Protein expression and purification
Alexotech offers a complete service regarding manufacturing of proteins including gene synthesis, protein expression and purification. We work close with our customers and can in depth design expression and purification to suit your needs. As a customer, you only have to provide the desired protein sequence. The technique is suitable both to acquire active proteins as well as proteins for immunizations. In addition, it is suitable for preparation of uniform labeling with 15N and/or 13C isotopes suitable for NMR studies.
Price: DNA synthesis: 0.79 USD/nucleotide
Expression and purification: 2100 USD*
*Alexotech guarantees >10 mg of >95% pure protein, if this is not accomplished the cost for expression and purification is waved. If for some reason, the fusion partner cannot be removed (very rare) the protein will be delivered with its fusion partner.
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A project is usually divided into the following steps:
The first step of a successful production starts in the computer. As a first approach the desired needs with the produced proteins should be defined and questions regarding which properties that should be preserved, should be answered. Should the structure/activity be preserved or is the protein intended for e.g. immunization where a native sequence as well as ultra-high purity may be of higher importance? Is solubility a problem and could a suitable fusion partner be included to get a higher probability of acquiring a soluble product? Alexotech has a long experience in structure based protein design and can help in this process. When a sequence and an approach have been cleared by the customer the project is initiated.
The gene of interest is synthesized de novo and cloned into an in-house expression vector. All proteins contain a removable affinity tag, as well as a proteolytic site of digestion, enabling their full removal resulting in a fully native sequence of the protein. Depending on the size and predicted solubility a protein fusion partner may be included.
A test for expression is made in 3 different E.coli strains. When suitable conditions for protein expression are found, a culture of 4 liters is grown in LB. In this step we offer the option of using minimal media containing 15N and/or 13C as sole nitrogen and carbon source to acquire uniform isotope labeling.
The expressed protein will be purified using the added affinity tag. Proteins that remain soluble after expression will be purified under non-denaturing conditions. Proteins forming inclusion bodies will be solubilized using UREA and purified under denaturing conditions. The process is followed by dialysis and subsequent removal of the fusion partner and affinity-tag.