CDH1 Human, Sf9

What is CDH1 and what is its biological significance?

CDH1 (E-cadherin) functions as both a cell adhesion molecule and as an APC/C (anaphase-promoting complex/cyclosome) coactivator required to recruit substrates during mitotic exit and the G1/G0 phase of the cell cycle . As a cancer predisposition gene, germline mutations in CDH1 are associated with hereditary diffuse gastric cancer (HDGC). Research shows that by age 80, CDH1 mutation carriers have a 70% cumulative incidence of gastric cancer for males and 56% for females, with females also having a 42% risk of breast cancer . The protein plays a crucial role in cellular adhesion, tissue formation, and the regulation of cell cycle progression through its interaction with multiprotein complexes.

Why are Sf9 insect cells preferred for human CDH1 expression?

Sf9 cells offer distinct advantages for expressing complex human proteins like CDH1. These cells provide high expression levels with posttranslational modifications approaching those of mammalian cells, which is critical for producing functional CDH1 protein . The system allows for simplified cell growth conditions that can be readily adapted to high-density suspension culture for large-scale expression. Research demonstrates that sufficient amounts of properly folded, functional human CDH1 can be produced in this system, making it ideal for structural and biochemical studies of CDH1-containing protein complexes such as the APC/C .

What are the comparative advantages of baculovirus-Sf9 systems over other expression platforms?

The baculovirus-Sf9 system offers multiple advantages for CDH1 expression compared to other platforms:

The baculovirus-Sf9 system provides an optimal balance of high protein yield, mammalian-like post-translational modifications, and reasonable production time, making it particularly suitable for complex proteins like CDH1 .

What is the optimal workflow for generating recombinant baculovirus containing the CDH1 gene?

The workflow for generating CDH1-expressing baculovirus depends on the specific system used. Using the BaculoDirect system provides an efficient approach:

• Clone the human CDH1 gene into a Gateway entry vector

• Perform a 1-hour Gateway LR recombination reaction between the entry clone and BaculoDirect linear DNA

• Transfect either Sf9 or Sf21 cells (not High Five cells due to lower transfection efficiency)

• Harvest P1 viral stock after 72-96 hours

• Amplify to generate high-titer P2 and P3 stocks

• Verify recombinant virus purity using the lacZ marker

• Determine viral titer using plaque assays or qPCR methods

The BaculoDirect system generates recombinant virus in minimal time using a strong polyhedrin promoter for high-level expression, with options for C-terminal or N-terminal 6xHis and V5 tags for easy detection and purification .

What expression parameters yield the highest functional CDH1 protein?

For optimal expression of functional human CDH1, several parameters must be carefully controlled:

Peak expression of CDH1 protein depends on these factors, and it's recommended to perform a small-scale optimization with time-course experiments to determine the precise optimum for each specific CDH1 construct .

What tagging strategies are most effective for CDH1 purification and detection?

Multiple tagging approaches have been successfully employed for CDH1 expression in Sf9 cells:

For structural studies of CDH1 in complexes like APC/C, researchers have successfully used His6-HA-CDH1 constructs with immunoprecipitation using specific antibodies (anti-CDC27 or anti-Flag) followed by peptide elution .

How can researchers troubleshoot low CDH1 expression yields?

When facing suboptimal CDH1 expression, a systematic troubleshooting approach should be undertaken:

• Viral titer evaluation: Ensure adequate titer through plaque assays or qPCR methods

• Cell health assessment: Sf9 cells should be >95% viable and in mid-log phase

• Infection parameter optimization: Test different MOI values (1, 5, 10) and harvest times

• Codon usage analysis: Consider CDH1 sequence optimization for insect cell expression

• Protein solubility evaluation: Determine if CDH1 forms inclusion bodies or aggregates

• Proteolytic degradation prevention: Add appropriate protease inhibitors during harvest

• Construct design modification: Test alternative signal sequences, tags, or fusion partners

• Culture conditions adjustment: Optimize media composition, temperature, and cell density

Each of these factors can significantly impact CDH1 expression and should be systematically evaluated when troubleshooting expression issues.

What purification strategies yield the highest quality CDH1 protein?

Effective purification of CDH1 from Sf9 cells typically involves a multi-step approach:

• Initial capture: Affinity chromatography using N- or C-terminal tags (6xHis tags show good results with CDH1)

• Intermediate purification: Size exclusion chromatography to separate monomeric CDH1 from aggregates

• Polishing: Ion exchange chromatography to remove remaining contaminants

• Buffer optimization: Inclusion of glycerol (10%) for stability throughout purification

• For CDH1-containing complexes: Immunoprecipitation with specific antibodies followed by peptide elution

• For highest purity structural studies: GraFix method (gradient fixation) combining glycerol density gradient centrifugation with mild chemical crosslinking

This approach has been successfully employed in structural studies of CDH1-APC/C complexes, yielding samples suitable for electron microscopy analysis .

How do researchers verify CDH1 functionality after purification?

Verification of purified CDH1 functionality requires multiple complementary approaches:

• Physical characterization: SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and integrity

• Structural assessment: Circular dichroism spectroscopy for secondary structure evaluation

• Stability analysis: Thermal shift assays to determine protein stability under various conditions

• Functional binding assays: Interaction studies with known binding partners such as APC/C components

• Substrate recruitment analysis: Binding studies with model substrates like Hsl1, which has been shown to bind stably to APC/C-CDH1 complexes

• Activity assessment: In vitro reconstitution of CDH1-dependent complexes and functional assays

• Structural integrity confirmation: Negative-stain electron microscopy to visualize CDH1 in multiprotein complexes

For APC/C coactivator function specifically, research has shown that CDH1 properly expressed in Sf9 cells can successfully recruit substrate proteins like Hsl1 to the APC/C complex in a D-box and KEN-box dependent manner .

