Protein Purification Optimization Kit Using SUMO Protease Elution
(Kit # 1000000258 )

Depositing Lab:   Christopher Bahl

Validation set of 96 unique, single-domain proteins that met the following criteria: structurally characterized (deposited with the Protein Data Bank), disulfide-free, mesophilic, and previously expressed in E. coli. The set was designed to be diverse in topology, size, and isoelectric points.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

Original Publication

AI Proteins, Inc. plasmids (unpublished)

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.

Kit Documentation

Please find all protocols in the manuscript.

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The Protein Purification Optimization Kit Using SUMO Protease Elution was a gift from Christopher Bahl (Addgene kit #1000000258).”

Protein Purification Optimization Kit Using SUMO Protease Elution - #1000000258

Inventory

Well	Plasmid	Resistance
A / 1	pAIP4_2D5J	Kanamycin
A / 2	pAIP4_1EDG	Kanamycin
A / 3	pAIP4_2GAS	Kanamycin
A / 4	pAIP4_1H6T	Kanamycin
A / 5	pAIP4_2H2Z	Kanamycin
A / 6	pAIP4_5IHW	Kanamycin
A / 7	pAIP4_2OKT	Kanamycin
A / 8	pAIP4_5YPV	Kanamycin
A / 9	pAIP4_1XSZ	Kanamycin
A / 10	pAIP4_6BIO	Kanamycin
A / 11	pAIP4_4WDC	Kanamycin
A / 12	pAIP4_5EF9	Kanamycin
B / 1	pAIP4_5MC7	Kanamycin
B / 2	pAIP4_3RZY	Kanamycin
B / 3	pAIP4_3K1H	Kanamycin
B / 4	pAIP4_4ZHW	Kanamycin
B / 5	pAIP4_1EYH	Kanamycin
B / 6	pAIP4_2H3L	Kanamycin
B / 7	pAIP4_4NFB	Kanamycin
B / 8	pAIP4_1AKO	Kanamycin
B / 9	pAIP4_4CFI	Kanamycin
B / 10	pAIP4_4RZ9	Kanamycin
B / 11	pAIP4_3V46	Kanamycin
B / 12	pAIP4_1UXZ	Kanamycin
C / 1	pAIP4_1TJ6	Kanamycin
C / 2	pAIP4_1BKR	Kanamycin
C / 3	pAIP4_1HKA	Kanamycin
C / 4	pAIP4_2JIK	Kanamycin
C / 5	pAIP4_5GUQ	Kanamycin
C / 6	pAIP4_1AKZ	Kanamycin
C / 7	pAIP4_2EYI	Kanamycin
C / 8	pAIP4_4QMD	Kanamycin
C / 9	pAIP4_4O6G	Kanamycin
C / 10	pAIP4_3EYE	Kanamycin
C / 11	pAIP4_4Q2S	Kanamycin
C / 12	pAIP4_2OHW	Kanamycin
D / 1	pAIP4_3ONH	Kanamycin
D / 2	pAIP4_1EW4	Kanamycin
D / 3	pAIP4_1KQX	Kanamycin
D / 4	pAIP4_3A7L	Kanamycin
D / 5	pAIP4_5KUW	Kanamycin
D / 6	pAIP4_5H28	Kanamycin
D / 7	pAIP4_3H6Q	Kanamycin
D / 8	pAIP4_5MDT	Kanamycin
D / 9	pAIP4_4QB4	Kanamycin
D / 10	pAIP4_5EIP	Kanamycin
D / 11	pAIP4_5HFD	Kanamycin
D / 12	pAIP4_2PPN	Kanamycin
E / 1	pAIP4_1NG6	Kanamycin
E / 2	pAIP4_3KT9	Kanamycin
E / 3	pAIP4_5M2Y	Kanamycin
E / 4	pAIP4_4A02	Kanamycin
E / 5	pAIP4_4REH	Kanamycin
E / 6	pAIP4_2A4D	Kanamycin
E / 7	pAIP4_1ZHV	Kanamycin
E / 8	pAIP4_5HFQ	Kanamycin
E / 9	pAIP4_3HNX	Kanamycin
E / 10	pAIP4_1TP9	Kanamycin
E / 11	pAIP4_3TOW	Kanamycin
E / 12	pAIP4_5NJO	Kanamycin
F / 1	pAIP4_1TJE	Kanamycin
F / 2	pAIP4_5Y33	Kanamycin
F / 3	pAIP4_5HIH	Kanamycin
F / 4	pAIP4_1JL1	Kanamycin
F / 5	pAIP4_1LU4	Kanamycin
F / 6	pAIP4_2NT3	Kanamycin
F / 7	pAIP4_1KAF	Kanamycin
F / 8	pAIP4_2WWE	Kanamycin
F / 9	pAIP4_1U8T	Kanamycin
F / 10	pAIP4_4ACJ	Kanamycin
F / 11	pAIP4_5XBC	Kanamycin
F / 12	pAIP4_1UKF	Kanamycin
G / 1	pAIP4_3ILS	Kanamycin
G / 2	pAIP4_1FL0	Kanamycin
G / 3	pAIP4_1ELK	Kanamycin
G / 4	pAIP4_2JHX	Kanamycin
G / 5	pAIP4_2RH3	Kanamycin
G / 6	pAIP4_2H7W	Kanamycin
G / 7	pAIP4_3CX2	Kanamycin
G / 8	pAIP4_2AWG	Kanamycin
G / 9	pAIP4_4HS5	Kanamycin
G / 10	pAIP4_6F2Q	Kanamycin
G / 11	pAIP4_3KB5	Kanamycin
G / 12	pAIP4_5F66	Kanamycin
H / 1	pAIP4_3OBS	Kanamycin
H / 2	pAIP4_1T3Y	Kanamycin
H / 3	pAIP4_4IPC	Kanamycin
H / 4	pAIP4_3D1B	Kanamycin
H / 5	pAIP4_3S60	Kanamycin
H / 6	pAIP4_2ESK	Kanamycin
H / 7	pAIP4_4J5Q	Kanamycin
H / 8	pAIP4_6WEY	Kanamycin
H / 9	pAIP4_1YW5	Kanamycin
H / 10	pAIP4_3W4Q	Kanamycin
H / 11	pAIP4_6IYE	Kanamycin
H / 12	pAIP4_9C74	Kanamycin

Data calculated @ 2025-06-20