Throughout my 3 years of studying biochemistry – especially regarding that of protein folding discussed in class – the curriculum has always emphasized the important implications of protein denaturation due to excessive heat, pH, etc. The biology curriculum has etched within our minds the importance of protein limitation as they constitute a significant percentage of enzymes – the ultimate catalyst for life. However, the 3 years of biology (this year included) has failed to enlighten about denaturation’s counterpart: renaturation and the powerful implications of protein refolding. The aforementioned topic has always held value, tingling my curiosity, for the study of renaturation can bring about powerful remedies in the bio-medical field (one that I plan to explore in the near future)
Currently, medical innovation have given rise to “elegant and well established recombinant DNA methodologies [which] have set the stage for the production of heterologous proteins in microbial hosts” (Lilie, Schwarz, Rudolph 1998); through the application of recombination, a plethora of host microbial organisms have been genetically modified to produce certain desired proteins (insulin being a fine example). Unfortunately, the limited regulation of recombination/protein production has resulted in multiple cases of over production; “An increase in the concentration of non-native polypeptides due to high expression levels seems to be responsible for aggregation of the recombinant protein. This assumption was quantified in a kinetic model that analyzed the yield of native protein as a function of the competition between folding and aggregation” (Lilie, Schwarz, Rudolph 1998). The kinetic model establishes the tendency for protein production rates to vary time to time, to the point where the number of proteins requiring chaparonin enzymes for proper folding outweighs the number of chaparonin (protein folding) complexes present. These “extra” protein segments then accumulate into insoluble, wasted protein aggregates, inflicting massive losses and immense reduction in protein production efficiency. However, researchers Hauke Lilie, Elisabeth Schwarz, and Rainer Rudolph of Germany have investigated the usage of protein revitalization and renaturation as a solution to these inclusion protein aggregates. Utilizing different methods of renaturing protein aggregates in E.coli hosts (CoFactor addition, Sulfur methods, etc.), Lilie, Schwarz, and Rudolph have found that renaturation requires “the control of parameters such as temperature, pH or redox conditions, the presence of low molecular weight compounds in the renaturation buffer” (Lilie, Schwarz, Rudolph 1998) for unimpaired functioning. In addition, several other components in renaturation baths are required to A) prevent future protein aggregation and B) to facilitate efficient and optimal conditions for protein refolding. According to the German team’s trials, “Specific cofactors, such as Zn2+ or Ca2+, can stabilize proteins already at the level of folding intermediates, thus, preventing off-pathway reactions. Besides such cofactors and prosthetic groups, a large series of low molecular weight additives are, in certain cases, very efficient folding enhancers: non-denaturing concentrations of chaotrophs, such as urea or GdmCl, for example, are essential for the renaturation of reduced chymotrypsinogen A and have been shown to promote folding of several other proteins” (Lilie, Schwarz, Rudolph 1998), providing a solution to the pesky inclusion protein aggregates and shedding light to a promising field of research and development. Thus, renaturation stabilizes the intermediates of proteins, preventing them from entering “off road” pathways. In addition, the process breaks down the inclusion aggregations, restructuring the protein fragments to its original format. Through the aforementioned procedure, A) protein production rates would reach its prime state, producing more of the essential proteins demanded (i.e. insulin) while also B) opening the doors for more applications utilizing the renaturation as a solution to the detrimental effects of denaturation.
After reading the esoteric article (wow that was laborious to get through!) presented by researchers Hauke Lilie, Elisabeth Schwarz, and Rainer Rudolph, several other questions regarding the renaturation process still linger. How will the rates of renaturation compare to rates of protein aggregate formation and denaturation? Can renaturation still function without the removal of the denaturant? How costly will the process of renaturation be, and will it present itself as a practical solution (financially-wise) for inclusion protein aggregates? Hopefully, these questions will be answered as research on the renaturation process proliferates through the biological community, and as I trudge deeper into the topical literature. Until next time!
Lilie Hauke, Schwarz Elisabeth, Rudolph Rainer, 1998. Advances in refolding of proteins produced in E. coli. Retrieved from http://web.mnstate.edu/provost/Advancesproteinrefolding.pdf