DOWNSTREAM PROCESSING

PRESENTATION

Tag-mediated single-step purification

and immobilization of
recombinant proteins toward
protein-engineered advanced
materials
VINAYA SRI S -21B241
• The review aims to showcase the progress made over the past
five years, emphasizing the development of efficient single-step
purification and immobilization strategies.

• Key points covered include the unique abilities of protein

purification/immobilization tags to establish specific interactions
with solid materials, leading to the creation of tailored functional
materials and hybrid materials.
• Affinity tags are highlighted for their wide material-binding
capabilities, making them ideal for protein purification, while
covalently binding tags excel in long-term protein
immobilization but are limited to protein-based materials.

• Additionally, the review touches on self-aggregating tags, which

when combined with other tags, enable the development of protein-
engineered materials with self-assembling, flexible, and responsive
properties. Overall, these advancements signify a promising future for
eco-friendly functional materials and their diverse applications.
INTRODUCTION:

• In recent years, there has been a growing demand for materials that
are biocompatible, bioactive, and environmentally friendly, leading to
collaborations between material science and genetic engineering
fields.
• This has resulted in the development of bioinspired engineered
materials that combine the advantages of natural and synthetic
materials.
•Protein-Engineered Materials: These materials combine advantages from
natural and synthetic materials, offering biofunctionality and customizability.

•Genetic Engineering for Designing Proteins: Through genetic engineering

and recombinant DNA technology, precise amino acid sequences can be
designed and inserted into host organisms to produce desired peptides/proteins.

•Applications in Various Industries: Recombinant proteins find applications in

biotechnology (biosensing, biocatalysis, bioremediation), biomedicine (drug
delivery, vaccine development, implants), food, textiles, and paper industries.
•Host Organisms for Recombinant Protein Production: Prokaryotic (e.g., E. coli, Bacillus
subtilis) and eukaryotic hosts (yeast, animal cells) are engineered for protein production, each with
advantages and limitations.

•Advantages of E. coli Expression System: E. coli is preferred for its efficiency in incorporating
foreign DNA, fast growth, and cost-effectiveness, despite challenges in purification due to
intracellular protein production.

•Overcoming Limitations with Fusion Tags: Fusion partners or "tags" (protein domains, small
peptides, enzymes) are fused to target proteins to enhance expression, solubility, and purification,
leading to high-yield production.

