Chapter 4
Probing the Role of Protein Folding in Inclusion Body Formation 1
Boris A. Chrunyk , Judy Evans, and Ronald Wetzel
Downloaded by CORNELL UNIV on October 21, 2016 | http://pubs.acs.org Publication Date: April 22, 1993 | doi: 10.1021/bk-1993-0526.ch004
Macromolecular Sciences Department, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406
Point mutations in recombinant human interleukin-1β(IL-1β) have been identified which exhibit dramatic changes in the level of inclusion bodies (IBs) in E. coli producing this protein. In both wild type and mutants, a smaller percentage of IL-1β is deposited into IBs when cells are grown at 32° C rather than 42° C. Some of the mutant proteins have been purified and examined for their stabilities and folding kinetics by monitoring the fluorescence of the lone tryptophan. In addition, aggregation associated with thermal treatment of the native protein, and with the refolding of the protein from the denatured state, was monitored. Several lines of evidence suggest that at least one sequence variant, Lys97->Val (K97V), may form IBs due to an alteration in the properties of a folding intermediate. Implications of the structural locations of this and other IB mutants will be discussed. The expression of heterologous proteins in bacterial hosts sometimes leads to the deposition of the product of interest into amorphous refractile particles known as inclusion bodies (IBs) (7,2). This often poses technical problems in protein recovery, since (a) denaturing conditions are often required for solubilization of IBs, and (b) once solubilized in denaturant, refolding conditions must be identified to allow recovery of active protein. At the same time, in those cases where refolding conditions can be established, inclusion body formation may actually prove advantageous. For instance, inclusion body formation can protect the protein from proteolytic digestion during expression. In addition, since in many cases the isolated inclusion body is highly enriched in the overexpressing protein, separation of IBs from soluble cellular contents can serve as an initial purification step. It would thus be useful to be able to control inclusion body formation, and acquiring an understanding of the mechanism(s) of IB formation would seem a valuable approach to this goal. Several external factors have been suggested to influence inclusion body deposition in bacterial cells (2). To date, however, the only external factor proven to influence the balance between soluble or insoluble expression in a variety of systems is temperature (5,4). Characteristics of the overexpressed protein itself undoubtedly contribute significantly to their deposition into inclusion bodies. For 1
Current address: Central Research Division, Pfizer, Inc., Groton, C T 06340
0097-6156/93/0526-0046$06.00/0 © 1993 American Chemical Society
Cleland; Protein Folding ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
4.
CHRUNYK E T AL.
Protein Folding in Inclusion Body Formation
47
example, most proteins are only marginally stable at 25°C (5) and eventually undergo cooperative unfolding at some higher temperature (T ). The unfolded state that is populated at equilibrium at temperatures above this T would be expected to be prone to aggregation due to the exposure of normally buried hydrophobic residues (6). Similarly, proteins which require disulfide bonds to achieve net folding stability may populate at equilibrium aggregation prone unfolded states under the reducing conditions of the cytoplasm. Alternatively, aggregation of transiently populated folding intermediates may be responsible for some IBs, for example if the intermediates are poorly soluble and/or build up to relatively high concentrations as controlled by folding kinetics. How might sequence determine the fate of a freshly synthesized polypeptide chain? Since many proteins can achieve their native folds in vitro without requiring accessory factors (7), the code for folding, in many cases, is determined by the amino acid sequence alone. Alterations in the sequence by as little as a single point mutation are known to produce dramatic effects on die thermodynamic stability of a protein, the kinetics of folding, or both (8), thus leading to the kinds of species discussed above as potential sources of aggregate formation. In discussing the origins and consequences of off pathway reactions it is useful to invoke a generalized folding scheme such as that shown in Figure 1. Here, P represents proteolytic digestion and Agg represents aggregate formation. In this scheme, there exists a potential for aggregate formation or proteolysis at any stage in the folding of the polypeptide chain. Mutations can alter the stability of the native protein or generate a proteolytic site, both of which can lead to decreased levels of soluble product. As suggested for P22 tailspike protein, mutations can also alter IB levels via effects on a folding intermediate (9,10). In their investigation of mutations in the trimeric Salmonella phage P22 tail spike protein, King and coworkers (9, 10) isolated temperature sensitive folding (tsf) mutations. At permissive temperatures (
