Biomedical subjects

Cha-Yong Choi

Publications and source records attributed to Cha-Yong Choi.

16 recordsLinked to original sources

PubMed2006

Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts.

This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours.

Cell-Free System↗

PubMed2006

The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract.

In our experiments to produce different combinations of recombinant proteins in a cell-free protein synthesis system derived from Escherichia coli, we found that certain pairs of ORFs were not expressed equally. Instead, only a single DNA species was expressed dominantly, while the expression of the others was almost completely repressed. This bias during the co-expression of the DNA pairs was eliminated when an identical downstream box sequence was added to the 5'-ends of the template DNA pairs. By introducing identical nucleotide sequences of the his-tag or the downstream box of chloramphenicol acetyltransferase (CAT-DB) in front of the target genes that were otherwise not expressed compatibly, both of the encoded proteins were produced at similar productivities. Moreover, in the presence of a common downstream box, multiple genes were simultaneously expressed in the same reaction mixture. We expect that the proposed approach will offer a powerful tool for the preparation of unbiased protein libraries, as well as for studying the structure and functions of interacting proteins.

Cell Extracts↗

PubMed2006

Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system.

In this study, as a part of our efforts to improve the robustness and economical feasibility of cell-free protein synthesis, we developed a simple method of preparing the cell extracts used for catalyzing cell-free protein synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support cell-free protein synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.

Cell-Free System↗

PubMed2006

Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system.

We developed a cell-free protein synthesis system that produces more than 1mg/ml of recombinant proteins in two hours. A basal system that supports the stable maintenance of ATP and amino acids was constructed by using high concentrations of CP (100 mM) and amino acids (3 mM). Approximately 0.6 mg/ml of protein was produced during the batch incubation of the basal system. We found that the accumulation of inorganic phosphate reduces the concentration of free magnesium ions and that there exists a critical concentration of magnesium at which the protein synthesis is halted. Based on this finding, we attempted to extend the duration of the protein synthesis by keeping the magnesium concentration sufficiently high throughout the reaction period. The protein synthesis reaction continued for at least 2 h when the reaction was repeatedly supplemented with magnesium, and approximately 1.2 mg/ml of active CAT or GFP was produced. The simple, fast, and highly productive cell-free protein synthesis system described herein should offer a versatile platform for the preparation of protein molecules in various post-genomic efforts.

Adenosine Triphosphate↗

PubMed2006

Oxalate enhances protein synthesis in cell-free synthesis system utilizing 3-phosphoglycerate as energy source.

Herein, we report our results showing that the productivity of cell-free protein synthesis can be enhanced through the regulation of the in vitro metabolism of an energy source. In a reaction mixture utilizing 3-phosphoglycerate (3PG) as an energy source, the supply of ATP was significantly enhanced when the reaction mixture was supplied with sodium oxalate, a potent inhibitor of phosphoenolpyruvate synthetase (PPS). The productivity of protein synthesis was also increased by approximately 70% upon the addition of oxalate. It was presumed that this enhancement in ATP supply resulted from the prevention of the pyruvate --> PEP reaction, which causes nonproductive ATP consumption. For the initial presence of 2.1 mM sodium oxalate, approximately 720 microg/ml chloramphenicol acetyltransferase (CAT) was produced after 3 h of incubation at 37 degrees C.

Adenosine Triphosphate↗

PubMed2006

Fragment library-based representation system for protein conformation.

The representation system for protein conformation has a crucial effect on the speed of various protein-related simulations, including ab initio protein structure prediction and protein-protein docking simulation. Usually, the finer a representation system, the longer is the computational time required to employ the representation system in simulations. On the other hand, very coarse lattice systems cannot be directly applied to the simulation problems with real proteins. We report a new, fragment library-based protein conformation representation system, prepared by clustering amino acid conformations from 154 proteins. This system was composed of 64 most representative fragments per each amino acid, and based on the unified residue approach in which two spheres per amino acid were used. It could represent the conformation of the 82 proteins in an independent test set with the mean and standard deviation RMSD of 1.01 and 0.09 A, respectively, based on the position of alpha carbons and the centers of mass of sidechains.

Models, Molecular↗

PubMed2006

A linear function for the approximation of accessible surface area of proteins.

