CELL CULTURE
rTrypsin
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Graphical representation of rTrypsin molecule
Trypsin Trypsin (EC 3.4.21.4) is an endopeptidase that preferentially hydrolyses ester bonds whose carboxyl groups are
contributed by Lysine (Lys) or Arginine (Arg) except when either is followed by proline (PRO). It is used for numerous
biotechnological processes such as tissue dissociation, cell harvesting and subculturing cells. Although native trypsin from
bovine or porcine pancreas has been available for many years, more stringent regulations are now engaged to ensure
safer biologicals and biopharmaceuticals. Novozymes offers rTrypsin, a recombinant product manufactured by microbial
fermentation. rTrypsin is animal component-free and manufactured to ISO 9001:2008 standards.
Advantages of rTrypsin• Produced by a recombinant manufacturing process, rTrypsin is not contaminated with animal enzymes such as
chymotrypsin or carboxypeptidases.
• Animal origin-free (AOF) manufacturing process eliminates the risk of viral and animal-related contaminants in
the final product
• Allows simple replacement of bovine or porcine trypsin
• Stability is not affected by Ca2+
• Consistent supply
Applications for rTrypsin• Cell dissociation and propagation of multiple cell lines including stem cells
• Vaccine production and processing
• Dissociation of dissected cells prior to cell fixing and sorting
• Cleavage of proinsulin to yield intact insulin
StabilityrTrypsin is available as a granulated product.
Temperature stabilityThe exceptional stability of rTrypsin permits easy and convenient storage and routine handling of the enzyme during
cell culture procedures.
Stability of rTrypsin is not affected by
free Ca2+ ions in the buffer
Excess free Ca2+ (1mM CaCl2)Without excess free Ca2+ (+1mM EDTA)
Excess free Ca2+ (1mM CaCl2)Without excess free Ca2+ (+1mM EDTA)
Stability of porcine trypsin is dependent on
free Ca2+ ions in the buffer
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SpecificityrTrypsin cleaves peptide bonds at the carboxyl side of
lysine and arginine as native trypsin. This permits simple
substitution of rTrypsin for porcine or bovine trypsin.
rTrypsin for dissociating cellsrTrypsin may be used to re-suspend cells from monolayers grown adhering to the interior surfaces of culture vessels.
rTrypsin performs well on stem cell lines such as iPH and hES as well as CHO, Vero and MDCK cells lines. Cells are most
commonly removed from the culture substrate by treatment with rTrypsin/EDTA/PBS buffer adjusted to pH 7. Depending
on the cell line, the suggested rTrypsin concentration should be adjusted to 0.2 mg/ml (corresponding to 200 U/ml
rTrypsin with an activity of 1000 USP/mg powder) and sterile filtered.
Available from Novozymes
USP = Trypsin activity unit using USP Crystallised Trypsin Reference Standard. The activity is determined relative to a protease A standard.
pH stability pH activity
rTrypsin is active at physiological pH 7.4, at 37°CrTrypsin is stable for 2hrs. at 37°C, pH range 5-9
rTrypsin Specificity at pH 9.0 on Suc-AAPX-pNA
Substrates: Suc-AAPA-pNA, Suc-AAPR-pNA,Suc-AAPD-pNA, Suc-AAPI-pNA, Suc-AAPM-pNA,Suc-AAPV-pNA, Suc-AAPL-pNA, Suc-AAPE-pNA,Suc-AAPK-pNA, Suc-AAPF-pNA.
Assay buffer: 100mM succinic acid, 100mM HEPES,100mM CHES, 100mM CABS, 1mM CaCl2,150mM KCl, 0.01% Triton X-100, pH 9.0.
Assay: 20μl rTrypsin/porcine trypsin(diluted in 0.01% Triton X-100) was mixed with100μl assay buffer in a microtiterplate well.The assay was started by adding 100μl pNA substrate(50mg pNA-substrate dissolved in 1.0ml DMSO andfurther diluted 45x with 0.01% Triton X-100).
The increase in OD405 was monitored as a measureof rTrypsin/porcine trypsin activity.
SA181 (hESC) after five passages using rTrypsin. Fixated cells were stained with an antibody detecting Oct4, a typical marker used to detect undifferentiated cells.
Product Name Product No. Unit size
rTrypsin 6395020 100g or 1 kg
For Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. CAUTION: Not intended for direct administration into humans or animals.
Product Name EC No. Substrate specificity Format Optimal Conditions Unit Activity Application
rTrypsin 3.4.21.4 Serine protease granulate pH 7.8-8.0 800 USP/mgHydrolysis of amide and ester bonds of lysine and arginine at carboxyl terminal
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© Novozymes A/S · No. 2014-12716-01