As a protein biochemist and a Ph.D. student, I was given the task to express a eukaryotic protein in a bacterial system. And to say that I was having a hard time would be an understatement. It took me many PCRs, cloning and transformations to get to the right construct that would eventually express the desired protein.
After spending a good two years in the darkroom developing western blots that showed me nothing but the molecular weight marker or the biotinylated protein bands from bacteria, the first time I saw a faint band of my protein was undoubtedly a day of extreme euphoria. I learned that there is no single sure way of expressing a protein. Proteins, like people, are unique. The individual properties of a protein, i.e. its three-dimensional structure, the number of disulfide bonds, the presence or absence of hydrophobic regions or transmembrane domains, and post-translational modifications like N or O- glycosylations, are all factors that make a difference and play a defining role in the ease or difficulty in protein expression. To express proteins in similar systems is definitely easier. However, the challenge arises when you have to switch to systems that are not essentially the same, which is when you need additional tools for expression. I want to share with you some of the tools that I learned, which helped me express tough proteins:
To understand this, we must first understand what codon optimization means. Simply put, it means introducing the right kind of codons to our construct so that they can be read with ease by the tRNAs in the host bacteria (if this is where you are expressing your protein) and translated into amino acids without difficulty. Therefore, it is a method of increasing the translational efficiency of the protein by interchanging codon sequences on the target gene to make it compatible with a reading by the tRNAs in the host strain.
Different biological systems have different numbers of tRNAs available for the codons and each system has a certain number of tRNAs that respond to each codon. However, if the ratio of the tRNAs to the codons is not right, it will hinder the expression of a recombinant protein in a different host system. Therefore, it is essential to codon optimize the gene, so that the gene has the correct codons which can be efficiently read by the available tRNAs present within the host system, thereby allowing optimum protein expression. Several companies like Gene Art, Artes, etc. codon optimize the desired gene to be expressed.
When we want to express a gene in order to produce a protein, it is essential we remember that we need to purify the protein unless we intend to work with the crude extract, i.e. whole cell extract. In order to purify the protein, we must attach certain tags to it so we can use methods such as affinity purification to isolate and purify it.
There is an excellent range of tags that have been thoroughly studied. They can be attached either to the N- terminus or to the C-terminus of the protein. Some such tags are the Strep-II tag, the His tag, GST, Trx, etc. These tags help the protein be separated from the vast number of other proteins in the crude extract that are being produced by the bacteria. They can bind to a column and be eluted with special buffers – the result is a pure batch of your protein of interest.
Chaperones and Foldases
A tough gene to express is like a tough nut to crack. You need additional force. For a protein to work optimally it is essential that it is folded the correct way. The cytoplasm has a reducing environment that disables the proper formation of disulfide bonds thereby affecting its folding. In order to aid the protein to fold correctly and avoid the formation of insoluble misfolded aggregates, we need helper plasmids with chaperones and foldases. Some expression strains have additional chaperones inside them while others do not. Some well-known chaperones and foldases (e.g. DsbA/C, Skp, FkpA, GroEL/ES, DnaK/J/GrpE) can be co-expressed in helper plasmids.
Certain tags can also help in the folding of the protein. One such example is the Maltose binding protein (MBP). This 42.5 kDa protein not only helps the protein to become more soluble but also aids folding. One more advantage of MBP is that it can be bound on an amylose column and help in purification too. Both periplasmic and cytoplasmic chaperones can be selectively chosen depending on where the protein is expressed in the host.
Where your protein is finally being expressed, be it in the cytoplasm, the periplasm, or extracellular in the medium has its consequences. If a protein has disulfide bonds (S-S) between cysteine residues, it will much prefer the oxidizing environment of the periplasm as opposed to the reducing one in the cytosol. An oxidizing environment will help the protein to form the right bonds and acquire the right structure.
For specific translocation of the protein to specific regions in the cell or outside, one needs special signal sequences at the N- terminus, which guide the protein to that specific area. For example, the PelB leader sequence derived from Pectobacterium carotovorum directs the protein to the periplasm of the gram-negative bacteria while the signal sequence from Streptomyces lividans leads to the extracellular secretion of proteins into the culture medium. You must decide beforehand where you want the protein to be sent and why.
Pathway for Protein Expression
Different pathways of protein expression are also important in determining the efficiency of protein folding. There are two major pathways for protein expression in E.coli – the Tat (Twin Arginine translocation pathway) and the Sec pathway (Secretion pathway). The Tat pathway leads to the transportation of already folded proteins whereas the Sec pathway allows folding after translocation into the periplasm (Post-translational).
Another pathway that converges with the Sec pathway is the SRP (Signal recognition particle), which allows for simultaneous folding and translocation of the protein (Co-translational) (Valent et al., 1998). For proteins with disulfide bonds, an oxidizing environment such as that in the periplasm is preferred in comparison to the reducing one in the cytosol so disulfide bonds are formed properly. Therefore, it is preferable to have signal sequences such as the PelB, which works via the Sec pathway, or a DsbA signal, which allows an effective SRP pathway (Schierle et al., 2003), (Thie et al., 2008).
Choosing the right expression strain is of utmost importance. Different strains have different features: for example, you can choose a bacterial strain that is protease deficient since the recombinant protein may be recognized as alien or might be misfolded in the host expression strain leading to its degradation. To avoid this, we must choose protease-deficient strains depending on where the protein localizes-cytoplasm or periplasm. There are many different and specialized strains available so take a look at what is available and choose your expression strain with care. A good website for looking up different E.coli strains and plasmids is Ecoliwiki.net.
Miscellaneous Factors: Media, Temperature & Induction
The right media is important for optimal cell growth and division. You can test different media ranging from complex, minimal or defined in small test cultures (30 ml) to see which yields the maximum expression of your protein. Other criteria like the right conditions of temperature and shaking are essential as well. Some proteins express well at lower temperatures while others at higher temperatures.
The right molarities and time period of induction with Isopropyl-?-D-1-thiogalactopyranoside (IPTG) must also be tested. Using small-scale tests cultures, the different conditions like temperature, induction, and media can be tested one by one, keeping others constant. Running an SDS PAGE and western blot of the different probes can help you distinguish the best conditions for expression.
Right Period of Cell Incubation
The right length of cell incubation is also crucial for proper cell maintenance and to prevent protein degradation. Sometimes, the cell culture can be left incubating for longer than necessary, which leads to cell lysis, the release of toxic proteases, and the loss of your desired protein. Therefore it is important to harvest your cells at the right time. A time-lapse study at different time points can be done by retrieving samples at different hours and testing for protein amounts. Too late or too early cell harvesting leads to the loss of both effort and protein.
These are some of the basic points I learned along the way to express proteins in bacteria and I do hope that these criteria help you when you are planning your experiments; a well-planned experiment definitely a lot of time saved. If you know more useful tips on protein expression, feel free to leave them in the comments section below. We would be delighted if any of the mentioned tips help you in your work!