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PubMed1987

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure.

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.

Allosteric Regulation↗
PubMed2025

Deep learning guided programmable design of Escherichia coli core promoters from sequence architecture to strength control.

Core promoters are essential regulatory elements that control transcription initiation, but accurately predicting and designing their strength remains challenging due to complex sequence-function relationships and the limited generalizability of existing AI-based approaches. To address this, we developed a modular platform integrating rational library design, predictive modelling, and generative optimization into a closed-loop workflow for end-to-end core promoter engineering. Conserved and spacer region of core promoters exert distinct effects on transcriptional strength, with the former driving large-scale variation and the latter enabling finer gradation. Based on this insight, Mutation-Barcoding-Reverse Sequencing approach was used and constructed a synthetic promoter library comprising 112 955 variants with minimal redundancy and a 16 226-fold expression range. A Transformer-based model trained on this dataset achieved a Pearson correlation of 0.87 with experimentally measured promoter strengths. When combined with a conditional diffusion model, the system enabled de novo generation of promoter sequences with defined strengths, achieving a design-to-measurement correlation of 0.95 and maintaining high accuracy (R = 0.93) across varied sequence contexts. The designed promoters consistently preserved their intended strength gradients, demonstrating robust plug-and-play functionality. This work establishes a scalable and extensible platform (www.yudenglab.com) for deep learning-guided programmable design of Escherichia coli core promoters, enabling precise transcriptional control.

Promoter Regions, Genetic↗
PubMed2001

VP5 and the N terminus of VP2 are not responsible for the different pathotype of serotype I and II infectious bursal disease virus.

Two serotypes have been identified in infectious bursal disease virus (IBDV), a member of the family BIRNAVIRIDAE: A reverse genetics system was used for generation of chimeras in genome segment A of the two serotypes, in which the complete viral VP5 gene and 3' noncoding region (NCR), or parts thereof, were exchanged. The engineered viruses were characterized in vitro and in vivo in comparison to serotype I and II IBDV. Our results show that IBDV chimeras exhibit a different phenotype in cell culture compared to the wild-type viruses. In in vitro-cultivated bursal-derived cells, chimeric viruses infected B lymphocytes, as does serotype I IBDV. Surprisingly, serotype II virus was also able to infect in vitro-cultivated bursal cells, but these were neither B lymphocytes nor macrophages. After infection of susceptible chickens all chimeras replicated in the bursa of Fabricius (BF), and three chimeric viruses caused mild depletion of bursal cells. In contrast, after infection of chickens with a chimeric IBDV containing exchanged VP5 as well as 3'-NCR, no depletion was detectable. The serotype II strain did not replicate in the BF nor did it cause depletion of bursal cells. Thus, the origin of VP5 does not explain the different pathotype of IBDV serotype I and II.

3' Untranslated Regions↗
PubMed2026

Decoding glioblastoma evolution and heterogeneity through mechanistic modeling: implications for clinical translation.

Glioblastoma (GBM) is one of the most aggressive and lethal primary brain tumors in adults, characterized by dynamic clonal evolution and extensive genomic, cellular, spatial, and microenvironmental heterogeneity. Multi-omics studies have revealed that GBM follows complex evolutionary trajectories involving genetic, epigenetic, transcriptional, and immune-microenvironmental remodeling as tumors grow, adapt to the brain microenvironment, and acquire therapeutic resistance. Increasing evidence suggests that GBM may originate from aberrant neural stem or progenitor cells, including those residing in the subventricular zone, and that glioblastoma stem cells (GSCs) contribute to tumor propagation, heterogeneity, and recurrence. A key conceptual challenge is to reconcile hierarchical cancer stem cell models, in which GSCs are viewed as relatively stable tumor-propagating subpopulations, with dynamic state plasticity models, in which stem-like properties can be reversibly acquired or lost during transitions among proneural-like, mesenchymal-like, invasive, and therapy-tolerant states. Recent advances in single-cell profiling, spatial transcriptomics, lineage tracing, organoid culture, 3D bioprinting, genetically engineered models, and artificial intelligence (AI)-assisted computational modeling have substantially improved the ability to study these processes. However, no currently available model fully recapitulates human GBM heterogeneity, recurrence, treatment history, and tumor-microenvironment interactions. Therefore, model selection should be guided by clearly defined mechanistic questions rather than by reliance on any single platform. This review summarizes current advances in in vitro, ex vivo, in vivo, and computational models for studying GBM evolution and heterogeneity, and discusses how integrated model pipelines may improve preclinical drug testing, treatment-response prediction, and precision neuro-oncology.

