Solid phase in vitro mutagenesis using plasmid DNA template.

Site-specific mutagenesis was accomplished using a solid support to generate single stranded vector and insert fragments which can be used to form gap-duplex plasmids through flanking, complementary double stranded regions. More than 80% mutants were obtained in both a single and a double primer approach. No special vectors or strains are needed and mismatch repair is avoided as the mutagenesis region is in a single stranded form when transformed into the Escherichia coli host cell. The fragments to be immobilized can be produced either by a polymerase chain reaction using general primers or by a site-specific restriction followed by a fill-in reaction. This novel method is rapid, simple and flexible and well suited for both manual and semi-automated in vitro mutagenesis protocols.     

In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.

Denatured Bacteriophage T4D DNA is able to stimulate aminoacid incorporation into TCA-precipitable material in an in vitro protein synthesis system according to base DNA sequences. Newly synthesized polypeptides remain associated with ribosomes and have a molecular weight in range of 15,000 to 45,000 Daltons.     

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.     

Sequence specific block of in vitro DNA synthesis with isopropyl phosphotriesters in template oligodeoxyribonucleotides.

We have synthesized four oligodeoxyribonucleotides each bearing an isopropyl phosphotriester at a defined position. These oligomers were used as templates for in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I large fragment. Results showed that the phosphotriester inhibits the DNA chain elongation and the level of the inhibition is dependent on the base 5' to the phosphotriester.     

Green synthesis of conductive polyaniline by Trametes versicolor laccase using a DNA template

The green synthesis of highly conductive polyaniline by using two biological macromolecules, i.e laccase as biocatalyst, and DNA as template/dopant, was achieved in this work. Trametes versicolor laccase B (TvB) was found effective in oxidizing both aniline and its less toxic/mutagenic dimer N‐phenyl‐p‐phenylenediamine (DANI) to conductive polyaniline. Reaction conditions for synthesis of conductive polyanilines were set‐up, and structural and electrochemical properties of the two polymers were extensively investigated. When the less toxic aniline dimer was used as substrate, the polymerization reaction was faster and gave less‐branched polymer. DNA was proven to work as hard template for both enzymatically synthesized polymers, conferring them a semi‐ordered morphology. Moreover, DNA also acts as dopant leading to polymers with extraordinary conductive properties (～6 S/cm). It can be envisaged that polymer properties are magnified by the concomitant action of DNA as template and dopant. Herein, the developed combination of laccase and DNA represents a breakthrough in the green synthesis of conductive materials.     

DNA template requirements for human mismatch repair in vitro

The human mismatch repair pathway is competent to correct DNA mismatches in a strand-specific manner. At present, only nicks are known to support strand discrimination, although the DNA end within the active site of replication is often proposed to serve this role. We therefore tested the competence of DNA ends or gaps to direct mismatch correction. Eight G.T templates were constructed which contained a nick or gap of 4, 28, or approximately 200 nucleotides situated approximately 330 bp away in either orientation. A competition was established in which the mismatch repair machinery had to compete with gap-filling replication and ligation activities for access to the strand discontinuity. Gaps of 4 or 28 nucleotides were the most effective strand discrimination signals for mismatch repair, whereas double strand breaks did not direct repair to either strand. To define the minimal spatial requirements for access to either the strand signal or mismatch site, the nicked templates were linearized close to either site and assayed. As few as 14 bp beyond the nick supported mismatch excision, although repair synthesis failed using 5'-nicked templates. Finally, asymmetric G.T templates with a remote nick and a nearby DNA end were repaired efficiently.     

Preparation of template plasmids for cell-free protein synthesis using a regenerated anion-exchange column

In this report, we describe how reactions of cell-free protein synthesis can be successfully conducted using plasmids prepared with regenerated anion-exchange columns. When washed, stripped, and equilibrated with appropriated buffers, regenerated columns were able to be used repeatedly to prepare plasmids with consistent yield and purity. The regenerated columns exhibited comparable performance to a fresh column with respect to the efficiency of protein synthesis using the plasmids prepared from them. Overall, we expect that the presented results will contribute significantly to economizing the technology of cell-free protein synthesis as a practical method for protein production in preparative scales.     

