The term ‘native protein’ is possibly one of the most misused terms in proteomics. The term implies that the tertiary and quaternary structure and biological activity of proteins are preserved. In reality, commonly used additives such as non-ionic detergents and even protease inhibitors can alter the native conformation of proteins.
The use of detergents
The polyoxyethylene sorbitan series detergents such as Tween 20 and 80 (and the lesser known Tweens 40, 60, 65 and 85) are widely used to prevent protein aggregation and other non-specific interactions while generally preserving biological activity. However, their high micellar molecular weight complicates their removal from protein solutions. Tween 20 is commonly used at concentrations over six-times its critical micelle concentration where over 85% of the detergent mass exists as 38 kDa micelles Citation[1]. For Triton X–100, the mean aggregate is 143 monomers and the mean micellar molecular weight is over 90 kDa Citation[2].
Further, the binding of detergents can radically change protein molecular mass. For example, the 460 kDa insulin receptor in the presence of Triton X–100 becomes a complex of over 1000 kDa Citation[3]. Cytochrome P-450 exhibits a molecular mass of 100 kDa in CHAPS, but over 300 kDa in sodium cholate Citation[4]. It is both interesting and important to note that lowering the detergent concentration can also effect protein mass. Adenylate cyclase is twice its molecular size in 0.01% Lubrol than it is in 0.1% Lubrol Citation[5].
Is this native?
Protease inhibitors & the ‘preservation’ of native state
Though considered a preservative, EDTA can actually drive the denaturation or partial denaturation of some proteins. EDTA has been shown to cause irreversible denaturation and aggregation of zinc-binding proteins Citation[6]. Even more disconcerting is the finding that the unfolding of zinc-binding domains by EDTA can lead to non-specific protein-protein interactions Citation[7]. For platelets incubated with EDTA, the irreversible loss of platelet aggregation and a diminished capacity to bind fibrinogen, corresponding to modification of the membrane glycoproteins has been reported Citation[8].
PMSF and AEBSF covalently bind to serines in the active sites of serine proteases. Because it is so stable, AEBSF can continue to modify proteins for several days, where it may non-specifically bind and modify Tyr, Lys, His and N-terminal residues Citation[9].
Are these native proteins?
Bringing proteins in from the cold
The native conformation of some proteins may exist over a relatively narrow temperature range. For example, streptococcal nuclease begins unfolding at temperatures below 4°C or above 38°C Citation[10]. Approximately 4% of the plasma proteins are cryoprecipitable Citation[11]. While the reversible cold denaturation of many proteins occurs at temperatures near −20°C, some proteins have been shown to denature at temperatures above freezing. In a recent review, Privalov compiled a list of over 50 enzymes that are inactivated over the 0–10°C range, thought to result from the reversible dissociation of these proteins into their subunits at low temperature Citation[12]. Hence, the widely practiced isolation and storage of proteins in the cold might not be appropriate for all proteins.
Further, there are growing concerns over lyophilization to preserve proteins for biologics, since freeze-drying can drive disulfide interchange and other protein modifications Citation[13].
Protein therapeutics
By 2008, the US FDA had approved over 130 proteins for therapeutic and diagnostic use Citation[14], and at least 400 other proteins were in the advanced stages of testing or clinical trials Citation[15]. In 2010, Baker et al. published a list of 29 FDA-approved recombinant and plasma-derived protein therapeutics for which 18 had reported immune response Citation[16].
For example, anti-erythropoietin antibody production has been reported during treatment with recombinant human erythropoietin Citation[17,18]. Formulations of recombinant human erythropoietin containing Tween 80 have been shown to contain micelle-associated protein thought to be the cause of increased immunogenicity and loss of therapeutic efficacy Citation[19].
While the consensus is that current evidence does not support the possible link between vaccines containing thimerisol and neurodevelopmental disorders such as autism, the hydrolysis of thimerisol yields thiosalicylic acid and free ethylmercury which can form adducts with protein free thiols Citation[20,Citation21]. In 2001, the European Union banned thimerisol and the FDA has removed the preservative from most vaccines.
Are these native proteins?
Native is a relative term
The hyperthermophile Methanopyrus kandleri discovered on the walls of deep sea hydrothermal vents can grow and reproduce at 122°C Citation[22]. In Antarctica, tardigrades can live for months at temperatures below −22°C and at least one species has been shown to survive brief exposure to −196°C in its hydrated state Citation[23]. In their anhydrobiotic state, tardigrades have been shown to survive the conditions of space Citation[24].
It would seem that ‘native’ is a relative term.
Acknowledgements
The author would like to thank David Green, Srikanth Kakumanu and JD Herlihy for their critical review of this manuscript.
Financial & competing interests disclosures
GB Smejkal is an Affiliate Assistant Professor at the University of New Hampshire, Hubbard Center for Genome Studies. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.
No writing assistance was utilized in the production of this manuscript.
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