Humans↗
PubMedDate not supplied

Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein.

A genetically-engineered mutant of cytochrome b5, incorporating a unique cysteine residue, was conjugated to maleimide-terminated oligo(N-isopropylacrylamide). The conjugation of the protein by reaction of the cysteine residue, precisely positioned by site-directed mutagenesis techniques, with an activated oligomer containing only one reactive end group in the oligomer chain permits the site-specific and stoichiometric conjugation of the oligomer with the protein. The protein-oligomer conjugate was shown to exhibit lower critical solution temperature (LCST) behavior, similar to the free oligomer. Furthermore, the LCST behavior of the protein-oligomer conjugate is reversible and allows selective precipitation of the conjugate above its LCST.

Acrylic Resins↗
PubMed2002

Engineering Vero cells to secrete human insulin.

Cell therapy may have the potential for the treatment of Type I diabetes. To date, cells suitable for this purpose have not been developed. This study investigates the feasibility of modifying Vero, a cell line that may be considered safe to implant into humans, for this purpose. Stable Vero transfectants containing full-length human preproinsulin complementary deoxyribonucleic acid (cDNA) were generated using a liposomal transfection reagent. Reverse transcriptase-polymerase chain reaction, immunocytochemistry, Western blotting, and enzyme-linked immunosorbent assays were used to assess the resulting cells. Proinsulin was expressed but was not processed to insulin by these cells. Proinsulin cDNA was genetically modified, resulting in a form that is furin sensitive. The resulting stably transfected Vero clones constitutively release approximately 34%/h (32.68 +/- 2.21 to 35.62 +/- 3.14%) of the product formed, approximately 62% (59.99 +/- 6.45 to 64.64 +/- 4.57%) of which is mature insulin. These Vero transfectants did not exhibit glucose-stimulated insulin secretion. As GLUT2 and glucokinase (GCK) are not constitutively expressed by these cells, human GLUT2 cDNA and GCK cDNA were cotransfected with furin-sensitive preproinsulin cDNA into Vero cells. Insulin and GCK proteins were detected in the cytoplasmic region of the resulting cells, whereas GLUT2 was predominantly expressed in the nucleus. Coexpression of GLUT2 and GCK did not result in glucose-stimulated insulin secretion. The results from this study demonstrate the feasibility of engineering a relatively "safe" nonbeta cell line to produce human insulin. Coexpression of GLUT2 and GCK, at the levels achieved here, is not adequate enough to induce glucose-stimulated insulin secretion in such cells; the subcellular location of transfected components may be relevant.

Animals↗
PubMed1991

Transgenic mice that express the human multidrug-resistance gene in bone marrow enable a rapid identification of agents that reverse drug resistance.

