Recombinant Chaetomium globosum Signal peptidase complex catalytic subunit SEC11 (SEC11)

What is the Signal Peptidase Complex Catalytic Subunit SEC11 in Chaetomium globosum?

SEC11 is the catalytic component of the signal peptidase complex (SPC) in Chaetomium globosum, responsible for cleaving N-terminal signal sequences of proteins targeted to the endoplasmic reticulum. This cleavage occurs during protein translocation through the translocon pore into the endoplasmic reticulum, either cotranslationally or post-translationally. SEC11 from C. globosum belongs to the peptidase S26B family and consists of 172 amino acids with a molecular weight of approximately 19 kDa .

What is the amino acid sequence of C. globosum SEC11?

The amino acid sequence of C. globosum SEC11 is:
MLSSLQNPRQAAAQLMNFGLILSTAFMMWKGLSVITDSPSPIVVVLSGSMEPAFQRGDLLLLWNRNLISETNVGEIVVYNVKGKDIPIVHRIVRKFGVGPDAKLLTKGDNNAADDTELYARGQDYLNRKDIVGSVVGYMPFVGYVTIMLSEHPWLKTVMLGIMGLVVVLQRE

What expression systems are commonly used for recombinant C. globosum SEC11 production?

Several expression systems have been utilized for the recombinant production of C. globosum SEC11:

• Yeast expression system: Provides eukaryotic post-translational modifications that may be important for proper folding and activity.

• E. coli expression system: Offers high yield and simplicity, though lacks eukaryotic post-translational modifications.

• In vivo biotinylation in E. coli: Utilizes AviTag-BirA technology, where BirA catalyzes the formation of an amide linkage between biotin and a specific lysine residue in the AviTag sequence.

• Baculovirus expression system: Enables production in insect cells, providing more complex eukaryotic post-translational modifications .

The choice of expression system depends on the specific experimental requirements, including the need for post-translational modifications, protein yield, and downstream applications.

How should I design primers for amplifying the SEC11 coding sequence from C. globosum?

When designing primers for amplifying the SEC11 coding sequence from C. globosum, consider the following methodological approach:

• Retrieve the complete coding sequence from genomic databases such as the Broad Institute's C. globosum annotation database (http://www.broadinstitute.org/annotation/genome/chaetomium_globosum.2/Home.html)[3].

• Design forward and reverse primers with appropriate restriction sites or recombination sequences (e.g., Gateway technology compatible sequences) for subsequent cloning.

• Include 18-25 nucleotides complementary to the target sequence with a GC content of 40-60%.

• Consider adding a Kozak consensus sequence (ACCATGG) before the start codon if expressing in eukaryotic systems.

• For C-terminal tagging, remove the stop codon in the reverse primer and ensure in-frame fusion with the tag sequence.

• For Gateway cloning specifically, follow a similar approach to that used for other C. globosum genes, where specific primers were designed for recombination into entry vectors like pDONR221 .

What are the optimal conditions for expressing recombinant C. globosum SEC11 in E. coli?

For optimal expression of recombinant C. globosum SEC11 in E. coli, follow these methodological guidelines:

• Vector selection: Use expression vectors with strong inducible promoters such as T7 (e.g., pDEST17), which allows for controlled expression with an N-terminal 6×His tag for purification .

• E. coli strain: BL21(DE3) or Rosetta(DE3) strains are recommended, with the latter providing additional tRNAs for rare codons that might be present in fungal genes.

• Culture conditions:

  • Grow cultures at 37°C until OD600 reaches 0.6-0.8

  • Induce with 0.1-1.0 mM IPTG

  • Shift temperature to 16-25°C post-induction to enhance proper folding

  • Continue expression for 16-20 hours

• Buffer optimization: Use buffers containing 20-50 mM Tris-HCl pH 8.0, 150-300 mM NaCl, and consider adding glycerol (5-10%) to stabilize the protein.

