Recombinant Human interleukin-16 protein (IL16), partial (Active)

What is the biological function of human IL-16?

Human IL-16 primarily functions as a chemoattractant for CD4+ cells, including lymphocytes, monocytes, and eosinophils. It stimulates migratory responses in these cell populations, with significant migration observed at concentrations starting from 5 ng/ml . IL-16 also primes CD4+ T-cells for IL-2 and IL-15 responsiveness and induces the expression of the interleukin-2 receptor (CD25) on T-lymphocytes . Additionally, IL-16 modulates T lymphocyte growth and can suppress the replication of human immunodeficiency virus (HIV) and simian immunodeficiency viruses (SIV) in vitro .

What is the structure of recombinant human IL-16 protein?

Recombinant human IL-16 protein is typically a monomeric protein with a molecular weight of approximately 13.4 kDa . While the naturally occurring biologically active IL-16 was originally proposed to be a homotetramer of 14 kDa chains, recombinant E. coli-derived IL-16 is predominantly present as a monomer . The protein consists of 130 amino acid residues and represents the C-terminal fragment of the full pro-IL-16 precursor protein .

What is the amino acid sequence of recombinant human IL-16?

The amino acid sequence of recombinant human IL-16 is: MPDLNSSTDS AASASAASDV SVESTAEATV CTVTLEKMSA GLGFSLEGGK GSLHGDKPLT INRIFKGAAS EQSETVQPGD EILQLGGTAM QGLTRFEAWN IIKALPDGPV TIVIRRKSLQ SKETTAAGDS . This sequence corresponds to the C-terminal fragment (typically positions 1203 to 1332 or Pro2-Ser130) of the full-length pro-IL-16 precursor protein .

What expression systems are used for producing recombinant human IL-16?

Recombinant human IL-16 is typically expressed in Escherichia coli (E. coli) expression systems . This bacterial expression system allows for the production of the biologically active C-terminal fragment of IL-16 with high purity (≥95%) and low endotoxin levels (≤1.00 EU/μg) . The resulting protein is suitable for various applications including SDS-PAGE analysis and functional studies .

How is the purity of recombinant IL-16 preparations assessed?

The purity of recombinant IL-16 preparations is typically determined by SDS-PAGE analysis under both reducing and non-reducing conditions . A purity level of ≥95% is standard for commercial preparations . Additional quality control measures include endotoxin testing using kinetic LAL (Limulus Amebocyte Lysate) assays, with acceptable levels being ≤1.00 EU/μg . Functional assays, such as chemotaxis assays with primary human T cells, are also used to confirm the biological activity of the purified protein .

What is the relationship between pro-IL-16 and the active IL-16 fragment?

Pro-IL-16 is a 631 amino acid residue precursor protein that lacks a signal peptide . The biologically active IL-16 is derived from the C-terminus of this precursor through proteolytic cleavage, likely at aspartate residue 510, yielding a 121-130 amino acid residue protein . This cleavage is thought to be mediated by proteases present in or on activated CD8+ cells . While pro-IL-16 is detected in cell lysates, the secreted, biologically active form is the cleaved C-terminal fragment . Interestingly, pro-IL-16 also has distinct functions, including roles in cell cycle progression in T-cells and transcriptional regulation .

How should recombinant IL-16 be reconstituted and stored for optimal activity?

For optimal activity, lyophilized recombinant IL-16 should be centrifuged before opening the vial . It should be reconstituted in a sterile solution, typically containing 10 mM sodium phosphate at pH 7.5 . Gentle pipetting and washing down the sides of the vial are recommended during reconstitution to ensure complete solubilization of the protein . After reconstitution, the protein should be stored at appropriate temperatures (typically -20°C to -80°C) in small aliquots to avoid repeated freeze-thaw cycles that could compromise its activity.

What functional assays are used to confirm IL-16 biological activity?

The biological activity of recombinant human IL-16 is primarily assessed using chemotaxis assays with primary human T cells . A significant increase in migration over basal levels is expected in response to IL-16, typically starting at concentrations of 5 ng/ml . The activity is determined by the protein's ability to induce chemotaxis of CD4+ T cells specifically . Additional functional assays may include measuring IL-2 receptor (CD25) upregulation on T lymphocytes, assessment of T cell proliferation in the presence of IL-2 or IL-15, and HIV replication inhibition assays .