The development of preclinical models for the rapid testing of agents that circumvent multidrug resistance in cancer is a high priority of research on drug resistance. A common form of multidrug resistance in human cancer results from expression of the MDR1 gene, which encodes a Mr 170,000 glycoprotein that functions as a plasma membrane energy-dependent multidrug efflux pump. We have engineered transgenic mice that express this multidrug transporter in their bone marrow and demonstrated that these animals are resistant to leukopenia by a panel of anticancer drugs including anthracyclines, vinca alkaloids, etoposide, taxol, and actinomycin D. Differential leukocyte counts indicate that both neutrophils and lymphocytes are protected. Drugs such as cisplatin, methotrexate, and 5-fluorouracil, which are not handled by the multidrug transporter, produce bone marrow suppression in both normal and transgenic mice. The resistance conferred by the MDR1 gene can be circumvented in a dose-dependent manner by simultaneous administration of agents previously shown to be inhibitors of the multidrug transporter in vitro, including verapamil isomers, quinidine, and quinine. Verapamil and quinine, both at levels suitable for human trials that produced only partial sensitization of the MDR1-transgenic mice, were fully sensitizing when used in combination. We conclude that MDR1-transgenic mice provide a rapid and reliable system to determine the bioactivity of agents that reverse multidrug resistance in animals.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Transgenic plants expressing cationic peptide chimeras exhibit broad-spectrum resistance to phytopathogens.

Here we describe a strategy for engineering transgenic plants with broad-spectrum resistance to bacterial and fungal phytopathogens. We expressed a synthetic gene encoding a N terminus-modified, cecropin-melittin cationic peptide chimera (MsrA1), with broad-spectrum antimicrobial activity. The synthetic gene was introduced into two potato (Solanum tuberosum L.) cultivars, Desiree and Russet Burbank, stable incorporation was confirmed by PCR and DNA sequencing, and expression confirmed by reverse transcription (RT)-PCR and recovery of the biologically active peptide. The morphology and yield of transgenic Desiree plants and tubers was unaffected. Highly stringent challenges with bacterial or fungal phytopathogens demonstrated powerful resistance. Tubers retained their resistance to infectious challenge for more than a year, and did not appear to be harmful when fed to mice. Expression of msrA1 in the cultivar Russet Burbank caused a striking lesion-mimic phenotype during leaf and tuber development, indicating its utility may be cultivar specific. Given the ubiquity of antimicrobial cationic peptides as well as their inherent capacity for recombinant and combinatorial variants, this approach may potentially be used to engineer a range of disease-resistant plants.

Amino Acid Sequence↗
PubMed1994

Effect of the sulfonylurea glyburide on superoxide dismutase activity in alloxan-induced diabetic rat hepatocytes.

In the present study we administrated glyburide (glibenclamide) to type 2 (NIDDM) diabetic rats and determined the effect of such treatment on liver superoxide dismutase (SOD) activity. Hepatic SOD activity was significantly reduced in diabetic animals. Glyburide treatment of diabetic rats for 4 weeks corrected the changes observed in diabetic liver. In addition, blood glucose levels of untreated diabetic rats decreased following glyburide treatment. Administration of glyburide to diabetic rats reversed the diabetes-induced changes, suggesting that glyburide may directly increase liver SOD enzyme activity.

Animals↗
PubMed1990

Effect of intravenous drug administration mode on drug distribution in a tumor slab: a finite Fourier transform analysis.

Cancer therapy using chemotherapeutic drugs frequently involves injection of the drug into the body through some intravenous mode of administration, viz, continuous (drip) infusion or single/multiple bolus injection(s). An understanding of the effect of the various modes of administration upon tumor penetration of drug is essential to rational design of drug therapy. This paper investigates drug penetration into a model tumor of slab geometry (between two capillaries) in which the overall transport rate of drug is limited by intra-tumor transport characterized by an effective diffusion coefficient. Employing the method of Finite Fourier Transforms (FFT), analytical solutions have been obtained for transient drug distribution in both the plasma and the tumor following three modes of administration, viz, continuous infusion, single bolus injection and equally-spaced equal-dose multiple bolus injections, of a given amount of drug. The qualitative trends exhibited by the plasma drug distribution profiles are consistent with reported experimental studies. Two concepts, viz, the dimensionless decay constant and the plasma/tumor drug concentration trajectories, are found to be particularly useful in the rational design of drug therapy. The dimensionless decay constant provides a measure of the rate of drug decay in the plasma relative to the rate of drug diffusion into the tumor and is thus characteristic of the tumor/drug system. The magnitude of this parameter dictates the choice of drug administration mode for minimizing drug decay in the plasma while simultaneously maximizing drug transport into the tumor. The concentration trajectories provide a measure of the plasma drug concentration relative to the tumor drug concentration at various times following injection. When the tumor drug concentration exceeds the plasma drug concentration, the drug will begin to diffuse out of the tumor. Knowledge of the time at which this diffusion reversal occurs is especially useful for optimum scheduling of subsequent bolus injections in a multiple bolus dosing regimen. There are no reported applications of the FFT method to solve repeated input functions in either the chemical engineering or pharmaceutical science literature. Thus, the application of FFT method to solve multiple bolus injections is a unique one. Use of this FFT based analysis as a predictor tool can limit the number of costly experiments which are being done now to achieve this purpose. Even though the model in its present form is simplified, the analysis thereof has nevertheless led to a better understanding of the various factors that must be taken into account for rational design of drug therapy.