• Solubility enhancement: If facing solubility issues, consider fusion partners like MBP or SUMO, or the addition of solubility enhancers like sorbitol and betaine to the culture medium.

What purification strategy should I employ for recombinant C. globosum SEC11?

A comprehensive purification strategy for recombinant C. globosum SEC11 includes:

• Affinity chromatography: For His-tagged SEC11, use Ni-NTA resin with an imidazole gradient (10-250 mM) for elution. For biotinylated SEC11, use streptavidin-coated resins with biotin or desthiobiotin for elution .

• Size exclusion chromatography: Further purify using a Superdex 75/200 column to separate monomeric SEC11 from aggregates and other contaminants.

• Ion exchange chromatography: If needed, use cation or anion exchange depending on the theoretical pI of SEC11.

• Quality control: Assess purity using SDS-PAGE (expected band at approximately 19 kDa) and confirm identity by Western blot using anti-His antibodies or mass spectrometry.

• Activity assessment: Verify catalytic activity using synthetic peptide substrates containing signal peptide cleavage sites.

How can I investigate the role of SEC11 in the secretory pathway of C. globosum?

To investigate the role of SEC11 in the C. globosum secretory pathway, employ the following methodological approach:

• Gene disruption/deletion: Utilize CRISPR-Cas9 system, similar to what has been used for other C. globosum genes such as CgPKS11 . Design guide RNAs targeting the SEC11 gene and a repair template for homologous recombination.

• Conditional expression systems: Create strains with SEC11 under the control of an inducible promoter to study the effects of SEC11 depletion on the secretory pathway.

• Secretome analysis: Compare the secreted protein profiles of wild-type and SEC11-depleted strains using proteomics approaches such as:

  • 2D gel electrophoresis followed by mass spectrometry

  • Label-free quantitative proteomics

  • Targeted analysis of specific secreted enzymes important for C. globosum, such as cellulases and other glycohydrolases

• Subcellular localization: Perform immunogold electron microscopy with antibodies against SEC11 or fluorescence microscopy with SEC11-GFP fusion proteins to determine the precise localization within the secretory pathway.

• Interactome analysis: Identify protein-protein interactions using co-immunoprecipitation coupled with mass spectrometry to elucidate the components of the signal peptidase complex in C. globosum.

How does SEC11 from C. globosum compare to homologs from other fungi in terms of structure and function?

To conduct a comprehensive comparative analysis of SEC11 from C. globosum with homologs from other fungi:

• Sequence alignment and phylogenetic analysis:

  • Retrieve SEC11 sequences from diverse fungal species

  • Perform multiple sequence alignment using MUSCLE or CLUSTALW

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Identify conserved domains and residues, especially those in the catalytic site

• Structural comparison:

  • Predict the 3D structure of C. globosum SEC11 using homology modeling (if crystal structure is unavailable)

  • Compare with available structures of homologs using structural alignment tools

  • Analyze conservation of catalytic residues and substrate binding sites

• Functional complementation assays:

  • Express C. globosum SEC11 in yeast SEC11 mutants to test functional conservation

  • Compare enzymatic parameters (Km, kcat) of recombinant SEC11 proteins from different fungi using identical substrates

  • Assess substrate specificity differences using a panel of peptides with various signal sequences

• Evolutionary analysis:

  • Calculate selection pressures (dN/dS ratios) across the gene to identify regions under positive or purifying selection

  • Correlate evolutionary patterns with functional differences and ecological niches of the source organisms

How can I use biotinylated C. globosum SEC11 for protein-protein interaction studies?

