Process for reconstituting the polymerase III* and other subassemblies of E. coli DNA polymerase III holoenzyme from peptide subunits

• US 6,221,642 B1
• Filed: 03/31/1999
• Issued: 04/24/2001
• Est. Priority Date: 08/31/1994
• Status: Expired due to Fees

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: OʼDonnell, Michael E.
• Primary Examiner(s): Weber, Jon P.
• Application Number:
  US09/282,917
• Time in Patent Office: 755 Days
• Field of Search: 435/193, 435/194
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The process of the invention provides for the reconstitution of the polymerase III* subassembly, Pol III*, of E. coli DNA polymerase III holoenzyme from substantially pure peptide subunits. In the first of two general schemes in which the subunits are added in a specified order, γ and τ are premixed before addition of δ and δ′. In the second general scheme, δ′ is first assembled onto γ (or τ); then the excess δ′ is removed before adding τ (or γ), following which δ is added. Reconstituted Pol III* had the same subunit composition as purified natural Pol III*, as well as similar activity. Other smaller subassemblies of the polymerase III holoenzyme may also be reconstituted by the process of the invention.

Claims

1. A process for reconstituting the γ

-less Pol III* subassembly from E. coli from subunits τ

, α

, ε

, θ

, δ

, δ

′

, χ

, and Ψ

, which comprises;

combining τ

, α

, ε

, θ

, δ

, δ

′

, χ

, and Ψ

, under conditions effective to form said γ

-less Pol III* subassembly, wherein said combining comprises;

combining χ

, Ψ

, δ

, and δ

′

under conditions effective to form a χ

Ψ

δ

δ

′

complex;

combining said χ

Ψ

δ

δ

′

complex with τ

under conditions effective to form a τ

χ

Ψ

δ

δ

′

complex;

combining said τ

χ

Ψ

δ

δ

′

complex with α

, ε

, θ

; and

recovering said γ

-less Pol III* subassembly.

2. A process for reconstituting an α

τ

χ

Ψ

δ

δ

′

subassembly from E. coli from subunits τ

, α

, δ

, δ

′

, χ

, and Ψ

, which comprises;

combining τ

, α

, δ

,δ

′

, χ

, and Ψ

in any order under conditions effective to form said α

τ

χ

Ψ

δ

δ

′

subassembly and

recovering said α

τ

χ

Ψ

δ

δ

′

subassembly.

3. A process of claim 2, wherein said combining comprises:

combining τ

, χ

, Ψ

, δ

, and δ

′

under conditions effective to form a τ

χ

Ψ

δ

δ

′

complex; and

combining said τ

χ

Ψ

δ

δ

′

complex with α

to form said α

τ

χ

Ψ

δ

δ

′

subassembly.

4. A process for reconstituting an α

τ

δ

δ

′

subassembly from E. coli from τ

, α

, δ

, and δ

′

, which comprises;

combining τ

, α

, δ

, and δ

′

in any order under conditions effective to form said α

τ

δ

δ

′

subassembly and

recovering said α

τ

δ

δ

′

subassembly.

5. A process of claim 4, wherein said combining further comprises:

combining τ

, δ

, and δ

′

under conditions effective to form a τ

δ

δ

′

complex; and

combining said τ

δ

δ

′

complex with α

under conditions effective to form said α

τ

δ

δ

′

subassembly.

6. A process for reconstituting an α

ε

θ

τ

δ

δ

′

subassembly from E. coli from subunits τ

, α

, ε

, θ

, δ

, and δ

′

, which comprises;

combining τ

, α

, ε

, θ

, δ

, and δ

′

in any order under conditions effective to form said α

ε

θ

τ

δ

δ

′

subassembly and

recovering said α

ε

θ

τ

δ

δ

′

subassembly.

7. A process of claim 6, wherein said combining further comprises:

combining τ

, δ

, and δ

′

under conditions effective to form a τ

δ

δ

′

complex; and

combining said τ

δ

δ

′

with α

, ε

, and θ

under conditions effective to form said α

ε

θ

τ

δ

δ

′

complex.