Drug Administration Routes↗
PubMed2003

Use of avian retroviral vectors to introduce transcriptional regulators into mammalian cells for analyses of tumor maintenance.

A key issue in cancer biology is whether genetic lesions involved in tumor initiation or progression are required for tumor maintenance. This question can be addressed with mouse models that conditionally express oncogenic transgenes, i.e., under the control of tetracycline (tet)-dependent transcriptional regulators. We have developed a system for studying tumor maintenance by using avian retroviral [i.e., replication-competent avian leukosis virus long terminal repeat with splice acceptor (RCAS)] vectors to deliver the reverse tet transcriptional transactivator (rtTA) gene to somatic mammalian cells. rtTA can regulate any transgene in which the protein coding sequence is preceded by a tet-operator (tet-o); RCAS viruses infect only cells engineered to express ectopically the avian retroviral receptor, TVA. One vector, RCAS-rtTA-IRES-GFP, also encodes GFP to identify infected cells. Infection of cells from beta-actin TVA transgenic mice with this vector permits efficient regulation of tet-responsive transgenes. Sarcomas arise when p53-deficient murine embryonic fibroblasts carrying beta-actin TVA and tet-o-K-ras4bG12D transgenes are infected with RCAS-rtTA-IRES-GFP and introduced into nude mice treated with the tet analog, doxycycline (dox); when dox is withdrawn, K-ras4bG12D levels fall, cells undergo apoptosis, and tumors regress. Regression can be prevented by means of a genetic complementation assay in which tumors are superinfected before dox withdrawal with other RCAS viruses, such as those carrying an active allele of K-ras. Many TVA and tet-regulated transgenic mice have been generated; thus, this method for somatic cell-specific and temporally controlled gene expression may have broad applications for the study of oncogenesis and tumor maintenance, as well as other cell functions and development.

Animals↗
PubMed2000

Theoretical design of antisense genes with statistically increased efficacy.

Endogenous expression of antisense RNA represents one major way of applying antisense nucleic acids. To express antisense RNA intracellularly, recombinant antisense genes have to be designed and introduced into cells where the target RNA is encountered. Efficient annealing between the antisense RNA and the target RNA is crucial for efficacy and is strongly influenced by RNA structure. Here we extend structural rules for the design of in vitro transcribed antisense RNAs to the design of recombinant antisense genes. Intracellularly expressed antisense RNA transcripts contain a central antisense portion and additional flanking vector-derived sequences. A computer algorithm was generated to compose large sets of antisense genes, to calculate secondary structures of the transcribed sequences and to select for favorable structures of antisense RNA in terms of annealing and efficacy. The biological test system to measure efficiency of antisense genes was human immunodeficiency virus type 1 (HIV-1) replication in 293T cells. When considering the lower intracellular steady-state levels of favorably structured endogenous transcripts, an antisense effect against HIV-1 replication was observed that was up to 60-fold stronger than that measured for predicted unfavorable species. The computational selection was successful for antisense portions of 300 nt but not 100 nt in length. This theoretical design of antisense genes supports their improved application under time- and labor-saving conditions.

