone marrow and a cDNA library was created. PCR and overlap PCR were then performed on the library to create scFvs. Phage bearing the scFvs were panned against American cockroach extract to isolate those scFvs that recognize cockroach allergens. ScFvs were used for Western blot analysis of cockroach extracts that had been separated by 1D and 2D gel electrophoresis. Results The scFvs recognize proteins in American cockroach extract by Western blot analysis. Nine major proteins bands migrate at 75 kD, 60 kD, 50 kD, 45 kD, 42kD, 37 kD, 31 kD, 20 kD, and 12 kD. These scFvs also recognize several proteins in German cockroach extracts, with the primary bands migrating at 300 kD, 75kD, and 12kD. Conclusions We have generated an scFv library that recognizes a large number of American cockroach proteins as well as several German cockroach proteins. Using this library we will identify and characterize novel German and American cockroach allergens that have similar antibody binding epitopes.

General method for the modification of different BAC types and the rapid generation of BAC transgenic mice.

Most genome projects have relied on the sequencing of bacterial artificial chromosomes (BACs), which encompass 100-300 kb of genomic DNA. As a consequence, several thousand BAC clones are now mapped to the human and mouse genome. It is therefore possible to identify in silico a BAC clone that carries a particular gene and obtain it commercially. Given the large size of BACs, most if not all regulatory sequences of a gene are present and can be used to direct faithful and tissue-specific expression of heterologous genes in vitro in cell cultures and in vivo in BAC-transgenic mice. We describe here an optimized and comprehensive protocol to select, modify, and purify BACs in order to generate BAC-transgenic mice. Importantly, this protocol includes a method to generate, within 2 days, complex plasmid cassettes required to modify BACs, and to efficiently modify different types of BACs selected from the two major BAC libraries available. Altogether, using a combination of genomic database analysis, overlap PCR cloning, and BAC recombination in bacteria, our approach allows for the rapid and reliable generation of "pseudo knockin" mice. genesis 38:39-50, 2004.

Human VPAC1 Receptor Selectivity Filter: IDENTIFICATION OF A CRITICAL DOMAIN FOR RESTRICTING SECRETIN BINDING

The human VPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) belongs to the class II family of G protein coupled receptors with seven transmembrane segments. It recognizes several VIP-related peptides and displays a very low affinity for secretin despite >70% homology between VIP and secretin. Conversely, the human secretin receptor has high affinity for secretin but low affinity for VIP. We took advantage of this reversed selectivity to identify a domain of the VPAC1 receptor responsible for selectivity toward secretin by constructing human VPAC1-secretin receptor chimeras. A first set of chimeras consisted of exchanging the entire N-terminal ectodomain or large parts of this domain. They were constructed by overlap PCR, transfected in COS-7 cells, and their ligand selectivity, expressed as the ratio of EC(50) for secretin/EC(50) for VIP (referred to as S/V), in stimulating cAMP production was measured. Two very informative chimeras respectively referred to as S144V and S123V were obtained by replacing the entire ectodomain or only the first 123 amino acids of the VPAC1 receptor by the corresponding sequences of the secretin receptor. Whereas S144V no longer discriminated between VIP and secretin (S/V = 1.2), S123V discriminated between the two peptides (S/V = 300) in the same manner as the wild-type VPAC1 receptor. The motif responsible for discrimination was determined by introducing small blocks or individual amino acids of secretin receptor in the 123-144 sequence of the S123V chimera. The data obtained from 14 new chimeras sustained that two nonadjacent pairs of amino acids, Gln(135) Thr(136) and Gly(140) Ser(141) in the C-terminal end of the N-terminal VPAC1 receptor ectodomain constitute a selective filter that strongly restricts access of secretin to the VPAC1 receptor.

Adenovirus serotype 30 fiber does not mediate transduction via the coxsackie-adenovirus receptor.

