From gene to protein

Why determine three dimensional structures

Most structure determination projects presently are performed to obtain information about a certain biological process, and the role of a gene-product therein. To better understand this process, knowledge about the 3 dimensional structure of the protein(s) involved is of utmost importance. Generally, a certain function is ascribed to a region of the protein on the basis of bioassays. This functional unit is called a domain.

With the availability of complete genome sequences of prokaryotes, archea and eukaryotes however protein domains are presently more classified on the basis of sequence homology. Thus a domain can be described without necessarily knowing its function. Obtaining structure information either by molecular modeling or experimental methods for these domains is one of the big challenges for the future. This novel approach is often called the structure based functional genomics.

Determination of domain boundaries

Irrespectively whether structure determination projects are motivated by biological questions or by genome analysis in both cases the determination of the boundaries of a domain is a critical step. This is generally performed on the basis of sequence conservation. Several WEB servers are available that each by different criteria, have classified all presently known (parts of) proteins in domain families and superfamilies. These classifications together with BLAST and FASTA searches, secondary structure prediction programs on the basis of primary amino acid sequences as well as more advanced structure prediction servers help to determine the domain boundaries. Sometimes however experiments, such as proteolytic digestions or functional assays need to be performed to exactly determine domain boundaries

Cloning of a protein domain

When the boundaries are known for the domain of interest, PCR primers are designed that permit amplification and cloning of the DNA that codes for the protein domain. The PCR product is cloned into a suitable (prokaryotic) expression vector. After conformation of the successful cloning of the PCR fragment using restriction enzyme analysis, and sequencing, expression and solubility of the recombinant protein can be tested in a suitable host cell

Purification of a protein

Most expression systems that are presently used (His-tag fusion, GST-fusion, the Intein-system, MBP fusion, thioredoxin-fusion) allow fast separation of the recombinant protein from the bulk protein of the host, followed by a cleavage of the protein of interest from its fusion partner. After this step the protein is generally not pure enough for structure determination, therefore additional purification steps are performed. The first step could be an ion exchange step, a hydrophobic interaction or both, followed by a gelfiltration. Most proteins are after these steps essentially pure and after concentration and buffer exchange using a amicon concentrator, structure determination experiments can be performed.

back
index page wetlab
nmr homepage