Algorithms↗
PubMed2001

HuM291(Nuvion), a humanized Fc receptor-nonbinding antibody against CD3, anergizes peripheral blood T cells as partial agonist of the T cell receptor.

BACKGROUND: Humanized Fc receptor (FcR)-nonbinding antibodies against CD3 are promising immunosuppressive agents that may overcome both the neutralizing response to and the cytokine release syndrome seen with conventional monoclonal antibodies against CD3. In addition, evidence from several murine models suggests that these recombinant antibodies may actively induce T cell unresponsiveness by a mechanism other than modulation of the T cell receptor (TCR) or T cell depletion. We hypothesized that FcR-nonbinding antibodies against CD3 could induce T cell unresponsiveness by acting as partial agonist ligands of the TCR and thus, inducing T cell anergy. METHODS: To test this hypothesis, we examined the signaling and functional effects of HuM291 (Nuvion), a FcR-nonbinding humanized antibody against CD3, on primary human T cells. RESULTS: Short exposure of human peripheral blood T lymphocytes to HuM291 caused a partial agonist type of signaling through the TCR characterized by incomplete phosphorylation of TCR zeta, failure to activate ZAP-70 and to phosphorylate LAT but activation of ERK-1/-2 and subsequent up-regulation of CD69 expression. These changes correlated with a dose-dependent induction of anergy in human, primary resting T cells, which was reversed by exogenous interleukin-2. CONCLUSIONS: The tolerogenic properties of FcR-nonbinding monoclonal antibodies against CD3 correlate with its ability to reproduce the biochemical and functional effects of TCR partial agonist ligands. Thus, generation of engineered antibodies against CD3 with low TCR oligomerization potential may provide a clinically applicable partial agonist-based strategy for the prevention of polyclonal T cell responses.

Antibodies, Monoclonal↗
PubMed2003

A translational bridge to cancer immunotherapy: exploiting costimulation and target antigens for active and passive T cell immunotherapy.

Building on significant advances in basic tumor immunology over the past decade, current translational efforts to develop novel antitumor T cell therapeutics continue to accelerate. Both passive T cell immunotherapy (e.g., adoptive T cell transfusions) and active immunotherapy (e.g., vaccination) may eventually become part of the arsenal to treat cancer. Successful approaches will need to repair host immunoincompetence in T cell function, circumvent immunosuppressive factors of the tumor microenvironment, and optimize target antigens with regard to clinical applicability, autoimmunity, and risk of antigen mutation. Here, we characterize two model systems for the ex vivo activation and expansion of human T lymphocytes and describe the potential for providing broadly applicable antitumor specificity by targeting universal tumor antigens. Polyclonal CD4+ T lymphocytes can be activated and expanded using anti-CD3 and anti-CD28 antibodies presented on magnetic beads, and CD8+T lymphocytes can be successfully expanded using a novel genetically engineered cell-based technology that presents anti-CD3 and anti-CD28 along with the costimulatory molecule CD137 (4-1BBL). As the prototypical and best-described universal tumor antigen, the human telomerase reverse transcriptase hTERT is vastly overexpressed in human tumors but absent in most normal tissues. Cytotoxic T lymphocytes (CTL) recognize peptides derived from hTERT and kill hTERT-positive tumor cells of multiple histologies. Phase I trials translating these discoveries to novel active and passive T cell therapies have been initiated, with an eye toward combining these strategies once safety is established.

Animals↗
PubMedDate not supplied

The genetically-engineered secretory B27/Q10 chimeric molecule inhibits HLA-B27 restricted alloreactive T-lymphocytes.

