Protein Purification Optimization Kit Using SUMO Protease Elution

(Kit #1000000258)

Depositing Lab : Christopher Bahl

96개의 고유한 single-domain proteins로 구성된 검증 세트로 다음과 같은 특징이 있습니다.

본 키트는 96-well plate format의 bacterial glycerol stocks으로 제공됩니다.

Description

We have developed a novel, highly automated, high-throughput process for the expression and purification of recombinant protein from E. coli.

To assess the generalizability of our method, we selected a validation set of 96 unique, single-domain proteins that met the following criteria: structurally characterized (deposited with the Protein Data Bank), disulfide-free, derived from mesophilic organisms, and previously expressed in E. coli. The set was designed to capture a wide range of structural topologies, molecular sizes, and isoelectric points.

This collection serves two purposes. First, the collection serves to benchmark high-throughput protein production methods and demonstrate broad applicability and will serve as a useful reference standard for others who are doing the same in the future. Second, because the structure for each of the proteins has been determined, they are a great tool for measuring different protein properties and can serve as an excellent set for those who are developing computational methods to assess and alter protein solution behavior; having a large set of very different proteins will provide an essential capability to enable this research.

Each gene of interest was cloned into a pET expression vector under the control of a T7 promoter. Constructs were designed to include an N-terminal His-tagged SUMO fusion to enable binding to Ni-NTA magnetic beads, with specific elution via SUMO protease. To ensure optimal protease cleavage, a Ser-Gly dipeptide was introduced at the N-terminus of the native protein sequence.

Expression was carried out in E. coli Lemo21 cells using autoinduction media at 25 °C for 16 hours. Cells were harvested and lysed by sonication. Purification was performed using Ni-charged magnetic beads, and proteins were eluted with a thermostable, engineered SUMO protease derived from Chaetomium thermophilum.

Eluted proteins were analyzed for yield and folding. Protein concentration was quantified using Qubit assays, and structural integrity was evaluated via circular dichroism spectroscopy.

The complete process—from DNA assembly to purified protein—was conducted using the automated platform in triplicate (biological replicates), with a turnaround time of four days.

Original Publication

AI Proteins, Inc. plasmids (unpublished)

Contents of Kit