CFB (260-764) Human, Sf9

What is CFB (260-764) Human, Sf9 and what are its key characteristics?

CFB (260-764) Human, Sf9 is a recombinant form of human Complement Factor B, specifically containing amino acids 260-764 of the native protein, produced in Spodoptera frugiperda (Sf9) insect cells using baculovirus expression systems. This recombinant protein is a single, glycosylated polypeptide chain with a molecular mass of approximately 58.1kDa . It is typically engineered with a C-terminal His-tag (9 amino acids) to facilitate purification through proprietary chromatographic techniques .

The protein is typically formulated in Phosphate Buffered Saline (pH 7.4) with 10-20% glycerol for stability . Being produced in an insect cell line, this recombinant protein contains post-translational modifications, particularly glycosylation patterns that differ from native human CFB but maintain functional activity for research applications.

What is the biological function of Complement Factor B in the human immune system?

Complement Factor B is a critical component of the alternative pathway of complement activation within the innate immune system. Its primary functions include:

• Circulating in blood as a single-chain polypeptide

• Undergoing cleavage by Complement Factor D to yield two fragments:

  • Ba: A non-catalytic chain that inhibits B lymphocyte proliferation

  • Bb: A catalytic serine protease subunit essential for complement activation

• Forming the C3bBb complex (alternative pathway C3 convertase) when the Bb fragment associates with C3b

• Amplifying the complement cascade by cleaving additional C3 molecules

• Contributing to opsonization, inflammation, and formation of the membrane attack complex

The gene encoding CFB is located on chromosome six in humans, in proximity to other complement-related genes in the major histocompatibility complex region .

What are the optimal storage and handling recommendations for CFB (260-764) Human produced in Sf9 cells?

For optimal stability and activity of CFB (260-764) Human from Sf9 cells, researchers should follow these evidence-based storage recommendations:

The protein is typically formulated in Phosphate Buffered Saline (pH 7.4) with 10-20% glycerol to enhance stability . When using the protein for experiments, it's advisable to aliquot the stock solution to minimize freeze-thaw cycles that can compromise structural integrity and functionality. For experimental work, maintain the protein on ice when thawed and use within the same day when possible.

How can researchers validate the functional activity of recombinant CFB (260-764) Human for complement studies?

Comprehensive validation of CFB (260-764) Human functional activity should employ multiple complementary approaches:

Enzymatic Activity Assessment

• Serine protease activity using synthetic peptide substrates

• C3/C5 convertase formation assays in reconstituted systems

• Quantification of C3a and C5a fragment generation by ELISA

Binding Interaction Studies

• Surface plasmon resonance to measure C3b binding kinetics

• Co-immunoprecipitation with C3b and Factor D

• ELISA-based protein-protein interaction assays

Functional Complement Assays

• Alternative pathway hemolytic assays using rabbit erythrocytes (most direct functional test)

• Zymosan or LPS activation assays to measure alternative pathway activation

• Complement-dependent cytotoxicity assays with appropriate target cells

Cleavage Analysis

• Verification of correct processing by Factor D (generating Ba and Bb fragments)

• SDS-PAGE and immunoblotting to confirm molecular weight of cleavage products

• Stability assessment of the C3bBb complex over time

Researchers should compare activity to native CFB standards and consider testing activity restoration in CFB-depleted serum, as these approaches collectively provide robust validation of physiologically relevant functionality.

How does the truncated form (260-764) of CFB affect its enzymatic properties compared to full-length protein?

The truncated CFB (260-764) differs significantly from full-length CFB in several functionally relevant ways:

Researchers should carefully consider these functional differences when designing experiments, particularly when studying activation mechanisms or regulatory interactions that involve the Ba domain. Validation against full-length protein is recommended for comprehensive studies of CFB biology.

What are the key considerations for designing experiments using CFB (260-764) Human in complement activation studies?

