Recombinant DPO4-type DNA polymerase variants with amino acid substitutions that confer modified properties upon the polymerase for improved single molecule sequencing applications are provided. Such properties may include enhanced binding and accurate incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.

Mutant polymerases are provided that have improved ability to incorporate modified nucleotides, including 3′-OH unblocked reversible terminators. The mutant polymerases may be used in a variety of applications, such as for polynucleotide sequencing, primer extension reactions, and template-independent enzymatic oligonucleotide synthesis.

The present disclosure describes Bst polymerase variants which exhibit DNA-dependent DNA polymerase and reverse transcriptase activity, and methods of use thereof. The Bst polymerase variants of the disclosure may be combined with a separate enzyme having reverse transcriptase activity to amplify target RNA sequences; however, the addition of a separate enzyme having reverse transcriptase activity is not necessary for the successful amplification of RNA targets using the Bst polymerase variants of the disclosure. Target DNA sequences may also be amplified using Bst polymerase variants of the disclosure.

A modified DNA polymerase includes a DNA polymerase fragment and a G-quadruplex binding peptide fused to an N-terminal of the DNA polymerase fragment.

The present invention provides improved DNA polymerases that may be better suited for applications in recombinant DNA technologies, in particular technologies involving plant-derived samples. Among other things, the present invention provides modified DNA polymerases derived from directed evolution experiments designed to select mutations that confer advantageous phenotypes under conditions used in industrial or research applications.

Disclosed are methods for isolating polymerase complexes from a mixture of polymerase complex components. The polymerase complexes can comprise a nanopore to provide isolated nanopore sequencing complexes. The methods relate to the positive and negative isolation of the polymerase complexes and/or nanopore sequencing complexes. Also disclosed is a nucleic acid adaptor for isolating active polymerase complexes, polymerase complexes comprising the nucleic acid adaptor, and methods for isolating active polymerase complexes using the nucleic acid adaptor.

This disclosure provides, among other things, a composition comprising: comprising a fusion protein comprising: (a) a DNA polymerase; and (b) a heterologous sequence-specific DNA binding domain. A method for copying a DNA template, as well as a kit for performing the same, are also described.

A portable fluidic platform for rapid and flexible end-to-end production of recombinant protein biologics includes a bioreactor system hosting stable and robust cell-free translation systems that is fluidically integrated with modular protein separation functionalities (e.g., size exclusion, ion exchange or affinity chromatography systems) for purification of the cell-free expressed product and which are configurable for process-specific isolation of different proteins, as well as for formulation. The bioreactor utilizes lysates from engineered eukaryotic (e.g., yeast) or prokaryotic (e.g., bacterial) strains that contain factors for protein folding and posttranslational modifications. Combination of various purification modules on the same fluidic platform allows flexibility of re-routing for purification of different proteins depending on specific target requirements. Protein synthesis and purification modules are integrated into self-contained disposable fluidic cartridge that eliminates cross-contamination between runs. The platform allows for flexible production of protein biologics within 24 hours (from DNA to purified product).

The present invention relates to the preparation of human basic fibroblast growth factor by using Bacillus subtilis and endonuclease. Specifically, the present invention provides a nucleic acid construct, which comprises an insert, and the insert comprises, from the 5′ end to the 3′ end, a polynucleotide sequence that encodes a short peptide affinity tag, a trans-splicing intein derived from Anabaena and an exogenous polypeptide; and wherein the short peptide affinity tag serves as an N-terminal extein of the trans-splicing intein, and the exogenous polypeptide serves as a C-terminal extein of the trans-splicing intein. The present invention further provides an expression vector and a host cell that comprise the construct, and a method for producing and purifying foreign proteins. The expression system and method of the present invention can significantly improve the expression efficiency of biologically active exogenous proteins, reduce the generation of inclusion bodies, simplify purification steps, greatly reduce purification costs, and are especially suitable for large-scale cultivation.

Provided herein relates to nucleic acid polymerase variants and kits including the same, where the nucleic acid polymerase variant has an improved function and activity of performing template-independent nucleic acids synthesis using ribonucleotides (rNTPs) in a thermotolerant manner.