Biotinylated C. globosum SEC11 produced through in vivo biotinylation using AviTag-BirA technology offers powerful approaches for protein-protein interaction studies:

• Pull-down assays:

  • Immobilize biotinylated SEC11 on streptavidin-coated beads

  • Incubate with C. globosum cell lysates or recombinant potential interactors

  • Wash thoroughly and elute bound proteins

  • Identify interactors by mass spectrometry or Western blotting

• Surface Plasmon Resonance (SPR):

  • Capture biotinylated SEC11 on streptavidin-coated sensor chips

  • Flow potential interaction partners over the surface

  • Measure association and dissociation kinetics

  • Calculate binding affinities (KD values)

• Proximity-dependent labeling:

  • Fuse SEC11 to a proximity-labeling enzyme (e.g., BioID or APEX2)

  • Express in C. globosum or heterologous system

  • Activate labeling to biotinylate proteins in proximity to SEC11

  • Purify biotinylated proteins and identify by mass spectrometry

• Crosslinking Mass Spectrometry (XL-MS):

  • Use chemical crosslinkers to stabilize transient interactions

  • Digest the crosslinked complexes

  • Identify crosslinked peptides by mass spectrometry

  • Map interaction interfaces between SEC11 and its partners

How should I interpret differences in SEC11 activity when comparing recombinant proteins produced in different expression systems?

When comparing the activity of SEC11 produced in different expression systems (yeast, E. coli, baculovirus) , consider these analytical approaches:

• Normalization strategies:

  • Base comparisons on equimolar amounts of purified protein rather than total protein

  • Use activity per molecule of enzyme rather than per mass unit

  • Ensure similar purity levels across preparations

• Post-translational modification analysis:

  • Perform mass spectrometry to identify and quantify modifications present in each preparation

  • Use specific glycosylation or phosphorylation detection methods if these modifications are suspected to affect activity

  • Correlate modifications with activity differences

• Structural integrity assessment:

  • Use circular dichroism (CD) spectroscopy to compare secondary structure content

  • Employ differential scanning fluorimetry (DSF) to compare thermal stability

  • Consider limited proteolysis to assess structural differences

• Systematic activity measurement:

  • Test activity across a range of conditions (pH, temperature, ionic strength)

  • Determine enzyme kinetics parameters (Km, Vmax, kcat) under identical conditions

  • Create a comprehensive comparison table with statistical analysis of replicate experiments

• Data interpretation framework:

  • Consider the native environment of C. globosum SEC11 (fungal ER)

  • Evaluate which expression system provides conditions closest to the native environment

  • Assess whether differences reflect true biological variability or are artifacts of the expression systems

What are the key considerations when analyzing the impact of SEC11 on the C. globosum secretome?

When analyzing how SEC11 affects the C. globosum secretome, consider these analytical approaches:

• Experimental design considerations:

  • Include appropriate controls (wild-type, SEC11 overexpression, SEC11 knockdown/knockout)

  • Use biological replicates (minimum n=3) to ensure statistical robustness

  • Standardize growth conditions and sampling times

• Quantitative proteomics approach:

  • Use label-free quantification or isotope labeling methods (SILAC, TMT, iTRAQ)

  • Apply appropriate normalization methods to account for differences in total protein amounts

  • Set significance thresholds (p-value, fold change) for identifying differentially secreted proteins

• Bioinformatic analysis pipeline:

  • Predict signal peptides in identified proteins using tools like SignalP

  • Categorize proteins functionally using GO terms, KEGG pathways, or CAZyme classification

  • Perform enrichment analysis to identify overrepresented functional categories

• Integration with transcriptomics:

  • Compare secretome changes with transcriptional changes using RNA-Seq

  • Distinguish between direct effects (signal peptide processing) and indirect effects (altered gene expression)

  • Create correlation plots of protein abundance vs. transcript levels

• Validation experiments:

  • Confirm key findings using targeted approaches (Western blot, enzymatic assays)

  • Test specific hypotheses about SEC11's role in processing particular secreted proteins

  • Consider the temporal dynamics of secretome changes following SEC11 perturbation

What strategies can address poor solubility of recombinant C. globosum SEC11 in E. coli?