When designing experiments with CFB (260-764) Human for complement activation research, consider these critical parameters:

Buffer Composition and Experimental Conditions

• Maintain physiological pH (7.4) and ionic strength

• Include appropriate divalent cations (Ca²⁺, Mg²⁺)

• Consider temperature effects (complement optimally functions at 37°C)

• Account for glycerol content in storage buffer (may affect activity)

Control Design

• Include native human serum as positive control

• Use heat-inactivated serum as negative control

• Consider CFB-depleted serum reconstituted with recombinant protein

• Include appropriate isotype controls for antibody-based detection

Component Requirements

• Supplement with purified C3 and Factor D when studying direct interactions

• Consider properdin addition for stabilizing the C3 convertase

• Remember the truncated form lacks Ba domain, potentially affecting interpretation

Detection Strategy

• Select appropriate readouts based on experiment goals:

  • Hemolytic assays for functional activity

  • ELISAs for protein-protein interactions

  • Flow cytometry for cell surface complement deposition

  • Western blotting for cleavage product analysis

Potential Limitations

• Glycosylation differences between Sf9-produced and native CFB

• Possible His-tag interference with certain interactions

• Lack of Ba domain may affect some regulatory interactions

Careful experimental design addressing these factors will enhance reliability and physiological relevance of results in complement activation studies.

What expression and purification strategies yield optimal CFB (260-764) Human production in Sf9 cells?

Optimizing CFB (260-764) Human production in Sf9 cells requires careful attention to expression conditions and purification protocols:

Expression Optimization

• Maintain healthy Sf9 cultures with >95% viability in appropriate media (Insect-XPRESS™ or similar)

• Seed cells at optimal density (1-2 × 10⁶ cells/ml) for transfection/infection

• For transient expression, optimize DNA:transfection reagent ratio (2μg DNA with 3μl FUGENE® shows good results)

• Culture at 27°C for standard expression; consider lower temperatures (22-24°C) for difficult proteins

• Determine optimal harvest time through time-course experiments (typically 48-72h post-infection)

• Monitor cell viability using Trypan blue staining and MTT assays

Purification Strategy

• Initial clarification:

  • Centrifugation followed by 0.45μm filtration

• Primary capture:

  • Immobilized metal affinity chromatography (IMAC) using the C-terminal His-tag

  • Ni-NTA or Co²⁺ resins with imidazole gradient elution

• Secondary purification:

  • Ion exchange chromatography

  • Size exclusion chromatography to remove aggregates and fragments

• Quality control:

  • SDS-PAGE for purity assessment (target >95%)

  • Western blotting for identity confirmation

  • Activity assays for functional validation

Following these methodologies will maximize yield of high-quality, functional CFB (260-764) Human for research applications.

How can researchers troubleshoot low yield or inactive CFB (260-764) Human production?

When encountering issues with CFB (260-764) Human production, implement this systematic troubleshooting approach:

Cell Culture Issues

• Verify cell viability and growth rate (should maintain >95% viability)

• Check cell density at transfection/infection (1-2 × 10⁶ cells/ml is optimal)

• Examine cultures for contamination

• Validate media quality (freshness, proper storage)

Expression System Problems

• Sequence the construct to confirm correct CFB sequence

• Verify promoter functionality

• For baculovirus systems, confirm viral titer and optimal MOI

• Consider co-expressing chaperones to improve folding

Protein Degradation

• Add protease inhibitors during purification

• Analyze expression time course (protein may degrade if harvested too late)

• Keep samples cold (4°C) during processing

• Consider modifying culture conditions (temperature, pH)

Activity Loss During Purification

• Check each purification fraction for activity loss

• Optimize buffer conditions (pH, salt concentration)

• Minimize exposure to extreme conditions

• Test alternative chromatography methods

Protein Misfolding

• Lower culture temperature to 22-24°C

• Optimize cell lysis conditions

• Ensure proper disulfide bond formation

• Verify glycosylation status

If initial troubleshooting does not resolve issues, consider using alternate expression systems or reengineering the construct to improve expression or stability.

What medium formulations are most effective for Sf9 culture and CFB protein expression?

The choice of medium significantly impacts Sf9 cell growth and recombinant protein expression. Different options offer various advantages:

For optimal OSF9-ADCFM formulation, research shows the following composition is effective:

• Chemically defined lipid concentrate (CDLC): 0.5% (v/v)

• Yeast extract (YE): 11.0 g/L

• Soytone (ST): 3.0 g/L

Additionally, ultrafiltration of peptones (10 kDa cutoff) significantly improves Sf9 cell growth . While commercial media generally provide better expression, cost-conscious laboratories may opt for in-house formulations with approximately 50% cost savings, albeit with somewhat reduced expression levels.

