A new process for recombinant enzyme production

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Never resting on our laurels: Making our unique Shrimp Alkaline Phosphatase even better.

Shrimp Alkaline Phosphatase (SAP) was the first heat-labile alkaline phosphatase on the market. Considered the gold standard for the dephosphorylation of DNA, SAP is completely inactivated within five minutes following treatment at 65°C.

In 1993, a tightly-controlled purification process was developed by ArcticZymes to purify native alkaline phosphatase from the water used to rinse frozen shrimps. But unfortunately, purifying an enzyme from its natural source had limitations. Specifically, variable and unpredictable quality of the source meant that there were small batch-to-batch variations of the enzyme, which in turn affected its activity.

Today’s market demands an improved and robust SAP product. In particular, diagnostic labs regularly have to work with minute samples of DNA. To enable various analyses, this DNA needs to be amplified by polymerase chain reaction (PCR). Then, after amplification, the DNA product has to be “cleaned-up” to remove unwanted excess reagents.

Clean-up methods involving the use of spin columns are time-consuming and risk the loss of precious samples. SAP is a proven alternative. The enzyme is able to degrade excess deoxynucleotide triphosphates (dNTPs) and, when used together with another enzyme known as Exonuclease I, clean-up of PCR products is simple, rapid, and cost-effective. However, in the diagnostic lot-to-lot variation in the assay components can greatly influence the performance of an assay.

To establish a more reliable source of SAP, ArcticZymes’ scientists tapped into their vast experience in biotechnology to develop a recombinant form of SAP. The end result, an enzyme that suffered very little batch-to-batch variation and is stable at room temperature for extended period of time.

ArcticZymes set out to develop a recombinant production method that would eliminate these small batch-to-batch variations.

Developing the perfect and robust production process

Together with researchers from the University of Tromsø, ArcticZymes’ scientists identified the genes that were important for the expression of SAP.

In order to produce the recombinant SAP, the responsible genes first had to be cloned into a suitable vector before the vector was introduced into a production host. To do this, the research team had to try a number of vectors and production hosts before finding a combination that produced the protein at a significant amount. However, when using this combination, the enzymatic function of the protein was affected because the structure (folding) of the protein was not correct.

To improve the folding, the research team made various modifications to the production host, and eventually they found a clone that produced a high yield of recombinant SAP while retaining the enzymatic function of the native SAP. Further testing of the enzyme revealed that >95% of its activity was retained even after several days at room temperature.

The final hurdle the scientists at ArcticZymes had to cross was purification of the recombinant protein at a high enough concentration. Relying on more than 10 years of experience with purifying the native SAP, a patented purification method was developed to obtain high concentrations that also had highly-reproducible quality.

After producing and purifying multiple batches of the enzyme, samples from each batch were tested for enzyme quality using superior approaches developed by ArcticZymes, which far surpass previous industry standards.

In-house testing indicated that there were no batch-to-batch variations and customer evaluation of recombinant SAP proved as good as, or better, than native SAP, and gave far better results in most real-life-tests than competing enzymes.

ArcticZymes is adept at finding solutions to improve products and processes, in providing the next generation of molecular grade enzymes.