Snake Venom Metalloproteinases (SVMPs)

• The cysteine switch mechanism inactivates snake venom metalloproteinases (SVMPs)
• Activation occurs when Zn2+ ions trigger the dissociation of the prodomain, exposing the active site, allowing protein degradation and contributing to venom toxicity

SVMP Toxins

• SVMPs are highly toxic components in snake venom that cause necrosis, hemorrhage, and blood coagulation disorders
• They degrade extracellular matrix proteins, causing bleeding, inflammation, and muscle damage

Diversity of SVMPs

• Snakes produce multiple SVMPs with varied structures and functions
• SVMPs are categorized into PI, PII, and PIII classes, differing in domain number and targeting different host proteins

Conservation Among SVMPs

• SVMPs exhibit moderate to high conservation, especially in the catalytic domain
• Domains like disintegrin-like and cysteine-rich domains are more variable, potentially evolving to target different host proteins

Role of Extra Domains in SVMPs

• PI SVMPs contain only a catalytic domain, sufficient for protein breakdown but lacking targeting functions
• PII and PIII SVMPs include disintegrin-like and cysteine-rich domains that enhance toxin interaction with host proteins
• The cysteine-rich domain affects substrate specificity and protein stability
• Extra domains increase efficiency in degrading fibrinogen and extracellular matrix proteins

Advantages of Insect Cell Expression

• Insect cells allow proper protein folding and post-translational modifications like glycosylation, crucial for SVMP activity
• Proper folding requires correct disulfide-bond formation, facilitated by insect cells
• Insect cells can handle complex proteins better than bacterial systems
• Insect cell expression reduces the risk of endotoxin contamination

Disadvantages of Insect Cell Expression

• Slower growth rate compared to bacteria, increasing time
• More expensive due to specialized media and equipment
• Lower protein yield because the secreted protein needs correct folding for proper activity

Alternative Expression Systems

• Bacterial expression (E. coli) is unsuitable due to its inability to perform post-translational modifications like glycosylation
• Mammalian cell expression is more expensive and complex than insect cells
• Yeast expression is an alternative, but it can result in undesirable hyperglycosylation

Anion Exchange Chromatography basics

• Separates proteins based on charge
• The column contains positively charged resins that bind negatively charged proteins
• Proteins are eluted by increasing salt concentration, which disrupts ionic interactions and releases proteins from the resin
• More negatively charged proteins elute later, and less charged proteins elute earlier

Zymogen Band on MP Activation

• Could be due to insufficient Zn2+activation or non-optimal concentration
• Contaminating inactive forms of zymogens may have misfolded and become resistant to activation, because the prodomain is inaccessible

Fibrinogen as an Assay

• Fibrinogen used because it is a key blood-clotting protein, linking degradation directly to hemorrhagic effects of SVMPs
• Many SVMPs target fibrinogen and fibrin, disrupting clot formation
• Fibrinogen is a well-established, quantifiable assay that provides clear evidence of SVMP activity

Alternative Assays

• Collagen degradation assay: measures the ability of SVMPs to break down connective tissue
• Plasma clotting assay: measures how SVMPs affect blood clot formation and breakdown
• Insulin B assay: The presence of active SVMP will break down the insulin B-chain, confirmed using SDS-PAGE gel

Insect Cells

• Insect cells facilitate proper protein folding and post-translational modifications, critical for SVMP activity
• Bacterial systems lack these capabilities, often leading to misfolded or inactive proteins

Steps to Construct SVMP expression

• MultiBac baculovirus/insect cell expression system was used
• Bacmid Preparation & Transfection: Genes added to DH10EMBacY E. coli, screened using blue/white selection, and transfected into Sf21 insect cells
• Amplification & Expression: Virus-containing cells harvested, and protein expression induced in Hi5 cells
• Purification: Proteins extracted and purified using Immobilized Metal Affinity Chromatography (IMAC), Anion Exchange Chromatography (AIEX), and Size Exclusion Chromatography (SEC)