Prior work by members of our laboratory and others demonstrated that adenovirus serotype 30 (Ad30), a group D adenovirus, exhibited novel transduction characteristics compared to those of serotype 5 (Ad5, belonging to group C). While some serotype D adenoviruses bind to the coxsackie-adenovirus receptor (CAR), the ability of Ad30 fiber to bind CAR is unknown. We amplified and purified Ad30 and cloned the Ad30 fiber by overlap PCR. Alignment of Ad30 fiber with Ad3, Ad35, Ad5, Ad9, and Ad17 revealed that Ad30, like Ad9 and Ad17, has a shortened fiber sequence relative to that of Ad5. The knob region of fiber was 45% identical to that of the Ad5 knob regions. We made a chimeric recombinant virus (Ad5GFPf30) in which the Ad5 fiber (amino acids [aa]47 to 582) was replaced with Ad30 fiber sequences (aa 46 to 372), and CAR-mediated viral entry was determined on CAR-expressing Chinese hamster ovary (CHO) cells. While CAR expression significantly increased Ad5GFP-mediated transduction in CHO cells (from 1 to 36%), it did not enhance Ad5GFPf30 gene transfer. Binding of radiolabeled Ad5GFPf30 or Ad30 wild-type virus was also not improved by the expression of CAR. These results suggest that Ad30 fiber is distinct from Ad5, Ad9, and Ad17 fibers in its inability to direct transduction via CAR.

CONSTRUCTION AND EXPRESSION OF A RESHAPED VH DOMAIN AGAINST HUMAN CD28 MOLECULES

ABSTRACT Compared with the amino acid sequence of a mouse anti-human CD28 VH domain antibody, the two most homologous sequences of human antibodies were pulled out from Genbank. One of them was used as the main template for the framework regions of the reshaped VH domain. While the original mouse antibody CDRs were inserted into the human acceptor FRs, some residues in human acceptor FRs, which were different from those of the original mouse FRs in corresponding positions, were then determined or, alternatively, mutagenized to their conservative properties in kappa classification. Based on the amino acid sequences of the designed VH domain, the nucleotide sequence was deduced by using E. coli bias codons. The sequence was split into ten 30 to 60 nucleotide fragments for synthesizing, then annealed and amplified by overlap PCR. Taq DNA polymerase was used in a buffer with high Mg2+ concentration to induce more random mutations, both in FRs and CDRs. A phage display library was constructed by cloning these PCR products. After three rounds of panning, several reshaped VH with high antigen binding activity were obtained. One of them had the same CDR amino acid sequences as that of the original mouse VH domain. Further study showed that it retained a high antigen binding affinity after being expressed in E. coli BL21 (DE3).

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity, Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 M Ser) and R166K (Arg 166 M Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.

The Structure of the β-Propeller Domain and C-terminal Region of the Integrin αM Subunit: DEPENDENCE ON β SUBUNIT ASSOCIATION AND PREDICTION OF DOMAINS

The Structure of the β-Propeller Domain and C-terminal Region of the Integrin αM Subunit: DEPENDENCE ON β SUBUNIT ASSOCIATION AND PREDICTION OF DOMAINS

Humanization of an antibody recognizing a breast cancer specific epitope by CDR-grafting

Background: Mucl-H23 is a cell surface mucin that is expressed on normal breast luminal epithelial cells and over-expressed in most breast tumors. In addition, Muc-1 expressed by malignant cells is glycosylated differently than Muc-1 expressed by normal cells. This difference in glycosylation exposes a peptide epitope on malignant cells which is not exposed on normal cells. Murine monoclonal antibody H23 recognizes this epitope and stains 91% of breast cancers, but only 156 non-malignant breast tissue samples. Objective: To create a human antibody that was equivalent to H23 for potential uses in imaging and/or the therapy of breast cancer. Study design: We decided to humanize H23 by CDR-grafting using overlap PCR, and to this end, designed and constructed a bacterial expression vector that would allow V-regions, cloned via unique restriction sites, to be expressed as Fab fragments. In this way, we hoped to be able to rapidly evaluate Fab constructs for binding to Muc-1 and to cells and tissue sections that expressed the antigen. Results: A fully humanized Fab fragment was able to bind Muc-1 peptide, as well as breast cancer cells known to express the epitope and tissue sections, generally showing the same reactivity as the native antibody. In addition, an analysis of sFab expressed with a [His]6 tag preceded by a factor Xa proteolytic cleavage site suggested that E. coli periplasmic peptidase was able to cleave the factor Xa site, therebyremoving the [His]6 tag. Conclusion: We have generated a human antibody that is capable of recognizing a tumor specific epitope expressed by 91% of breast cancers.

