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Fusion polypeptides and uses thereof

A technology of fusing polypeptides and combining polypeptides, which is applied in the field of molecular biology and can solve the problems of no research on methods to improve the activity of ligases, etc.

Inactive Publication Date: 2012-07-18
MASSEY UNIVERISTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite this, little research has been done to improve the activity of ligases such as DNA ligase

Method used

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  • Fusion polypeptides and uses thereof
  • Fusion polypeptides and uses thereof
  • Fusion polypeptides and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0383] The construction of embodiment 1-plasmid and the production of fusion polypeptide

[0384] This example describes the construction of plasmids for the production in E. coli of fusion polypeptides comprising T4 DNA ligase (ligase) or E. coli ligase (LigA) fused to various DNA-binding polypeptides, as shown in the table below listed in 1. The orientation of polypeptides comprising ligase activity and DNA-binding activity relative to each other is indicated by an order wherein the polypeptides are described by the name of the fusion polypeptide, e.g. p50-ligase refers to a fusion polypeptide whose comprising a p50 DNA-binding polypeptide fused to the N-terminus of a T4 DNA ligase polypeptide (optionally via a linking polypeptide), whereas ligase-p50 refers to a fusion polypeptide comprising a T4 DNA-binding polypeptide fused to the N-terminus of a p50 DNA-binding polypeptide Enzyme polypeptide (again, optionally via linker polypeptide).

[0385] Table 1: Ligase-DNA bindi...

Embodiment 2-T4

[0404] The analysis of the connecting activity of embodiment 2-T4DNA ligase fusion protein

[0405] Gel-Based Activity Assays

[0406] For sticky-end ligation, by amplifying plasmid pCA24N-ompC with the aid of primers pCA24N.for (5'-GATAACAATTTCACACAGAATTCATTAAAGAG-3' [SEQ ID No. 19]) and pCA24N.rev (5'-CCCATTAACATCACCATCTAATTCAAC-3' [SEQ ID No. .20]), to generate a 1,277bp PCR product. The PCR product was cleaved with the aid of the restriction enzyme Spel, resulting in two linear fragments of very similar size (638bp and 639bp). The two products of the cleavage reaction were co-purified and incubated in the presence or absence of the various ligase proteins. 150 ng of substrate DNA was incubated with 20 pmol of enzyme for 10 min at 16°C. The reaction was terminated by heating to 65°C for an additional 15 minutes. Ligase activity was determined by purifying samples using QiagenMinElute columns and then running them on agarose gels. Activity was measured as the appearance...

Embodiment 3

[0414] Example 3-Analysis of the linking activity of Escherichia coli LigA fusion protein

[0415] Gel-Based Activity Assays

[0416] For sticky-end ligation, 170 ng of Spel-digested ompC substrate (as described in Example 2) was incubated with 20 pmol of each LigA enzyme for 17 hours at 16°C. Reactions were heat-killed (65°C, 15 minutes) and then run on agarose gels. In addition to LigA-p50 and p50-LigA fusion polypeptides, native LigA ligase and three control samples were assayed.

[0417] Positive control - commercially available T4 DNA ligase (Fermentas)

[0418] Negative control - no ligase added

[0419] · Commercial control - 1 μl E. coli LigA (New England Biolabs)

[0420] For blunt-end ligation, 120 ng of SfiI / SmaI-digested tig substrate (as described in Example 2) was incubated with 20 pmol of each enzyme for 17 hours at 16°C. Reactions were heat killed (65°C, 15 minutes) and then run on an agarose gel.

[0421] result

[0422] The sticky-end and blunt-end lig...

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Abstract

The invention relates to fusion polypeptides comprising a polynucleotide-binding domain, such as a DNA-binding domain, and a ligase domain, such as a DNA ligase domain, methods for the production of such fusion polypeptides, and uses of the fusion polypeptides, for example in a range of molecular biological techniques as well as applications in the diagnostics, protein production, pharmaceutical, nutraceutical and medical fields.

Description

technical field [0001] The present invention relates to the field of molecular biology, especially fusion polypeptide and its application. In particular, the invention relates to fusion polypeptides comprising a polynucleotide-binding domain, such as a DNA-binding domain, and a polynucleotide-ligase domain, such as a DNA ligase domain. Also provided are methods for producing the above-mentioned fusion polypeptides, as well as applications of the fusion polypeptides, for example, in the field of molecular biology techniques. Background technique [0002] Polynucleotide ligases, such as DNA ligases, belong to the most widely used molecular biological enzymes. A wide variety of molecular biology methods rely on the efficient activity of DNA ligases. [0003] Applications of ligases from a range of sources have been investigated: in molecular biology, and also in a growing number of industries in which molecular biology methods are employed, including the medical, pharmaceutic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/37C07K14/005C07K14/47C07K14/195C12N9/00C12N15/10C12N15/52C12N15/62
CPCC07K2319/81C12N9/93C07K2319/80C07K2319/85C07K14/005C07K14/195C07K19/00C12N15/62
Inventor 韦恩·迈克尔·帕特里克罗伯特·亨利·威尔逊
Owner MASSEY UNIVERISTY
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