Seattle Genova offers a range of custom enzyme production services. Our product range includes restriction enzymes, polymerases, nucleases, ligases, reverse transcriptase, nuclease, phosphatase and kinase, which are used for a variety of molecular biology applications such as target digestion, amplification, nucleic acid manipulation, DNA sequencing, polymerization, and reverse transcription.
The enzyme is a of which are responsible for thousands of metabolic processes in living cells. Based on the enzymatic reactions, a classification system categorizes enzymes into six major groups, which are oxidoreductase, transferase, hydrolase, lyase, isomerase and ligase. Each group of enzymes is labelled with an Enzyme Commission number (EC number) from EC 1 to EC 6. Hydrolase is the largest group of enzymes, which is followed by transferase and oxidoreductase. The proportion of enzymes in the rest three groups is relatively less.
Measurement of the enzyme molecular weight
➢SDS-PAGE: the most widely adopted method to separate enzymes from mixtures according to their electrophoretic mobility differences induced by the length of a polypeptide chain or molecular weight.
➢Western Blot: the designation of enzymes by their immunologic reaction with antibodies of known specificity.
Assessment of the enzyme purity
Measurement of the enzyme concentration
➢BCA assay: Bicinchoninic acid assay (BCA) is used to quantify the enzyme.
Amino-terminal (N-terminal) sequence analysis
➢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 verified by DNA sequencing.
Enzyme production and endotoxin removal process
The first step usually involves the 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 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 enzyme protein in the cell lysate and supernatant is analyzed via SDS-PAGE and/or western blotting using an anti-tag antibody.
On a large scale, 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.
Enzyme Purification process
After the cell culture period, the enzyme protein is purified via affinity chromatography. The concentration and purity of the purified protein 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.
The production scale, timeline, and properties of the enzymes should all be considered upon request.
Endotoxin Removal and detection
Each new production lot of protein is assessed for endotoxin using the Limulus Amoebocyte Lysates (LAL) assay. Our standard endotoxin specification is an industry-leading <0.1 EU/ug.
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