Purification Strategies for Soluble Proteins and Inclusion Body Proteins

    Protein purification is based on the similarities and differences among proteins. Similarities are used to remove non-protein substances, while differences are used to isolate the target protein from other proteins. The concepts of soluble proteins and inclusion body proteins are generally associated with prokaryotic expression. Soluble proteins are proteins that can dissolve in water or other solvents as small molecules. Inclusion bodies are membrane-bound, high-density, insoluble protein particles formed when exogenous genes are expressed in prokaryotic cells (particularly during high-yield expression in E. coli). Inclusion bodies typically form in the cytoplasm; if a secretory vector is used, they may form in the periplasm.

     Biologically active proteins in cells often exist in soluble form or as molecular complexes, and functional proteins are always folded into specific three-dimensional structures. Proteins within inclusions are aggregates in an unfolded state and lack biological activity. Inclusion formation is related to the rate of protein synthesis in the cytoplasm; when the concentration of newly synthesized peptides is high, there is insufficient time for folding, resulting in the formation of non-crystalline, amorphous protein aggregates. Additionally, inclusion formation is influenced by the host bacterium’s culture conditions, such as medium composition, temperature, pH, and ionic strength.

1. Extraction of Recombinant Proteins from Prokaryotic Expression

    Depending on the protein expression method, either the culture supernatant (secretory expression) or the bacterial cells (intracellular or periplasmic expression) are collected. Secretory expression, where the supernatant is collected, is less common. The collected bacterial cells are resuspended in a specific buffer and then subjected to lysis. Lysis methods include ultrasonication and high-pressure homogenization. For periplasmic expression, proteins can be fully released through osmotic pressure or repeated freeze-thaw cycles; After processing, centrifugation is required to separate the supernatant from the pellet (inclusion bodies).

    Specifically, the collected culture supernatant (secreted expression) and the supernatant separated after centrifugation are both soluble proteins; the pellet obtained after centrifugation consists of inclusion body proteins.

2. Soluble Protein Purification Strategy—CIPP Purification Strategy

    First, clarify the requirements and select an appropriate purification strategy based on those needs.

    The most classic approach in the field of chromatographic purification is the CIPP (Capture, Intermediate Purification, Polishing) purification strategy proposed by Cytiva. This theory posits that the purification of biomacromolecules requires a balance between resolution, speed, capacity, and recovery.

    The main steps are:

Capture: Separate, concentrate, and stabilize the target product while removing major impurities;

Intermediate Purification: Remove a large amount of impurities;

Polishing: The most challenging purification step, which removes hard-to-remove impurities to obtain a high-purity product.

Frequency of Use of Various Chromatographic Techniques in CIPP Purification Strategies

3. Purification Strategies for Inclusion Body Proteins

Denaturation and Renaturation of Inclusion Body Samples: Preliminary pretreatment, inclusion body lysis, and renaturation.

Preliminary Pretreatment:

Cell lysis (ultrasonication, high-pressure homogenization)

Washing of inclusion bodies to remove certain lipids, membrane proteins, and contaminating proteins

Buffer systems: PB, PBS, Tris

Surfactants: Tween 20, Triton X-100

Additives: Urea, EDTA, guanidine hydrochloride

Inclusion Body Dissolution:

Buffers: PB, PBS, Tris

Dissolution Reagents: 8 M urea, 6 M guanidine hydrochloride, 7 M guanidine hydrochloride

Reducing Agents: DTT, β-ME, TCEP

Detergents: SDS, CHAPS, N-lauroylsarcosine

Extreme pH: Low, High

Purification Methods for Denatured Proteins: Affinity purification, ion exchange purification, gel filtration purification, reverse purification

Inclusion Body Renaturation:

Renaturation Buffers: References, broad-spectrum screening

Renaturation Methods: Dilution (dialysis is considered dilution), on-column renaturation

Renaturation Conditions: Concentration, temperature, stirring rate, time, buffer exchange (and exchange method)