When facing solubility issues with recombinant C. globosum SEC11 in E. coli, implement these methodological solutions:

• Expression optimization:

  Parameter	Standard Condition	Optimization Strategy
  Temperature	37°C	Lower to 16-20°C after induction
  IPTG concentration	1.0 mM	Reduce to 0.1-0.5 mM
  Media	LB	Try auto-induction media or TB
  Induction time	Early log phase	Induce at mid-log phase (OD600 0.6-0.8)
  Expression duration	4-6 hours	Extend to 16-20 hours at lower temperature

• Protein engineering approaches:

  • Truncate potential disordered regions identified through bioinformatic prediction

  • Create fusion constructs with solubility-enhancing partners (MBP, SUMO, TrxA, GST)

  • Introduce surface mutations to increase hydrophilicity without affecting the catalytic site

• Lysis and buffer optimization:

  • Test various lysis methods (sonication, high-pressure homogenization, enzymatic lysis)

  • Screen buffers with different pH values (6.5-8.5) and ionic strengths (150-500 mM NaCl)

  • Add solubility enhancers (5-10% glycerol, 0.1% Triton X-100, 1 mM DTT, 50-500 mM arginine)

• Co-expression strategies:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Co-express with other components of the signal peptidase complex if known

• Refolding from inclusion bodies:

  • If all solubility efforts fail, purify inclusion bodies

  • Solubilize with 6-8 M urea or 6 M guanidine HCl

  • Refold by rapid dilution or dialysis against decreasing denaturant gradients

  • Add stabilizers during refolding (0.4 M arginine, 0.1 M proline, low concentrations of detergents)

How can I optimize the enzymatic activity assay for recombinant C. globosum SEC11?

To develop a robust enzymatic activity assay for recombinant C. globosum SEC11, follow these methodological guidelines:

• Substrate selection and design:

  • Design peptide substrates containing known or predicted signal peptide cleavage sites

  • Incorporate fluorogenic or chromogenic reporters for easy detection

  • Include both fungal and standard model signal sequences to compare specificity

• Assay optimization parameters:

  Parameter	Range to Test	Rationale
  pH	5.0-8.0	Cover the range of ER pH in fungi
  Temperature	25-37°C	Balance enzyme stability with activity
  Divalent cations	0-10 mM Ca²⁺, Mg²⁺, Mn²⁺	Test potential cofactor requirements
  Detergents	0.01-0.1% mild detergents	Mimic membrane environment
  Reducing agents	0-5 mM DTT or β-ME	Optimize disulfide status

• Detection method development:

  • For fluorogenic substrates: Optimize excitation/emission wavelengths and gain settings

  • For FRET-based substrates: Establish appropriate donor/acceptor pairs

  • For mass spectrometry: Develop targeted MRM methods for specific cleavage products

• Controls and validation:

  • Include heat-inactivated enzyme as negative control

  • Use known signal peptidase inhibitors as specificity controls

  • Create site-directed mutants of catalytic residues as additional controls

  • Validate with known substrates from related enzymes if available

• Kinetic parameter determination:

  • Perform time-course experiments to ensure linear range

  • Use substrate concentration series to determine Km and Vmax

  • Calculate catalytic efficiency (kcat/Km) for different substrates to assess specificity

What approaches can resolve difficulties in detecting protein-protein interactions involving C. globosum SEC11?

When encountering challenges in detecting protein-protein interactions involving C. globosum SEC11, consider these methodological solutions:

• Stabilization of transient interactions:

  • Use chemical crosslinkers with different spacer lengths (DSS, BS3, formaldehyde)

  • Apply membrane-permeable crosslinkers for in vivo studies

  • Consider photo-activatable crosslinkers for greater specificity

  • Employ protein interaction stabilizers like molybdate or tungstate

• Alternative detection technologies:

  • Try split reporter systems (split-GFP, split-luciferase) for in vivo detection

  • Implement more sensitive detection methods like AlphaScreen or HTRF

  • Use microscale thermophoresis (MST) for detecting interactions in solution

  • Consider hydrogen-deuterium exchange mass spectrometry for mapping interaction interfaces

• Reconstitution approaches:

  • Reconstruct the entire signal peptidase complex in vitro using purified components