Since the solvent accessible surface area of proteins has been utilized as an important factor in the prediction of their thermodynamic properties, a simple and analytical method for its determination would contribute to protein structure prediction and analysis methods. We report the development of a simple and analytical method for the estimation of the accessible surface area of protein, consisting of linear functions of distances between unified atoms. While our formulation was much simpler than previously developed methods, it showed comparable performance, with the mean relative error on total solvent accessible surface area of 0.49 and 2.16% for 89 denatured and native protein structures, respectively. Moreover, the partial derivatives of accessible surface area to the position of unified atoms could also be derived in a simpler form.

Algorithms↗

PubMed2005

Enhancing the efficiency of cell-free protein synthesis through the polymerase-chain-reaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues.

A method for the rapid generation of intact proteins in a cell-free protein synthesis system was developed. The productivity of the recombinant proteins from the polymerase-chain-reaction-amplified templates was enhanced remarkably using an optimized translation enhancer sequence. The extra amino acid residues derived from the translation enhancer sequence were effectively removed by utilizing the appropriate detergent and peptide cleavage enzyme in the reaction mixture. These results demonstrate the versatility of cell-free protein synthesis in providing optimized and customized reaction conditions for the efficient production of the desired proteins.

Cell-Free System↗

PubMed2005

Effect of energy source on the efficiency of translational termination during cell-free protein synthesis.

We studied how the fidelity of translation termination is affected by the method of ATP regeneration during cell-free protein synthesis. During the in vivo expression of hEPO, whose termination is directed by the UGA codon, we found that substantial proportions of the translational products showed a larger molecular weight than expected. Similar results were obtained in a cell-free synthesis reaction using phosphoenol pyruvate (PEP) or 3-phosphoglycerate (3PG) for ATP regeneration. However, when the energy source was switched to creatine phosphate (CP), the readthrough of the UGA codon was completely repressed and only the target protein of the correct size was expressed in a high yield. To the best of our knowledge, this is the first report describing the relationship between the regeneration of nucleotide triphosphates and protein readthrough, and we also believe that the discovery would pave the way to the selective and efficient expression of target proteins in cell-free protein synthesis systems.

Adenosine Triphosphate↗

PubMed2005

Cell-free synthesis of recombinant proteins from PCR-amplified genes at a comparable productivity to that of plasmid-based reactions.

The functional stability of mRNA is one of the crucial factors affecting the efficiency of cell-free protein synthesis. The importance of the stability of mRNA in the prolonged synthesis of protein molecules becomes even greater when the cell-free protein synthesis is directed by PCR-amplified DNAs, because the linear DNAs are rapidly degraded by the endogenous nucleases and, thus, the continuous generation of mRNA molecules is limited. With the aim of developing a highly efficient cell-free protein synthesis system directed by PCR products, in this study, we describe a systematic approach to enhance the stability of mRNA in cell-free extracts. First, exonuclease-mediated degradation was substantially reduced by introducing a stem-loop structure at the 3'-end of the mRNA. The endonucleolytic cleavage of the mRNA was minimized by using an S30 extract prepared from an Escherichia coli strain that is deficient in a major endonuclease (RNase E). Taken together, through the retardation of the endonucleolytic and exonucleolytic degradations of the mRNA molecules, the level of protein expression from the PCR-amplified DNA templates becomes comparable to that of conventional plasmid-based reactions. The enhanced productivity of the PCR-based cell-free protein synthesis enables the high-throughput generation of protein molecules required for many post-genomic applications.

Cell Extracts↗

PubMed2005

Cell-free synthesis and in situ isolation of recombinant proteins.

We present a method for rapid expression and isolation of recombinant proteins. Cell-free protein synthesis in the presence of affinity beads enables in situ isolation of translation products, which simplifies the procedures for the preparation of purified protein samples. In the present study, we have made an attempt to carry out in situ isolation of histidine-tagged proteins by using Ni-NTA magnetic agarose beads. The presence of Ni-NTA beads gave no drastic effects on the efficiency of protein synthesis and successfully captured the synthesized proteins. Purified proteins were obtained after subsequent washing and elution steps. In particular, most of the endogenous bead-binding proteins were removed by pre-treating S30 extract with affinity beads and the purity of the target proteins was enhanced up to 95%. The methods described here will provide a basis for fast and convenient preparation of purified proteins from multiple genetic sequences.

Cell-Free System↗

PubMed2005

Effect of temperature and ATP supply on the efficiency of programmed nonsense suppression.