How does the glycosylation pattern of Sf9-produced CFB (260-764) compare to native human CFB?

The glycosylation of CFB (260-764) produced in Sf9 cells differs substantially from native human CFB in ways that may impact research applications:

Key Differences

Functional Consequences

• Altered binding kinetics with complement components

• Different stability in experimental systems

• Modified immunogenicity in certain applications

• Potentially affected enzymatic activity

These differences necessitate careful validation when using Sf9-produced CFB for studies where glycosylation may influence the biological properties being investigated. For structure-function studies, researchers should consider how these modifications might impact their specific experimental questions.

What are the known interactions between CFB (260-764) Human and other complement components?

CFB (260-764) Human participates in multiple crucial interactions within the complement cascade:

Key Molecular Interactions

• C3b Binding

  • Mg²⁺-dependent interaction

  • Critical for proconvertase (C3bB) formation

  • Involves multiple contact points in the serine protease domain

  • Essential first step in alternative pathway activation

• Factor D Processing

  • Factor D cleaves CFB only when properly bound to C3b

  • Cleavage site becomes accessible upon conformational change

  • Results in release of Ba fragment and generation of active Bb

• Properdin Stabilization

  • Properdin binds to C3bBb complex

  • Extends convertase half-life from minutes to hours

  • Critical for sustained alternative pathway activation

  • Interacts primarily with the Bb portion

• C3 Substrate Processing

  • The active C3bBb convertase cleaves additional C3 molecules

  • Creates positive feedback amplification loop

  • Catalytic site in Bb domain responsible for this activity

• Regulatory Protein Interactions

  • Factor H competes with Factor B for C3b binding

  • Factor I inactivates C3b, preventing convertase formation

  • CR1 accelerates convertase decay

Understanding these molecular interactions is crucial for designing targeted experimental approaches and interpreting results in complement system research. The truncated nature of CFB (260-764) may influence some of these interactions compared to native protein.

How might CFB (260-764) Human from Sf9 cells be utilized in developing complement-targeted therapeutics?

The availability of well-characterized recombinant CFB (260-764) Human opens several avenues for therapeutic development:

Target Validation

• Serve as a tool for screening potential complement inhibitors

• Enable structural studies to identify druggable pockets

• Provide material for epitope mapping of therapeutic antibodies

• Support development of activity assays for drug screening

Therapeutic Applications

• Alternative pathway inhibition for complement-mediated diseases

• Development of decoy molecules based on CFB structure

• Creation of diagnostic tools for complement dysregulation

• Generation of neutralizing antibodies against specific CFB domains

Methodological Approaches

• Structure-based drug design targeting the serine protease domain

• Phage display screening against CFB to identify novel binding molecules

• Fragment-based drug discovery focusing on active site interactions

• Rational design of peptide inhibitors mimicking natural regulators

The recombinant protein allows for detailed mechanistic studies that can inform targeted therapeutic approaches, particularly for conditions involving alternative pathway dysregulation such as certain forms of hemolytic uremic syndrome, age-related macular degeneration, and C3 glomerulopathies.

What emerging technologies might enhance the production and characterization of CFB (260-764) Human?

Several cutting-edge approaches could advance CFB (260-764) Human research:

Production Enhancements

• CRISPR-engineered Sf9 cell lines with humanized glycosylation

• Synthetic biology approaches to optimize codon usage and expression

• Continuous bioprocessing for improved yield and consistency

• Automated microbioreactor systems for rapid optimization

Advanced Characterization Methods

• Hydrogen-deuterium exchange mass spectrometry for conformational analysis

• Cryo-electron microscopy for structural determination in convertase complexes

• Native mass spectrometry for studying intact protein complexes

• Single-molecule FRET for analyzing dynamic conformational changes

Functional Analysis Innovations

• Microfluidic complement activation platforms

• Label-free biosensors for real-time monitoring of complement activation

• Advanced proteomics to map the "complementome" interactions

• Nanoscale biophysical techniques to study membrane interactions

These technological advances would provide deeper insights into CFB structure-function relationships and potentially lead to improved recombinant proteins with more native-like characteristics for research and therapeutic applications.