BioActive™ Protease Protein Production

Seattle Genova offers our customers excellent protein production services that keep the bioactivities of your proteins just as they were in nature. To achieve this, we can offer both nature protein purification services and recombinant protein production services. We have developed very specialized protocols to make sure the produced proteins will maintain their biological activities. And our well-developed bioassays can ensure comprehensive activity validations before we release the proteins.

Background of Protease

Proteases conduct a non-reversible procedure to hydrolyze peptide bonds, such as reducing the initiating methionine from the recently synthesized cytoplasmic peptide, removing signal peptides and removing targeting signal peptides. 

Established on the mechanism of catalysis, proteases are distributed into five classes, metalloproteases, serine, cysteine, threonine and aspartic proteases in mammals. For aspartic proteases and metalloproteases, an active water molecule is utilized as a nucleophile to attack the substrate. For the rest three categories, they use an amino acid residue (Cys, Ser or Thr) as the nucleophile. In the Degradome Database, within the 569 human proteases, metalloproteases and serine proteases are the main 2 populated protease families.

 Research has shown that proteases function in all phases of tumour progression, comprising growth, survival, angiogenesis and invasion. Due to their significant roles in regulating multiple biological processes, protease is highly governed by several mechanisms, such as operating at the gene expression level, endogenous inhibitors and turning on/off zymogens.

Aspartate Protease & Regulator-enzyme

Aspartic proteases are a group of protease enzymes that use two highly conserved aspartic acid residues in the active site for a catalytic split of their peptide substrates. Unlike serine or cysteine proteases these proteases do not construct a covalent intermediate during cleavage. Nearly all available aspartyl proteases are inhibited by pepstatin. HIV protease is different in that it is a homodimer and each of the monomeric units contributes an aspartic acid.

Cysteine Protease & Regulator-enzyme

Cysteine proteases (CPs) are responsible for various biochemical processes arising in living organisms and they have been implicated in the development and progression of numerous diseases that comprise abnormal protein turnover. The crucial physiological role of Cysteine proteases is the metabolic degradation of peptides and proteins. The activity of Cysteine proteases is governed among others by their specific inhibitors: cystatins.

Metalloprotease & Regulator-enzyme

Metalloproteases are the most varied of the four main protease types, with more than 50 families assessed to date. Most metalloproteases expect zinc, but some aim for cobalt, which activates the water molecule. The metal ion is organized to the protein by amino acid ligands. The recognized metal ligands are His, Glu, Asp and Lys. 

Serine Protease & Regulator-enzyme

Serine proteases (SPs) are a family of proteases that operate a uniquely generated serine residue in the substrate-binding pocket to catalytically hydrolyze peptide bonds. All of the serine proteases comprise three residues at their active site: a serine, a histidine, and an aspartate. Serine proteases fall into two broad classifications based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

Proteases in cancer-enzyme

Proteases in normal cells are significant in carrying out biological processes. In living systems, a balance between proteases and their anti-proteases, and disruption of balance to numerous diseases like cancer. Steps starting from tumour initiation, growth, metastasis and finally invasion into some other site involve all five categories of proteases: serine, cysteine, aspartate, threonine and matrix metalloproteases. 

Production Process

Gene synthesis and Cloning 

We do sequence analysis and codon optimization for the expression system that will be used. The designed sequences are then synthesized to generate a DNA template.

The target gene is cloned into expression vectors for the mammalian, insect or E.coli cells. Multiple N-terminal signal peptides for protein secretion and N-terminal or C-terminal tags for protein affinity purification are available to choose from. The recombinant construct is confirmed by DNA sequencing.

Pilot-scale gene expression

The plasmid cDNA is extracted from the cells and transfected into 100 ml of E. coli or 50 ml of mammalian or insect suspension cells. The target protein in the cell lysate and supernatant is analyzed by SDS-PAGE and/or western blotting using an anti-tag antibody.

Bioactivity Testing

We can develop bioassays to validate the activity of the proteins we prepared. Only those proved to be active are qualified for release. Otherwise, we will start over to try different protocols until the bioactive protein is prepared.

Large-scale gene expression 

The expression vectors are prepared with endotoxin-free plasmid extract kits and are transfected into 2 litres of E. coli or 500 ml of mammalian or insect suspension cells. 

Purification 

After the cell culture period, the target protein is purified via affinity chromatography. The concentration and purity of the purified proteins are determined using a bicinchoninic acid (BCA) assay and SDS-PAGE with Coomassie blue staining, respectively. The protein is desalted into 0.2 µm filtered 1x phosphate-buffered saline (PBS), pH 7.4. The protein can then be aliquoted, lyophilized, and labelled upon request.

Bulk production (optional)

Large-scale gene expression and purification based on the customer’s desired amount of purified protein.

Features 

√Talented staff

√High Purity

Our proteins meet industry-leading purity specifications.

√Biological Activity

Proprietary methods for accurate protein folding ensure biologically relevant proteins.

√Lot-to-lot Consistency

Multiple lots are created for proteins, all with matching specifications. 

√Lower Endotoxin

Our standard endotoxin specification is an industry-leading <0.1 EU/ug.

√Long-term storage feasible

Proteins are delivered in a lyophilized form which ensures extra-longg terms of storage.

√Standard Quality control

Deliverable

√SDS-PAGE data

√WB data

√Bioactivity Validation data

√Purified protein

References

1.López-Otín, Carlos; Bond, Judith S. (November 2008). "Proteases: Multifunctional Enzymes in Life and Disease". Journal of Biological Chemistry. 283 (45): 30433–30437.

2.Rawlings ND, Barrett AJ (February 1993). "Evolutionary families of peptidases". The Biochemical Journal. 290 (Pt 1) (Pt 1): 205–18.

3.Oda K (2012). "New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases". Journal of Biochemistry. 151 (1): 13–25.