  • Use liposome reconstitution to provide a membrane environment

  • Consider nanodiscs or styrene-maleic acid lipid particles (SMALPs) for membrane protein studies

• Binding condition optimization:

  Parameter	Variations to Test	Rationale
  Salt concentration	50-500 mM	Modulate electrostatic interactions
  pH	6.0-8.0	Affect charge distribution and binding
  Detergents	Various types and CMCs	Maintain native-like membrane environment
  Divalent cations	1-10 mM Ca²⁺, Mg²⁺, Zn²⁺	Support potential metalloprotein interactions
  Reducing conditions	0-5 mM DTT or TCEP	Maintain appropriate disulfide status

• Expression tag considerations:

  • Test multiple tag positions (N-terminal, C-terminal, internal)

  • Use small tags (His, FLAG, HA) to minimize steric hindrance

  • Consider tag removal using specific proteases if the tag interferes with interactions

  • For challenging interactions, try in vivo proximity labeling approaches like BioID or APEX

How can SEC11 from C. globosum contribute to our understanding of fungal secretory pathways and biotechnological applications?

The study of C. globosum SEC11 opens several promising research avenues:

• Comparative secretory pathway biology:

  • Investigate differences between fungal and mammalian signal peptidase complexes

  • Examine evolutionary adaptations in secretory pathways across fungal lineages

  • Explore how variations in SEC11 contribute to the diverse ecological niches of different fungi

• Biotechnological applications:

  • Engineer SEC11 variants with altered substrate specificity for biotechnological protein processing

  • Develop SEC11-based systems for improved heterologous protein secretion

  • Leverage knowledge of SEC11 to enhance production of valuable fungal secondary metabolites like chaetoglobosin A

• Integration with systems biology:

  • Create comprehensive models of the C. globosum secretory pathway

  • Investigate the impact of SEC11 on the production of cellulose-degrading enzymes

  • Examine connections between protein secretion and secondary metabolism

• Development of antifungal strategies:

  • Evaluate SEC11 as a potential antifungal target, especially for fungi related to C. globosum

  • Design specific inhibitors based on structural and functional analysis

  • Assess the impact of SEC11 inhibition on fungal growth and development

• Synthetic biology applications:

  • Incorporate C. globosum SEC11 into synthetic secretory systems

  • Engineer signal peptide processing for controlled release of bioactive compounds

  • Develop biosensors based on SEC11 activity for detecting specific environmental conditions

What role might SEC11 play in the regulation of chaetoglobosin A biosynthesis in C. globosum?

Given the importance of chaetoglobosin A (ChA) in C. globosum biology , exploring the potential role of SEC11 in its biosynthesis presents an intriguing research direction:

• Pathway component processing:

  • Investigate whether SEC11 processes any enzymes involved in ChA biosynthesis

  • Examine if the CgcheA gene cluster contains proteins requiring signal peptide cleavage

  • Determine if SEC11 activity influences the localization of ChA biosynthetic enzymes

• Regulatory network analysis:

  • Explore potential cross-talk between secretory pathway stress and secondary metabolism

  • Investigate if SEC11 dysfunction triggers compensatory mechanisms that affect ChA production

  • Compare with known regulatory mechanisms like those involving the polyketide synthase CgPKS11

• Experimental approaches:

  • Create SEC11 conditional mutants and measure ChA production levels

  • Perform transcriptome and proteome analysis to identify effects on the ChA biosynthetic pathway

  • Use metabolic flux analysis to trace precursor incorporation into ChA under different SEC11 expression levels

• Comparative analysis:

  • Examine if other fungi producing cytochalasins show similar connections between SEC11 and secondary metabolism

  • Investigate if the relationship between protein secretion and secondary metabolism is conserved across fungal species

• Integration with cellular development:

  • Explore connections between SEC11, ChA production, and developmental processes like sporulation

  • Examine whether SEC11-dependent secreted proteins influence C. globosum growth patterns similar to the effects observed with CgPKS11 deletion