How can recombinant CDH1 be used for studying cancer-associated mutations?

Sf9-expressed CDH1 provides an excellent platform for studying HDGC-associated mutations through several approaches:

• Site-directed mutagenesis to introduce specific mutations identified in HDGC patients

• Parallel expression and purification of wild-type and mutant CDH1 proteins

• Comparative structural analyses to identify mutation effects on protein folding and stability

• Binding studies with known interaction partners to reveal functional defects

• Assessment of mutations' impact on CDH1's role in APC/C substrate recruitment and ubiquitination

• High-throughput screening of multiple mutations by creating parallel baculovirus constructs

• Functional reconstitution assays to determine if mutations affect complex formation

Given that CDH1 mutations confer a 70% lifetime risk of gastric cancer in males and 56% in females by age 80, with females also facing a 42% breast cancer risk, this approach provides crucial insights into the molecular mechanisms underlying HDGC syndrome .

How does CDH1 participate in protein complexes like the APC/C?

Research using Sf9-expressed CDH1 has revealed critical details about its role in the APC/C complex:

• CDH1 serves as a coactivator that recruits substrates to the APC/C during specific cell cycle phases

• Substrate binding occurs in a region intercalated between CDH1 and DOC1 (another APC/C subunit)

• The interaction between APC/C and CDH1 is stabilized by substrate binding

• Different substrates (Hsl1, Sororin, Securin) show varying binding stabilities to the APC/C-CDH1 complex

• CDH1 directly recognizes degradation signals (D-box and KEN-box motifs) in substrate proteins

• EM structural studies have precisely mapped CDH1 and substrate binding locations on the APC/C

• CDH1 functions as a bridge between the core APC/C machinery and its substrates

Electron microscopy studies of reconstituted complexes have demonstrated that when substrates like Hsl1 bind to APC/C-CDH1, they create a distinctive density intercalated between CDH1 and DOC1, revealing the molecular basis of CDH1's substrate recruitment function .

What structural analysis techniques work best with Sf9-expressed CDH1?

CDH1 expressed in Sf9 cells is compatible with numerous structural analysis techniques:

• Electron microscopy: Particularly valuable for studying CDH1 in large complexes like APC/C

• X-ray crystallography: If CDH1 can be crystallized alone or with binding partners

• Small-angle X-ray scattering (SAXS): For solution structure determination

• Hydrogen-deuterium exchange mass spectrometry: To probe conformational dynamics

• Cross-linking mass spectrometry: To map interaction surfaces within protein complexes

• Single-particle analysis: For high-resolution structure determination of CDH1-containing complexes

• The GraFix method: Particularly useful for stabilizing CDH1 in multiprotein complexes for EM studies

Cryo-negative staining EM has been successfully employed to map the location of CDH1 and its bound substrates within the APC/C complex, revealing critical structural insights into how this cancer-linked protein functions in cellular regulation .

How can the biGBac method improve CDH1 complex expression?

For studying CDH1 in the context of larger protein complexes, the biGBac method offers significant advantages:

• Enables assembly of up to 25 cDNAs into a single baculoviral expression vector in only two steps

• Uses computationally optimized DNA linker sequences for efficient assembly of linear DNA fragments

• Employs a flexible "mix and match" approach allowing generation of baculoviruses at any assembly stage

• Enables parallel generation of multiple multigene expression constructs

• Successfully used for expression of cell-cycle complexes containing up to 17 different subunits

• Particularly valuable for generating complexes containing CDH1 with its interaction partners

The biGBac method significantly improves the throughput of generating multigene constructs, allowing researchers to explore CDH1's role in various protein complexes through systematic mutagenesis approaches that were previously infeasible .

What considerations are important when scaling up CDH1 production?

Scaling up CDH1 production for larger structural studies requires careful attention to several factors:

• Culture format transition: Move from shake flasks to larger bioreactors while maintaining growth conditions

• Oxygen transfer optimization: Ensure adequate aeration in larger vessels

• Infection strategy refinement: Use high-titer virus stocks to achieve consistent MOI across larger volumes

• Harvest timing precision: Determine optimal expression time at scale

• Purification scale-up: Adapt chromatography steps for larger sample volumes

• Quality control implementation: Establish robust metrics for consistent protein quality

• Storage condition optimization: Develop buffer compositions for long-term stability

• Batch reproducibility protocols: Create standard operating procedures for consistency

When scaling up expression of insect cell proteins, maintaining appropriate cell density (1.5-2.5 × 10^6 cells/mL) at infection and ensuring proper aeration are particularly critical factors for success .

How can CDH1 expression be optimized for structural biology applications?

For structural biology applications, CDH1 expression requires specific optimizations:

• Construct design: Remove flexible regions that may interfere with crystallization or high-resolution imaging

• Expression conditions: Fine-tune MOI and harvest time to maximize properly folded protein yield

• Stabilizing additives: Identify buffer components that enhance CDH1 stability without interfering with structural techniques

• Complex formation: For CDH1's role in APC/C, optimize reconstitution of complete complexes

• Sample homogeneity: Implement additional purification steps to ensure sample uniformity

• Protein concentration: Determine optimal concentration conditions that prevent aggregation

• Cryoprotection: For cryo-EM studies, identify suitable conditions that preserve structural integrity

Research has demonstrated that properly prepared CDH1-containing complexes can yield high-quality structural data, as evidenced by successful electron microscopy studies that revealed CDH1's position within the APC/C and its role in substrate recruitment .