Term |
Explanation/Definition |
| Cell density at induction |
This refers to the stage of cell growth at which protein expression is
induced. Prior to expression, cells are
generally grown until they reach an optimal stage of growth, at which time expression is induced. The growth of the cells is
measured by their density, which is usually monitored by the spectroscopic absorbance of the cell culture at a given wavelength,
often 600nm or thereabouts. The absorbance, or optical density, is usually denoted as Aλ or ODλ,
where λ is the wavelength of interest. |
| Cell disruption method |
The technique used to lyse cells. There are several different techniques which can be used to lyse cells,
eg. sonication, French press, freeze/thawing. |
| Cell lysis |
The process by which cell walls are broken down to release the contents of the cell, eg. DNA, cellular proteins,
the protein of interest. |
| Chain length |
The number of amino acid residues in the protein sequence |
| Class |
Refers to the structural class of the protein as defined by the SCOP database. Eg. Alpha, beta, alpha/beta,
small proteins. More information on the SCOP database can be found here.
|
| Denaturant |
A chemical agent which disrupts protein structure, causing it to unfold. Denaturants are often used to dissolve
aggregates and/or inclusion bodies, and include salts such as guanidine hydrochloride, organic agents such as urea,
and also detergents such as sarkosyl. |
| Disulfide bonds |
Refers to the number of disulfide bonds formed by the protein in its native state. |
| Expression |
The translation of a gene to produce a protein. In recombinant methods expression is usually
directed to produce large quantities of a target protein. |
| Expression host |
The organism in which the protein is being expressed eg.E.coli. |
| Expression strain |
The strain of organism in which the protein is being expressed. |
| Expression vector |
The vector into which the gene of interest has been cloned. |
| Fusion tags |
Some proteins may be expressed with a specific tag to facilitate purification or protein activation.
Purification tags are generally encoded within the same plasmid vector and the protein is expressed as a single polypeptide chain
composed of two proteins joined by a short linker. The tag may often be removed after purification or refolding by cleavage
using a specific protease which recognises a specific unique sequence in the linker region. |
| Inclusion bodies |
Dense amorphous aggregates of misfolded protein, usually insoluble. |
| Induction |
The initiation of protein expression, usually by the addition of a specific compound or a change in
conditions (eg.temperature) which activates the promoter region of the target gene. Such compounds include isopropyl-D-thiogalactopyranoside
(IPTG), arabinose and nalidixic acid. |
| Lytic agent |
Enzymes or chemicals used to assist cell lysis. Such agents usually weaken, permeate or break down
cell walls. |
| Molecular chaperones |
Proteins which assist and facilitate the correct folding of the target protein. If used, molecular
chaperones are usually included in the refolding buffer. |
| Molecularity |
The oligomeric state of the protein. eg. monomer, dimer, trimer etc. |
| Organism |
Refers to the species from which the protein was originally derived. |
| Post-refolding analysis |
Techniques and assays used to assess the quality of the protein after renaturation. |
| Pre-refolding purification |
Purification steps performed using the denatured unfolded protein, prior to refolding of the protein.
ie. purification steps performed under denaturing conditions. |
| Protein construct |
Some proteins may not be produced in their full length form, or may be attached to other proteins to create
chimeras. Therefore, the original sequence of the protein may be different to that which is actually refolded,
and the actual construct which is refolded should be identified.
- Full length wild-type protein – refers to a construct in which the full protein is expressed and refolded.
- Domain only – refers to a construct in which only a portion of the protein is expressed and refolded.
- Chimeric protein – two proteins (or parts of two proteins) are expressed as a fusion protein in a single polypeptide.
- Variant(s) – the protein produced contains one or more amino acid substitutions its sequence.
|
| Redox agent |
Compounds which assist the correct formation of disulfide bonds. They may be included in the
refolding buffer, or added to the protein prior to or after the refolding process. |
| Refolding/Renaturation |
The process by which a protein which has been denatured/unfolded adopts its to its native functional structure.
The basic principle of protein refolding is the removal of denaturant from the system. Proteins are refolded by an
exchange of buffers - from denaturant-containing buffer (solubilization buffer)
to no denaturant (refolding buffer). |
| Refolding additives/chemical chaperones |
Inorganic compounds added to the refolding buffer to assist the correct folding of the protein.
Such compounds may function by stabilizing the native conformation of the protein (eg.arginine, glycerol), acting as chelators (eg.EDTA), or
preventing aggregation (eg. trimethylamine N-oxide (TMAO), PEG-3500) |
| Refolding buffer |
The buffer in which the protein is refolded. The refolding buffer contains no denaturant,
or in some cases very little denaturant. This buffer may also contain refolding additives (eg.arginine, glycerol)
or molecular chaperones which assist the correct folding of the protein. |
|
Refolding method |
The method used to refold the protein. |
| Refolding pH |
The pH of the refolding buffer. |
| Refolding temperature |
The temperature at which refolding is performed |
| SCOP family |
The classification of the protein with other structurally related proteins as defined by the SCOP database.
More information about the SCOP database can be found here. |
| Solubility |
Refers to whether the protein is present in a soluble (supernatant) or insoluble (pellet) form after
expression and cell lysis. Sometimes the protein may be partially soluble and hence present in both supernatant
and pellet. |
| Solubilization |
The process by which insoluble inclusion bodies are dissolved. Solubilization usually involves
the use of a denaturant. |
| Solubilization buffer |
Buffer used to dissolve the isolated insoluble inclusion bodies.
The solubilization buffer almost always contains a denaturant or is of alkaline pH. |
| Wash buffer |
The buffer used to wash the inclusion bodies
after cell lysis and prior to solubilization. The wash buffer
often contains detergents (eg.Triton X-100) or low concentrations of denaturant (eg.urea) to remove loosely-bound
contaminating proteins and other cellular debris. |