Strategies and open questions in solid-phase protein chemical synthesis

The review gives a large overview of the strategies used for protein synthesis by chemoselective peptide segment ligation on a solid support. It discusses also important aspects that remain to be explored to further develop the technology such as the role of the solid support on reactant diffusion rates, on ligation kinetics, as well as on the folding and functionality of the proteins attached to the solid support.     

Integrated solid-phase synthesis and purification of PEGylated protein

We describe an integrated method for solid-phase protein PEGylation and the purification of mono-PEGylated protein thus synthesized. Lysozyme was used as model protein in this study. Methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) was first immobilized on a stack of microporous hydrophobic interaction membranes housed in a module. The membrane-bound m-PEG propionaldehyde was then contacted with lysozyme solution, which also contained sodium cyanoborohydride as a reducing agent. The PEGylated lysozyme thus synthesized remained attached to the membrane, whereas unreacted protein could easily be removed from the module. PEGylated protein was then eluted from the membrane in a partially purified form using salt-free buffer. Two separate steps were thus integrated into a single process: protein PEGylation, followed by purification of mono-PEGylated protein. This solid-phase method is likely to be suitable for PEGylating any protein because it is based on the immobilization of the activated PEG and not the protein being PEGylated.     

Ecdysterone-induced protein synthesis in vitro

Ecdysterone added in vitro to wing tissue from diapausing Antheraea polyphemus pupae induced the synthesis of several epidermal cell proteins. This is one of few instances in which any steroid hormone in physiological concentrations has been able to induce specific protein synthesis in target tissue in vitro soon after hormone stimulation. Hormone-treated tissue was incubated with ³H-leucine while control tissue was incubated with ¹⁴C-leucine. Polyacrylamide gel electrophoretic distribution of labelled wing tissue proteins after ecdysterone stimulation in vitro for various periods of time was determined. The ${}^3\text{H}{}^{14}\text{C}$ ratio emphasized the areas of increased protein synthesis due to ecdysterone. These areas of increased protein synthesis were reproducible with several ecdysterone concentrations and with different incubation times. Induction of protein synthesis occurs at an earlier time period when the hormone dosage is higher, i.e. the lower the dosage, the longer it is necessary for exposure of tissue to hormone. α-Ecdysone, known to initiate the moulting process in vitro in some insect species, also induced protein synthesis. Cortisol, a mammalian steroid hormone, produced no hormone specific protein synthesis. Therefore, the results seen with ecdysterone and α-ecdysone are not the result of non-specific steroid stimulation. When no hormone was added to the incubation medium (control), only one area of the polyacrylamide gel demonstrated protein synthesis. Therefore, there are a few proteins being synthesized in vitro in wing tissue, removed from diapausing animals without hormone stimulation, which may be related to the ‘injury phenomenon’. Protein banding patterns were also determined and compared with the radioactivity profile. The study of such early biochemical and physiological responses of target tissue to hormones will aid in our understanding of a hormone's mechanism of action, since the earlier an event occurs, the more likely that it is the primary result of hormone stimulation.     

In vitro prototyping of limonene biosynthesis using cell-free protein synthesis

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-build-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 h from 0.2 to 4.5 mM (23–610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.     

The UvsY protein of bacteriophage T4 modulates recombination-dependent DNA synthesis in vitro

An in vitro system containing the T4 gene 43, 45, 44/62, 32, dda, and uvsX proteins catalyzes DNA synthesis that is dependent on the synapsis step of homologous genetic recombination. The rate of DNA synthesis in this system is highly dependent on the concentration of the uvsX recombinase (a recA-like protein). Here we report the effect of the T4 uvsY protein, a recombination accessory protein, on this reaction. Low concentrations of uvsY protein greatly stimulate DNA synthesis at low concentrations of uvsX protein, but these same concentrations inhibit DNA synthesis at high concentrations of uvsX protein. As a result, the addition of small amounts of uvsY protein lowers the minimum concentration of uvsX protein needed for the reaction 8-fold, and it lowers the uvsX protein concentration for maximum activity 4-fold. The uvsY protein can affect either the initiation or elongation phase of DNA synthesis, depending on the concentration of uvsX protein present. The implications of these results for the function of the uvsY protein in T4 DNA replication in vivo are discussed.