Monitoring Protein Expression

• Monitored using Yellow Fluorescent Protein (YFP) analysis
• Daily samples were lysed, and fluorescence was measured at excitation 430 nm, emission 528 nm to determine protein production levels

Activation Step

• Zn2+ was used to activate SVMP by disrupting the cysteine switch, releasing the prodomain to convert the zymogen to an active form
• Different Zn2+ concentrations were tested for complete activation

Immobilized Metal Affinity Chromatography (IMAC)

• Separates proteins based on their histidine (His) tags binding to nickel (Ni2+)-charged resins
• Non-His-tagged contaminants are washed away
• The protein is eluted using imidazole, which competes with the His-tag for binding, allowing collection of the target protein

Anion Exchange Chromatography purpose

• Separates proteins based on their charge
• SVMP proteins, which are negatively charged, bind to the positively charged resin
• Proteins are eluted gradually by increasing salt concentration

SDS-PAGE purpose

• Used to confirm protein expression and purity throughout the experiment
• After expression: To check successful production of SVMP constructs
• After purification steps: To see if impurities were removed
• After activation: To ensure zymogen was properly converted into the mature enzyme

Fibrinogen Degradation Assay definition

• Tested the enzymatic activity of activated SVMP constructs
• Measured ability to degrade fibrinogen, a key blood-clotting protein
• More fibrinogen degradation indicates higher SVMP activity

EDTA and SVMP Activity

• EDTA chelates Zn2+ ions, essential for SVMP activity
• By inactivating SVMPs, EDTA serves as a negative control, confirming observed activity is due to the enzyme

Zymogen Band Significance

• Incomplete activation: Proteins may not have fully lost their prodomain
• Inefficient Zn2+ binding: Activation conditions may not have been optimal
• Protein aggregation: Some zymogens may have misfolded, preventing activation

His-Tag Function

• A His-tag was added to SVMP constructs for purification using Immobilized Metal Affinity Chromatography (IMAC)
• The histidine residues bind strongly to nickel (Ni2+)-charged resins

Protein Separation using IMAC

• Separates via metal ions (Ni2+) bound to a column resin
• His-tagged proteins bind to the column after loading the sample
• Unwanted proteins washed away with a low-imidazole buffer
• Target protein eluted by increasing imidazole, which competes with His-tags for binding

AIEX post IMAC

• IMAC alone can leave impurities
• AIEX was used for further purification separating proteins based on charge
• Since SVMPs are negatively charged, they bind to the positively charged resin

Chromatography pH

• Correct pH determined via the isoelectric point (pI) of the protein
• Chosen pH is usually 2-3 units higher
• If a pH(8) > pI (~5), the protein will be negatively charged

SEC usage

• SEC serves as a final polishing step to ensure the protein is in correct oligomeric state
• Separates proteins based on size using a gel filtration column

Chromatography Salt

• Salt (e.g., NaCl) competes with the protein for binding

Dialysis reason

• Dialysis was performed to exchange the buffer and remove excess imidazole
• The protein was placed in a membrane bag, submerged in buffer

Potential IMAC contaminants

• Partially expressed proteins with weak His-tags
• Nonspecifically bound proteins from insect cell lysate
• Protein degradation products
• AIEX removes these through charge

Impurity peak presence and chromatography

• Suggests presence of non-target proteins, degradation fragments, or aggregates
• Additional purification steps or different buffer conditions may be needed

Chromatography Cryoprotectant

• Glycerol added to prevent protein aggregation or denaturation during freezing
• Varied concentrations used based on construct stability
  • MP/Dis/Crich construct → 9% glycerol
  • MP/Dis construct → 7.5% glycerol
  • MP construct → 5.5% glycerol

IMAC function

• IMAC pulls out all the media and concentrates the protein
• SEC requires lower amounts of volume (500ul)
• IMAC uses His-tags

SVMPs cryo-structures

• Solved for other PIII SVMPs but not for any Echis
• Ones that have been determined have the same three domains