Improved self-cleaving precipitation tags for efficient column free bioseparations.

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.

The Inducible Intein-Mediated Self-Cleaving Tag (IIST) System: A Novel Purification and Amidation System for Peptides and Proteins.

An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH).

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

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 (ßGal) 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 applications: from protein purification and labeling to metabolic control methods.

The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification.

Column-Free Purification Methods for Recombinant Proteins Using Self-Cleaving Aggregating Tags.

Conventional column chromatography processes to purify recombinant proteins are associated with high production costs and slow volumetric throughput at both laboratory and large scale. Non-chromatographic purifications based on selective aggregating tags have the potential to reduce costs with acceptable protein yields. A significant drawback, however, is that current proteolytic approaches for post-purification tag removal after are expensive and non-scalable. To address this problem, we have developed two non-chromatographic purification strategies that use either the elastin-like polypeptide (ELP) tag or the ß-roll tag (BRT17) in combination with an engineered split intein for tag removal. The use of the split intein eliminates premature cleavage during expression and provides controlled cleavage under mild conditions after purification. These self-cleaving aggregating tags were used to efficiently purify ß-lactamase (ß-lac), super-folder green fluorescent protein (sfGFP), streptokinase (SK) and maltose binding protein (MBP), resulting in increased yields compared to previous ELP and BRT17-based methods. Observed yields of purified targets for both systems typically ranged from approximately 200 to 300 micrograms per milliliter of cell culture, while overall recoveries ranged from 10 to 85 percent and were highly dependent on the target protein.

Purification of Antibacterial CHAPK Protein Using a Self-Cleaving Fusion Tag and Its Activity Against Methicillin-Resistant Staphylococcus aureus.

Therapeutic LysK-CHAP is a potent anti-staphylococcal protein that could be utilized as an antibiotic substitute. Intein-mediated protein purification is a reasonable and cost-effective method that is most recently used for recombinant therapeutic protein production. Intein (INT) is the internal parts of the protein that can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. INT sequence and their characteristic of self-cleavage by thiol induction, temperature, and pH changes are used for protein purification. The current study presents the expression of CHAPK262 domain of LysK gene that is fused with INT/chitin-binding sequence while evaluating its purification procedure and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The coding gene sequence of LysK-CHAP (CHAPK262) in pET22-b was amplified with polymerase chain reaction (PCR); the digested product was then cloned into the pTXB1 vector. Electrophoresis confirmed the cloning accuracy of the gene. The pTXB1-CHAPK262 plasmid was transformed to the Escherichia coli ER2566 (E. coli ER2566) expression strain and analyzed for expression of the recombinant protein by SDS-PAGE and Western blotting methods. Finally, CHAPK262 was purified by chitin affinity column using INT tag technology and confirmed by SDS-PAGE. Lytic activity of the purified protein was investigated by disk diffusion method. Cloning of CHAPK262 into the pTXB1 vector, which comprised INT/chitin-binding sequence, was successfully achieved. The SDS-PAGE data also revealed successful expression of the CHAPK262-INT fusion protein and Western blotting method validated the accuracy of the protein. Moreover, purification of CHAPK262 protein was induced by dithiothreitol (DTT) and confirmed by SDS-PAGE. Finally, inhibition zone in MRAS culture medium confirmed antibacterial activity of the protein. Application of intein-mediated antibacterial protein is an appropriate and streamlined method for one-step purification of CHAPK262 as a therapeutic and antibacterial protein. Self-cleaving tags like intein are cost-effective and could be used as a proper purification method for industrial purposes.