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Cloning and expression

MicroMol offers a combined strategy for cloning and expression of a desired sequence which might be a tremendous benefit for the partner from the biotechnical and  pharmaceutical industry but also for ambitioned projects within the university or the medical sector. According to the posibilities of MicroMols lab facility we suggest  - according to the customer´s needs - to express a sequence in various expression systems. In the first step we isolate the sequence of interest by PCR. Upon sequence verification we clone the sequence in the desired expression system. In this regard we offer the possibilty to express the protein in a bacterial system, in a yeast sytem or in a eukaryotic system (for expression in insect cells or in higher eukaryotes). Hereby we suggest to use one of our expression vectors offering a specific tag for protein determination. Subsequently we introduce the sequence of interest in the desired organism and check the expression by SDS-PAGE and if possible by Western Blot and/or ELISA with an appropriate antibody. If the customer wants to purify his protein on its own we deliver the expression vector directly to the customer.

Expert: Wolfgang Rudy (www.micromol.com)

Protein purification and characterization

MicroMol has the possibility to purify the desired protein from bacterial cells, from yeast as well as from eukaryotic cells. MicroMol uses a series of standard expression systems. In detail these are for bacterial expression (W3110, HB101, BL21) , for yeast (Pichia Pastoris) and for eukaryotes (SF9, COS, CHO, HEK293). However MicroMol is able to adapt the expression to the needs an organism the customer wants. In the first step the expression vector is introduced in the appropriate host and poositive clones are gained by appropriate selection procedures. Upon selection of the recombinant organism the recombinant protein is produced in the appropriate environment and subsequently purified using Affinity Chromatographie. Hereby we suggest doing the purification upon the tag (His, GST) expressed in combination with the recombinant protein. However if there is the possibilty or wish, the purification can also be done by a protein specific antibody or by conventional chromatography. Subsequently the puritiy and reactivity of the protein are determined using conventional staining procedures (Cooomassie Brilliant Blue, Silver staining) as well as immunological techniques such as ELISA and Western Blot.
If the customer wants to stop at that point MicroMol delivers a defined amount of the purified protein to the customer.

 

Expert: Wolfgang Rudy (www.micromol.com)

Development of antibodies

If the customer decides to have his own immunological tool to determine his protein MicroMol is the optimal partner for realization. There are several steps to get a highly reactive antibody. One of these possibilties is to use the whole recombinant protein as immunogen and in many cases this is a good choice. MicroMol however has very good experience with another strategy: We would offer and suggest to define a series of unique immunogenic peptides (at least 2) from the protein sequence, couple them to an appropriate carrier and use them as an immunogen for the desired host. During the immunization scheme MicroMol checks the antibody. Getting the final serum MicroMol prepares the antiserum and checks the final antibody titer and affinity. Subsequently we purify the reactive fraction from the antiserum by peptide dependent Affinity Chromatography  and determine the purity and concentration of the antiserum. In a final series of experiments MicroMol tests the functional  reactivity of the purified antibody by Western Blot an ELISA.
According to the customers wish a certain amount of the serum will be treated in that way and subsequently delivered to the customer.

This figure exemplifies what MicroMol can do for you. But we do even more - we invent the products you need.

Expert: Wolfgang Rudy (www.micromol.com)

Assay Development

If the customer wants to develop a sensitive test system with his novel Immunotools MicroMol might also be for that step the convenient partner. Upon purification of the antibodies by Affinity Chromatography we modify one of the specifities with an appropriate indicator (eg. Horse Raddish Peroxidase). This fraction will represent the detector whereas
the other unlabelled fraction functions as catcher. MicroMol is able to use these tools for the establishment of a specific Enyzme linked immunosorbent assay (ELISA). The customer – who delivered MicroMol in the beginning just with a sequence or cDNA - will finally end up in a fully evaluated test system.