Recombinant thyroid peroxidase-specific Fab converted to immunoglobulin G (IgG) molecules: evidence for thyroid cell damage by IgG1, but not IgG4, autoantibodies.

A recombinant autoantibody Fab (SP1.4) to thyroid peroxidase (TPO), cloned from intrathyroidal B cell immunoglobulin genes, interacts with an epitope on TPO recognized by all patients with autoimmune thyroid disease. To compare the biological properties of IgG1 and IgG4 TPO autoantibodies, we converted Fab SP1.4 to full-length immunoglobulins. The SP1.4 heavy and kappa light chain variable region genes, spliced by overlap PCR to a mammalian signal peptide, were transferred to expression vectors for human IgG1, IgG4, and kappa L chains. Plasmids containing the IgG1 (or IgG4) heavy chain and the kappa L chain were cotransfected into SP2/0 mouse myeloma cells. Cells secreting TPO autoantibodies were cloned, and IgG1-SP and IgG4-SP were affinity purified from medium using protein G. Their subclass specificities were confirmed by enzyme-linked immunosorbent assay and fluorometry after binding to Chinese hamster ovary cells expressing cell surface TPO. Further confirmation of SP1.4 Fab conversion to full-length molecules was the ability of protein A to precipitate IgG1-SP and IgG4-SP complexed to [125I]TPO. IgG1-SP1.4, IgG4-SP1.4, and Fab SP1.4 had similar high affinities for TPO (Kd = approximately 2 x 10(-10) mol/L). Complexes of [125I]TPO and IgG1-SP (but not IgG4-SP) bound to peripheral blood mononuclear cells (PBMC), but not to a B cell line. Flow cytometry demonstrated Fc receptors Fc gamma RI, Fc gamma RII, and Fc gamma RIII on PBMC, but only Fc gamma RII on the B cell line. Together, these data indicate that IgG1-SP/TPO complexes bind to either Fc gamma RI on monocytes or RIII on natural killer cells. In assays for antibody-dependent cytotoxicity using PBMC, 51Cr release was higher for thyroid cells preincubated with IgG1-SP (13.4%) than with IgG4-SP (2.5%) or with culture medium alone (-0.7%). No specific 51Cr release was observed when either fibroblasts or Chinese hamster ovary cells expressing cell surface TPO were used as target cells. In conclusion, a human TPO-specific Fab converted to IgG1, but not IgG-4, can mediate cytotoxic effects on human thyroid cells in vitro. These observations support the clinical relevance of TPO autoantibody subclass distribution and emphasize the likelihood that, as opposed to being simple markers of thyroid damage, TPO autoantibodies may play a role in the induction of thyroid dysfunction in vivo.