Chemical diversity of protein molecules can be expanded through in vitro incorporation of unnatural amino acids in response to a nonsense codon. Chemically misacylated tRNAs are used for tethering unnatural amino acids to a nonsense-mutated target codon (nonsense suppression). In the course of experiments to introduce S-(2-nitrobenzyl)cysteine (NBC) into a targeted location of human erythropoietin, we found that NBC incorporates more efficiently at lower temperatures. In addition, at a fixed reaction temperature, more NBC was incorporated with a reduced supply of ATP. Since the rate of peptide elongation was remarkably higher at the elevated temperature or with enhanced supply of ATP, these results indicate that the efficiency of nonsense suppression is inversely correlated to the peptide elongation rate. Therefore, maximal yield of nonsense-suppressed proteins is obtained at a compromised elongation rate. The present result will offer a primary guideline to optimize the reaction conditions for in vitro production of protein molecules containing unnatural amino acids.

Anticodon↗

PubMed2004

Enhanced in vitro midbrain dopamine neuron differentiation, dopaminergic function, neurite outgrowth, and 1-methyl-4-phenylpyridium resistance in mouse embryonic stem cells overexpressing Bcl-XL.

Embryonic stem (ES) cells provide a potentially unlimited source of specialized cells for regenerative medicine. The ease of inducing stable genetic modifications in ES cells allows for in vitro manipulations to enhance differentiation into specific cell types and to optimize in vivo function of differentiated progeny in animal models of disease. We have generated mouse ES cells that constitutively express Bcl-XL, an antiapoptotic protein of Bcl-2 family. In vitro differentiation of Bcl-XL overexpressing ES (Bcl-ES) cells resulted in higher expression of genes related to midbrain dopamine (DA) neuron development and increased the number of ES-derived neurons expressing midbrain DA markers compared with differentiation of wild-type ES cells. Moreover, DA neurons derived from Bcl-ES cells were less susceptible to 1-methyl-4-phenylpyridium, a neurotoxin for DA neurons. On transplantation into parkinsonian rats, the Bcl-ES-derived DA neurons exhibited more extensive fiber outgrowth and led to a more pronounced reversal of behavioral symptoms than wild-type ES-derived DA neurons. These data suggest a role for Bcl-XL during in vitro midbrain DA neuron differentiation and provide an improved system for cell transplantation in a preclinical animal model of Parkinson's disease.

1-Methyl-4-phenylpyridinium↗

PubMed2004

Expression system for enhanced green fluorescence protein conjugated recombinant antibody fragment.

Recent development of recombinant antibody technology has enabled fusion of recombinant antibody fragment with fluorescent proteins for various applications such as flow cytometry, fluorescence immunoassay, and fluorescent microscopy. In this study, we generated various forms of green fluorescence protein (EGFP)-fused anti-c-Met antibody fragment. Among these fusion proteins, EGFP fusion to the light chain showed high expression in a soluble form of protein in E. coli, and high binding activity to c-Met. A feasibility of the constructs was further examined by replacing the Fab gene by a Fab library of catalytic subunit of protein kinase A (PKA) to construct the Fab library in EGFP fused form. We also constructed the conventional Fab library. After a series of biopanning, we found that the binding capability of EGFP-anti-PKA Fab was comparable with anti-PKA Fab. Sequence analysis of the selected clones showed > or =99% identity in amino acid sequence and shared the same CDR sequence. These results demonstrate that EGFP fusion to the light chain using our vector system does not influence the selection of reactive Fab and that this vector system is useful for EGFP fusion to Fab to develop a one-step detection system.

Amino Acid Sequence↗

PubMed2003

Cloning and analysis of a DNA fragment stimulating avermectin production in various Streptomyces avermitilis strains.

To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis.

Bacterial Proteins↗

PubMedDate not supplied

Providing an oxidizing environment for the cell-free expression of disulfide-containing proteins by exhausting the reducing activity of Escherichia coli S30 extract.

We developed a novel method of producing proteins containing multiple disulfide bonds in a cell-free protein synthesis system. To provide an optimized redox potential during the synthesis of truncated plasminogen activator (rPA), we pretreated the E. coli S30 extract with an excess amount of oxidized glutathione based on the anticipation that the reducing potential of the S30 extract would be exhausted through the reduction of the oxidized glutathione molecules. As expected, it was found that the reducing activity of the S30 extract was remarkably decreased through the pretreatment, and active rPA was produced when the pretreated S30 extract was used after removing the residual glutathione molecules. In particular, compared to the method involving the iodoacetamide treatment of S30 extract, the present protocol was effective in producing active rPA during the batch reaction of cell-free protein synthesis.

Disulfides↗