OBJECTIVES: Intracellularly persisting bacterial infections and high association with HLA-B27 are the hallmarks of reactive arthritis. Soluble HLA-B27 molecules are induced by bacterial infection; however their biological role in arthritis is unknown. It was the aim of this study to generate soluble HLA-B27 molecule and to analyze its effect on cytotoxic HLA-B27 alloreactive CD8+ T-lymphocytes in order to better understand potential functional links between persistent infection and HLA-B27 association. METHODS: Using PCR Exons 1 through 4 of HLA-B*2705 were fused to Exon 5 of the soluble murine MHC class I variant Q10 and stably transfected into Hela-cells. Transfectants were analyzed using specific PCR, RT-PCR and intracellular and extracellular staining with anti-HLA-B27 monoclonal antibody ME1. Secretion of B27Q10 in the supernatant was examined by isoelectric focusing (IEF). The effect of B27Q10 on T-cells was analyzed using either HLA-B27- or HLA-A2-restricted alloreactive T-cells in a standard 51Cr-release assay. RESULTS: PCR and RT-PCR demonstrated the DNA and mRNA of B27Q10 in the transfectants. By intracellular and extracellular staining with ME1 B27Q10-molecule was detected intracellularly but was not expressed in the cell membrane. Using IEF soluble B27Q10-molecules were found in supernatants of transfectants in a concentration of up to 1.342 microg/ml. Soluble B27QJO-molecule inhibited specifically the cytotoxicity of HLA-B27-restricted alloreactive T-cells by about 30%. CONCLUSION: The secretory non-membrane-expressed molecule B27Q10 inhibits HLA-B27 specific T-cells. The inhibition of cytotoxic T-cells by bacteria induced soluble HLA-B27 may thus enable bacterial persistence.

Animals↗
PubMed1992

[Application of PCR and RT-PCR method to molecular biology study in nephrology].

Recent advance in molecular biology and genetic engineering has made a profound influence on basic and clinical medicine. Development of PCR (polymerase chain reaction) has transformed our trend of thought in molecular biology. Applications of PCR method has extended to nephrology study. RT (reverse transcription)-PCR method was elaborated to identify mRNA even in a small number of cells. It is also important and advantageous that PCR facilitates the utilization of a non-radioisotopic detection system. Newly developed chemiluminescent procedure yields as high sensitivity just as radioactive probes. Further applications are now being investigated including in situ PCR. PCR and RT-PCR methods are of great value for studying a molecular biological background of intricate kidney functions and diverse renal disorders.

Cloning, Molecular↗
PubMed1995

IL-2 gene-transduced human HLA-A2 melanoma cells can generate a specific antitumor cytotoxic T-lymphocyte response.

This study was designed to assess whether transfer of the interleukin-2 (IL-2) gene into human tumor cells could generate cytotoxic T lymphocytes (CTL) directed specifically against the autologous tumor. Two HLA class I+ melanoma cell lines, one (TOM) A2+ and the other (CLB-M) A2-, obtained from living patients were transduced with the IL-2 gene. The patients' peripheral blood lymphocytes (PBL) were incubated with irradiated IL-2 gene-transduced autologous tumor cells for up to four weeks. After seven days, PBL from both patients showed non-specific cytotoxic activity against the K562 cell line. When the co-culture incubation time was prolonged to 28 days, PBL from patient TOM (A2+) developed a lytic activity directed specifically against the autologous tumor cells. In contrast, after 28 days of incubation, PBL from CLB-M (A2-) displayed only non-specific cytotoxic activity. Inhibition experiments demonstrated that the specific lytic function observed with TOM cells transduced with the IL-2 gene could be reversed following incubation with monoclonal antibodies directed against HLA class I and CD8. The evidence that K562 cells were incapable of blocking the PBL killing capacity in a cold-target inhibition assay further confirmed that engineered TOM cells induced the generation of specific CTL. This study indicates that retroviral vector mediated transfer of the IL-2 gene into human melanoma cell lines can lead to the amplification of the autologous cytotoxic compartment and to the generation of specific antitumor CTL, and that the A2 allele may play an important role in the process of tumor recognition.

Animals↗
PubMedDate not supplied

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.

Acrylamides↗