Autoantibody-mediated capture and presentation of autoantigen to T cells via the Fcε receptor by a recombinant human autoantibody Fab converted to IgE

Fcε receptor (CD23)-mediated capture of IgE-antigen complexes by B cells provides a powerful antigen presenting system. Our goal was to develop a system using high affinity, human, organ-specific monoclonal autoantibodies for antigen capture by B cells. For this purpose, we converted a recombinant human autoantibody to TPO from a Fab (SP1.4) to an IgE molecule. Sera from all patients with autoimmune thyroid disease contain autoantibodies with the same epitope as SP1.4. The SP1.4 and L chain V region genes were spliced by overlap PCR to a mammalian, non-immunoglobulin signal peptide and transferred to expression vectors for human IgG1 and κ, respectively. After inserting the IgE constant region genes into the H chain vector, the κ and IgE H chain vectors were expressed in SP2 0 cells. SP1.4-IgE retains its high affinity ( K d) for TPO (∼ 2 × 10 −10 M), recognizes the same epitope as Fab SP1.4 and, importantly, binds to a different epitope than does Fab TR1.9. Binding of preformed complexes of SP1.4-IgE and biotinylated TPO to EB virus transformed B cells (EBVL) was weakly detectable by flow cytometry and was displaced by unlabeled TPO. SP1.4-IgE/ 125I-TPO complex binding to EBVL was much more clearly evident, was also inhibited by the addition of unlabeled TPO, and was greatly reduced by preincubation of the EBVL with anti-CD24. Further, autologous EBVL preincubated with SP1.4-IgE/TPO complexes stimulated proliferation of TPO-specific T cells. IgE autoantibody-mediated antigen focusing to B cells is unlikely to operate in vivo but is, instead, a powerful investigative tool. In conclusion, SP1.4-IgE is the first monoclonal human autoantibody to be developed for IgE-mediated antigen presentation to T cells by EBVL. Recombinant human autoantibodies converted to IgE, possibly in combinations if their epitopes permit simultaneous binding to the same molecule, provide a unique system to generate human T cell lines and clones specific for peptides naturally processed from internalized high affinity autoantibody/autoantigen complexes.

Expression and characterization of recombinant alpha-galactosidase in baculovirus-infected insect cells.

A cDNA encoding coffee bean alpha-galactosidase was subcloned into baculovirus expression vectors, pVL-1393 and pAc-GP67B, for intracellular and extracellular expression in Spodoptera frugiperda (Sf9) insect cells, respectively. The expressed protein (recombinant alpha-galactosidase) was immunologically reactive with antisera raised against its native counterpart isolated from coffee beans and was biologically active towards the substrate p-nitrophenyl alpha-galactopyranoside. The subcellular distribution of recombinant alpha-galactosidase expressed from different vectors was analyzed by Western blotting, immunofluorescent labeling, and electron microscopy. In addition, recombinant alpha-galactosidase was compared to the native enzyme with respect to glycosylation, thermostability, and pH profile. Furthermore, a recombinant alpha-galactosidase molecule with a His6 tag at its C-terminus was constructed by an overlap PCR method so that the enzyme expressed in Sf9 cells can be purified by a simple affinity chromatography procedure.

A mutant baculovirus with a temperature-sensitive IE-1 transregulatory protein.

We have mapped the mutation responsible for the temperature-sensitive (ts) phenotype of tsB821, a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus (H. H. Lee and L. K. Miller, J. Virol. 31:240-252, 1979), to a single nucleotide which changes alanine 432 of the multifunctional regulatory protein IE-1 to a valine. Mapping was done with a combination of marker rescue and transient expression assays, hybrid gene construction by overlap PCR gene splicing, and nucleotide sequence analysis. Cells infected with tsB821 at high multiplicities of infection showed a spectrum of responses from severe cytopathic effects, including apoptosis, to a lack of obvious signs of infection. Protein synthesis in tsB821-infected cells at the restrictive temperature appeared similar to uninfected cell protein synthesis, but viral DNA replication and budded virus production were observed, albeit in a delayed manner. The dependence of early and late promoter activity on the wild-type IE-1 gene, ie-1, was observed in transient expression assays. However, the dependence of early promoter activity on ie-1 was strongest in the absence of other viral genes. Thus, other viral genes appear to be able to compensate, at least in part, for the lack, or low levels, of ie-1 in transient expression assays using early promoters. The mutant should prove useful in further defining the function(s) of IE-1.