Neq DNA Polymerase Expression via Split Mini-Intein Trans-Splicing

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Solution Overview

Problem

There is no reported method for expressing, purifying, or using thermostable B-type DNA polymerase from the hyperthermophilic nanoarchaeon Nanoarchaeum equitans, which lacks intein and possesses a split mini-intein, for applications like PCR, due to the lack of understanding and utilization of its genetic and biochemical properties.

Innovation Solution

A recombinant vector system is developed to express and purify active Neq DNA polymerase by linking the extein-encoding regions of the large and small fragments of Neq DNA polymerase, enabling the production of a thermostable enzyme with high DNA polymerization and proofreading activity, suitable for PCR and PCR in the presence of dUTP.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If the split mini-intein structure of Neq DNA polymerase is utilized for expression, then the enzyme can be produced as separate large and small fragments, but the fragments alone lack DNA polymerization activity and require complex reconstitution processes

Engineering Contradiction:

ImproveExpression of Neq DNA polymerase as separate fragmentsVSAvoidComplexity of reconstitution process

Core Design Contradiction:

Ease of manufactureVSDevice complexity

Solution Approach 1:

The Neq DNA polymerase gene is divided into two separate genes (Neq L and Neq S) that encode the large and small fragments respectively. These fragments are expressed separately in E. coli and then reconstituted to form the active enzyme, allowing simplified expression of each fragment while maintaining the ability to produce functional polymerase through controlled assembly

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The split mini-intein sequence acts as an intermediary that facilitates the self-splicing and reconstitution of the large and small fragments. The intein domains in each fragment mediate the formation of the active enzyme through protein splicing, eliminating the need for complex external reconstitution procedures

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If thermostable DNA polymerases from other hyperthermophiles are used for high-fidelity PCR, then proofreading activity is achieved, but the enzymes require complex purification processes and are not yet available from Nanoarchaeum equitans

Engineering Contradiction:

ImproveProofreading activity and high-fidelity PCRVSAvoidPurification process complexity

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The Neq DNA polymerase system utilizes self-splicing intein domains that automatically mediate the assembly of active enzyme from the expressed large and small fragments. This self-reconstituting property eliminates the need for complex purification and assembly procedures, making the enzyme readily available while maintaining high-fidelity proofreading activity

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The enzyme system is optimized for high-temperature operation with optimal activity at 70°C and pH 8.0, matching the hyperthermophilic conditions of Nanoarchaeum equitans. These parameter optimizations enhance both the reliability of PCR reactions and the ease of manufacture by allowing simple heat-based purification methods

Inventive Principle:

Principle #35Parameter changes

3Productivity

If the complete Neq DNA polymerase sequence is expressed as a single polypeptide, then active enzyme is produced directly, but the gene structure is complex with embedded split mini-intein sequences that complicate cloning and expression

Engineering Contradiction:

ImproveDirect production of active enzymeVSAvoidGene structure complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The complex single-gene structure with embedded split mini-intein sequences is segmented into two separate, simpler genes (Neq L and Neq S). Each gene contains an extein-encoding region and an intein-encoding region that are independently cloned and expressed, significantly simplifying the cloning process while enabling direct production of active enzyme through controlled reconstitution

Inventive Principle:

Principle #1Segmentation

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The active Neq DNA polymerase exhibits high DNA polymerization and proofreading activity, making it suitable for various nucleic acid amplification reactions, including PCR, with optimal activity at 70°C and pH 8.0, and stability at high temperatures, thus addressing the need for a thermostable enzyme from Nanoarchaeum equitans.

Implementation Method 1

the large and small fragments of which are spliced to form an active DNA polymerase

Methodology Applied

Scientific EffectProtein trans-splicing:

Implementation Method 2

Deoxyribonucleic acid polymerases (DNA polymerases; E.C. number 2.7.7.7) are enzymes that synthesize DNA in the 5′ to 3′ direction on template DNA

Methodology Applied

Scientific EffectDNA polymerization:

Implementation Method 3

these enzymes have 3′→5′ exonuclease activity (this activity is known as proofreading activity) along with DNA polymerization activity

Methodology Applied

Scientific Effect3′→5′ exonuclease activity:

Data Source

PatentUS7749732B2Method for preparing active nanoarchaeum equitans DNA polymerase and the active DNA polymerase prepared by the method

Publication Date: 2010.07.06 RES & BUSINESS FOUND SUNGKYUNKWAN UNIV

AI summary

Disclosed are a method of preparing an active Nanoarchaeum equitans B-type DNA polymerase (Neq DNA polymerase), an active Neq DNA polymerase prepared according to the method, and a polymerase chain reaction (PCR) using the active Neq DNA polymerase. The active Neq DNA polymerase may be used in various nucleic acid polymerization reactions, such as PCR.