 

Expert: Andreas Dreusch (http://www.micromol.com)

Peptide Scanning

Peptide Scanning is a method MicroMol has developed during the course of several granted concepts. This method offers the possibilty to determine specific epitopes of a protein using a specific antibody or antiserum. For every customer who wants to have detailed epitope information of a distinct protein, MicroMol can do that with an appropriate antibody and delivers this information within a reasonable time frame.
Therefore Peptide Scanning will be a powerful method for nearly every branch of the life science industry dealing with antibodies, sera and proteins.

Expert: Andreas Dreusch (http://www.micromol.com)

Analysis of Cell Banks

The recombinant expression of proteins depends on the quality of the cells used for production.
Not only cross-contamination with another cell line, but contamination with bacteria, fungi and viruses or at least the identity and integrity of the expression construct of the recombinant cell lines are critical.
Thus, characterization and safety testing of the cell banks is an essential part of the safety concept for cell-derived products.
If a bacterial or metazoan cell-line is intended for the production of active pharmaceutical ingredients (API) the rules of ICH Topic Q5D (Quality of Biotechnological Products: Derivation and Characterisation of Cell Substrates Used for Production of Biotechnological/Biological Products) apply.
These rules force the producer to have a sound documentation about the source of the cell-line and its properties. The propagation of the line shall follow a cell banking system comprising master cell banks, working cell banks and where appropriate a production cell bank.

The objective of our testing scheme is to confirm the identity, purity and genetic stability of the cell line as required by ICH Q5D.

To eliminate the potential risk of introducing adventitious agents to the product, testing should performed at the level of the master cell bank, the working cell bank, and the post-production cells.

 

Expert: Andreas Dreusch (http://www.micromol.com)

Analysis of microbial Cell Banks

For bacterial and partially yeast expression systems the recommended testing package comprises:

• Identity testing
• Purity testing
• Strain characterization
• Phage detection

All methods performed at MicroMol have been validated in-house.

Identity Testing
The identity of the bacterial species can be tested by biochemical parameters, pulsed-field gel electrophoresis, PCR, phage-typing or isoenzyme analysis. The extent to which a strain has to be defind depends on the approving authority and the specific demands.

Purity testing
For this topic we provide a set of bacteriological test to enrich and test for adventitious bacteria.

Strain characterization
Here we recommend tests for identity of the expression construct, the expression properties and the plasmid retention behaviour.

Phage detection
One of our specialities is the detection of phages in prokaryotic cell banks. We can provide this test for samples directly or with enrichment on many typical expression strains. Furthermore we test expression hosts for the presence of unknown lysogenic phages. We also can detect phages from production environments, as we do identification of phages by PCR, strain preferences and other methods.

 

Analysis of metazoan Cell Banks

For human, murine and other types of cell banks we provide:

Identity testing
This is done by isoenzyme analysis

Bioburden
test for bacterial or fungal contamination and absence of bacteriostatic or fungistatic agents

Mycoplasma
PCR assay for mycoplasma and absence of mycoplasmastatic agents

Virus tissue culture safety tests
absence of cytopathogenic effects, hemadsorption or hemagglutination.

The demands for such investigations have to be guided by the preparation a thoroughly elaborated testing scheme. They depend on the individual expression strain, as well as on the purpose of the product. Contact us for further discussion about our services for this area.

Expert: Andreas Dreusch (http://www.micromol.com)

ELISA and PCR-based detection of viruses

The sensitive and specific determination of viruses is a major problem for various parts of the life science industry. According to the experience obtained in other projects MicroMol offers a strategy for the fast and easy implementation of anti virus determination. Hereby we determine specific sequences of the viral proteom by peptide scanning and subsequently develop peptide specific antibodies against these regions. MicroMol uses these immunotools to develop poweful catcher/detector systems which are able to determine the virus specific and with great sensitivity.

We also develop PCR based test systems for viruses. We bring to our customers the whole service from the supply of reference material over the construction of synthetic PCR control targets to the full validation of the tests. Tests for H1 Parvovirus, Killham rat virus, Hanta virus, Sendai virus, Pneumonia virus of mice, Reovirus and Influenza have been established as a routine.

Expert: Andreas Dreusch (http://www.micromol.com)