Musings from my graduate school days … or have you ever met a recombinant protein you didn’t like?

In graduate school, I learned the secret recipe of a colleague’s “Magic Inclusion Body Buffer.” I still have that coveted recipe card in my now-vintage, handwritten recipe-card box, painstakingly indexed.

What great hopes we had then for the expression of specific recombinant plant proteins in bacterial E. coli cells. Imagine! Protein made in the convenience of your home lab, in as many spinning culture flasks as you could fit in your incubator rather than my time-consuming collection of the tiniest of flower material, daily, for weeks and weeks. Gone were those laborious and long trips to the Cornell University greenhouses over the hot summer months—only to find that there were barely any flowers in bloom to harvest.

YES, recombinant proteins! By the miraculous advancement of science we now had the ability to mass produce the protein of interest in only hours, right in the wonderfully air-conditioned lab. Even better, recombinant protein production could also include a protein fusion tag for easy purification. This was truly “Better Living thru Chemistry.”

Alas, although the concept of protein overexpression sounded great, it was not all the free and soluble protein that you could hope for. The use of that “Magic Inclusion Body Buffer” recipe turned out to be a necessary evil. The mega amount of protein expression turned into too much INSOLUBLE protein—not enough soluble or secreted protein to use for downstream experiments. The only hope for the recombinant fusion protein project was to extract the inclusion bodies with the extremely harsh chemicals in the “Magic Inclusion Body Buffer”, including Guanidine HCl, with subsequent slow dilution to reduce the concentration for recombinant protein refolding. Unfortunately this was too harsh for my delicate protein to refold as expected, rendering it useless and leaving me with months and months of work with nothing to show for it.

Current researchers are now using a plethora of fusion protein expression systems, including purification tags like glutathione S-transferase (GST), His6, thioredoxin, and maltose binding protein. Systems can be optimized to produce greater ratios of soluble to insoluble protein using tightly-regulated inducible promoters, different cell strains and media component optimization. There are many tricks of the trade needed to produce complex and even toxic proteins in a bacterial culture system successfully. Yet, aggregation and inclusion body formations still plague systems, even with optimization. Overexpression taxes the E. coli chaperone protein folding systems (DnaK/Hsp70/ DnaJ/GrpE/GroEL/Hsp60/GroES).

So some researchers have turned to solubilizaton and refolding methods to recover proteins. Another alternative, one that my “Magic Inclusion Body Buffer” colleague resorted to, involved the refolding of overexpressed catalytic protein subunits while mixed with overexpressed chaperone proteins. Success at last! He finally achieved catalytically-active proteins. I eventually changed systems as well, but instead of E. coli, I successfully produced the plant proteins in a baculovirus-infected insect cell culture system.

But I still keep that recipe card, just in case…