•Targeted Tag Removal with Virus Proteases: Discovery of virus proteases allows for the
targeted removal of fusion tags, improving the biological activity of the final protein product.
PROTEIN PURIFICATION/ IMMOBILIZATION
TAGS
1. Protein purification is crucial in biotechnology and biomedicine.
2. Affinity protein purification uses specific ligands for tag binding.
3. Peptides hold potential as affinity tags because they offer low toxicity and stability.
4. Tag-ligand interaction strength determines purification efficiency.
5. Interactions can be reversible (non-covalent) or irreversible (covalent).
6. KD values measure the strength of reversible interactions.
7. Affinity tags promote reversible binding; covalent tags facilitate irreversible binding.
8. Solid-binding peptides (SBPs) are selective and bind with high affinity.
9. Tag/Catcher system enables covalent immobilization under physiological conditions.
10.Alternative purification tags include magnetic and self-aggregating tags for innovative
approaches.
AFFINITY TAGS
• Poly-His-tagging is widely used for protein purification due to its versatility and cost-effectiveness.
• His-tag displays affinity to matrices beyond metal ions, such as iron oxide nanoparticles.
• Enhanced IMAC matrices improve selectivity for His-tag via surface epitope molecular imprinting.
• His-tag is versatile, used in diverse purification and immobilization strategies, including binding to nickel
ions.
• Alternative peptides like Glu6 and (HE)7 offer affinity to iron oxide, expanding purification options.
• (HE)7 and NCTR25 tags offer improved purification efficiency and protein solubility compared to His-tag.
• Cationic poly-arginine tag provides an alternative to His-tag for purification via cation exchange resin.
• Tryptophan-based tags (NW)3 and (WF)3 show promise in affinity-based purification systems.
• Cost-effective purification systems like Car9 and SB7 exhibit high affinity to silica, reducing purification
costs.
• Novel affinity matrices based on dextran and streptavidin-binding peptide tags enable efficient protein
purification.
AFFINITY TAGS
• Carbohydrate-binding Modules (CBMs) offer specific binding affinity
to carbohydrate polymers, used in various applications.
• Compact CBM64 variants show versatility and extensive binding
capacity to cellulose-based matrices.
• MhPA14-tag and CL7/Im7 systems offer superior affinity and
purification efficiency compared to traditional methods.
• Reusable affinity matrices like ABD and DBD facilitate reversible or
irreversible immobilization onto Sephadex.
• Novel ALFA-tag and MIPs overcome limitations of conventional
epitope tags, promising efficient protein detection and purification.
Non-covalent molecular imprinting technology to create functional
biosynthetic polymers via conjugation of natural and synthetic
moieties.
Covalently binding tags
• Covalently binding tags enable rapid, specific, and irreversible protein
binding to ligands.
• Systems like HaloTag and SNAP-tag catalyze covalent attachment to
synthetic ligands for immobilization.
• The HaloTag system offers efficient protein immobilization and
purification from crude extracts.
• A novel approach based on splitting bacterial domains into Tag peptides
and Catcher proteins facilitates irreversible protein conjugation.
• The Tag/Catcher system allows rapid, irreversible protein linkage
without the need for chemical modification.
Covalently binding tags
• SpyTag/SpyCatcher and other engineered pairs enable fast covalent
bonding for protein assembly.
• Orthogonal Tag/Catcher pairs like SnoopTag/SnoopCatcher offer
versatility in protein architecture construction.
• Chemoenzymatic approaches like Sortase A-mediated immobilization
enable oriented protein immobilization on solid supports.
• Microbial transglutaminases (MTGs) catalyze covalent bond
formation for site-specific protein immobilization.
• New reactive tags and MTGs provide improved tools for efficient
covalent immobilization and biomaterial formation
Alternative purification tags
1.Alternative purification systems aim to simplify the process
economically and ecologically.
2.Self-aggregating tags induce protein aggregation, allowing
separation from crude extracts.
3.Elastin-like polypeptide (ELP) tags form reversible precipitates
with proteins, facilitating purification.
4.Tandem repeats of affinity ligands can enhance the capture
efficiency of ELP-tagged proteins.
5.pH-responsive CspB50 tags enable reversible precipitation at
acidic pH for column-free purification.
Alternative purification tags
1.ELP and CspB50 tags offer non-chromatographic purification methods with
comparable yields and purity.
2.Magnetic-absorption-based purification tags, like MagR, allow easy
recovery of proteins from crude extracts.
3.MagR-tagged proteins are absorbed to magnetic beads, enabling single-step
purification and immobilization.
4.MagR variants, like dMagR and clMagR, offer improved thermostability
and pH tolerance for enzyme immobilization.
5.Magnetic interactions between MagR variants and recovery supports are
less affected by chemical environments, maintaining enzyme activity over a
broad range of conditions.
Multiple protein tag fusion used to generate bioinspired
composites.
Self-assembly and functionalization of engineered protein-based
hydrogels.
Recent advances and applications
1.Nanoparticles Advantages: Magnetic nanoparticles enable single-step purification,
scalable, and easily automated processes.
2.Surface Functionalization: Coating MNPs enhances stability and specificity,
improving binding efficiency.
3.Innovative Matrices: MIPs and protein-based hydrogels offer selectivity and
affinity, alternative to traditional methods.
4.Hybrid Materials: Combining materials at the nano/molecular scale leads to
superior properties for various applications.
5.Protein-based Self-assembly: Recombinant proteins self-assemble for enhanced
functionality, simplifying processes.
6.Challenges and Future: Understanding complex interactions remains a challenge,
requiring further research for optimization.
• The sustainable production of proteins/peptides crucial for
these materials relies on advanced recombinant protein
production and purification/immobilization technologies.

• Tag-mediated strategies for protein

purification/immobilization have emerged as eco-friendly
and cost-effective alternatives, simplifying the fabrication
process of protein-engineered materials.
• Hybrid affinity-covalently binding tags have bridged this gap,
enabling efficient one-step purification/immobilization and
fostering the development of innovative protein-engineered
materials. Self-aggregating responsive tags have also
contributed to materials with self-assembling and flexible
properties.

• Advanced covalently binding tags like those in the Tag/Catcher

system have enhanced the design of stable and multifunctional
protein architectures adaptable to diverse applications.