High-throughput purification of recombinant proteins using self-cleaving intein tags

Abstract

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.

Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin

“…This observation coincides with previous results [19,23] in which the intein C-terminal cleavage rate increased when the pH was lowered to 6.0. Therefore, we speculate that this phenomenon maybe general, and acidic conditions may benefit the self-cleavage reaction of other intein fusion proteins.…”

A Brief Reminder of Systems of Production and Chromatography-Based Recovery of Recombinant Protein Biopharmaceuticals

“…The polyhistidine- (His-) tag mixed with IMAC (Immobilized Metal Affinity Chromatography) is a commonly used method for recombinant protein purification [49]. The His-tag attached to the N or C terminus of the target protein comprises a sequence of histidine residues (typically six or more).…”

“…There is an alternative to this protease-based tag elimination system that was designed to lower costs. Specific costly proteases might be replaced with, e.g., self-cleaving intein tags [49].…”

“…The method presented here has numerous advantages; generally, it can be used for many recombinant proteins, since His-tag does not possess an electric charge and it is neither immunogenic nor toxic [51]. Moreover, the reagents are commercially available and only a single column is required [49]. On the other hand, the aforementioned heavy metals can leach out from the column, lowering the productivity of the purification.…”

Inteins as tools for tagless and traceless protein purification

“…To expand its utility, this intein has been demonstrated with rapid cloning methods, including Invitrogen's Gateway® and Topo® cloning systems, as well as ligation independent cloning (LIC) . Most recently, this intein has also been applied to high‐throughput purification methods in a 96‐well filter plate using two different affinity tags; the conventional chitin‐binding domain (CBD) affinity tag and the non‐chromatographic ELP tag …”