What is the role of IL-16 in HIV pathogenesis and potential therapeutic applications?

IL-16 has been shown to suppress HIV-1 replication in vitro by repressing the transcription of the HIV-1 long terminal repeat . This finding suggests potential therapeutic applications in HIV research. Studies have examined differences in HIV-1 pathogenesis in different tissues infected with CCR5- and CXCR4-tropic HIV-1, with IL-16 potentially playing a role in these differences . The exact mechanisms by which IL-16 inhibits HIV replication and its potential as a therapeutic target require further investigation. Researchers studying this aspect should consider the interplay between IL-16, CD4 expression, and viral entry mechanisms.

How does IL-16 influence immune cell recruitment in inflammatory conditions?

IL-16 functions as a chemoattractant for multiple immune cell types, including CD4+ lymphocytes, monocytes, eosinophils, dendritic cells, and Langerhans cells . This chemoattractant activity suggests an important role for IL-16 in immune cell recruitment during inflammatory responses. Research indicates that IL-16 may be present in biological fluids associated with allergic responses, such as breast milk from allergic mothers . Additionally, IL-16 can induce pro-inflammatory cytokine production in monocytes, potentially amplifying inflammatory responses . When designing experiments to study IL-16's role in inflammation, researchers should consider the complex interplay between IL-16 and other cytokines, particularly in tissue-specific inflammatory contexts.

What are the cellular sources of IL-16 and how is its expression regulated?

IL-16 is primarily produced by CD4+ and CD8+ T cells . The pro-IL-16 mRNA is expressed in various tissues including spleen, thymus, lymph nodes, peripheral leukocytes, bone marrow, and cerebellum . The regulation of IL-16 expression appears to be linked to T cell activation, with the active IL-16 being released following proteolytic cleavage of pro-IL-16 by proteases present in or on activated CD8+ cells . The gene for IL-16 precursor has been localized to chromosome 15 . Researchers studying IL-16 expression should consider both transcriptional regulation of the pro-IL-16 gene and post-translational processing mechanisms that generate the active cytokine.

What are common issues in measuring IL-16 biological activity and how can they be addressed?

When measuring IL-16 biological activity, researchers may encounter challenges related to the protein's low affinity for CD4 and its predominantly monomeric form in recombinant preparations compared to the potentially tetrameric native form . To address these issues, optimization of protein concentration is crucial, as recombinant IL-16 exhibits chemotactic activity for lymphocytes at high concentrations but lacks chemotactic activities for monocytes at lower doses . Additionally, ensuring proper protein folding during reconstitution and maintaining sterile conditions are essential for reliable activity measurements. Inclusion of appropriate positive controls in chemotaxis assays is recommended to validate experimental systems.

How can researchers distinguish between the effects of pro-IL-16 and the active IL-16 fragment in experimental systems?

Distinguishing between the effects of pro-IL-16 and the active IL-16 fragment requires careful experimental design. Researchers can use antibodies specific to different regions of the protein to differentiate between the full-length precursor and the cleaved C-terminal fragment in immunological assays. Additionally, comparing the effects of recombinant active IL-16 fragment with those of full-length pro-IL-16 in parallel experiments can help delineate their distinct functions. It's important to note that pro-IL-16 appears to function primarily intracellularly as a scaffold for transcriptional regulation and cell cycle control , while the active C-terminal fragment functions extracellularly as a cytokine and chemoattractant .

What considerations are important when studying IL-16 in different experimental models?

When studying IL-16 in different experimental models, researchers should consider several factors: (1) Expression of CD4 on target cells, as IL-16's effects are CD4-dependent ; (2) The presence of proteases capable of generating active IL-16 from pro-IL-16 in the model system ; (3) The interplay between IL-16 and other cytokines, particularly IL-2 and IL-15, with which IL-16 acts synergistically ; (4) Species-specific differences in IL-16 structure and function when using animal models; and (5) The tissue-specific expression patterns of IL-16 and its receptors, as expression has been detected in various tissues including immune organs and the cerebellum . Cross-validation using multiple experimental approaches is recommended to confirm IL-16-mediated effects.