An Engineered Sso7d Variant Enables Efficient Magnetization of

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An engineered Sso7d variant enables efficient magnetization of yeast cells Carlos A Cruz-Teran, Kaitlyn Bacon, Nikki McArthur, and Balaji M Rao ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00084 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 10, 2018

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ACS Combinatorial Science

An engineered Sso7d variant enables efficient magnetization of yeast cells Carlos A. Cruz-Teran, Kaitlyn Bacon, Nikki McArthur, and Balaji M. Rao* Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC

*

Address Correspondence to: Box 7905, Engineering Building I, North Carolina State University, Raleigh, NC 27695, Phone: 919-513-0129 Fax: 919-515-3465 Email: [email protected]

Running Title: Sso7d variant enables yeast magnetization

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Abstract Magnetization using cheap and minimally toxic materials such as iron oxide nanoparticles can enable easy separation of cells from culture medium and is relevant to several industrial applications. Here we show that cell surface expression of a mutant protein that binds iron oxide can enable efficient magnetization of yeast cells. We screened a combinatorial library of mutants derived from the Sso7d protein scaffold, to isolate proteins that exhibit preferential binding to iron oxide. One of the isolated mutants, SsoFe2, was chosen for further characterization. Yeast cells expressing SsoFe2 as fusions to a cell wall protein – but not other Sso7d mutants with similar overall protein charge or amino acid composition – preferentially bind iron oxide when present in a solution with high protein concentration, and in the presence of 1000-fold excess of competitor yeast cells. Moreover, co-expression of cell surface SsoFe2 enables efficient magnetic capture and separation of yeast cells expressing an enzyme (glucose oxidase) on the cell surface from yeast culture medium, and solutions with high protein concentration and/or containing other metal oxides. Therefore, SsoFe2-enabled magnetization can enable a range of industrial and biotechnology applications where easy separation of cells or organelles from complex media is desirable.

Keywords: Sso7d, magnetization, cell capture, yeast surface display, magnetic separation

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Magnetization of yeast cells by functionalizing them with a magnetically responsive material such as iron oxide is highly relevant to several applications in biotechnology1. For instance, ease of separation of magnetized yeast cells from a complex medium using a magnet can reduce processing times and costs in several industrial applications, including biofuel production, biomining, and bioremediation2. Additionally, easy separation may enable reuse of cells in applications where yeast serves as a whole cell biocatalyst. Importantly, iron oxide nanoparticles show little toxicity towards yeast, making it an attractive material for yeast magnetization 3. Nonspecific adsorption of magnetite or maghemite on to the yeast surface has been used to magnetize yeast1. However, interaction of the yeast cell surface with maghemite or magnetite can be inhibited by the presence of competitors such as proteins commonly found in cell culture medium. Magnetization of yeast has also been achieved by generating an iron oxide layer on the cell surface using an in situ chemical reaction4 or formation of magnetic particles in the intracellular space through iron sequestration5. As an alternative to these methods, here we show that yeast cell surface expression of an engineered protein with affinity for iron oxide can be used for efficient magnetization of yeast. The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus has been shown to be a versatile scaffold for generating binding proteins for a wide spectrum of targets6. Pertinently, Sso7d mutants with affinity for silica surfaces have been previously isolated from a combinatorial library using yeast surface display, wherein mutant proteins are tethered to the cell surface as fusions to the Aga2 subunit of the cell wall protein a-agglutinin7. Therefore, we screened a yeast display library of ~ 108 Sso7d protein mutants using multiple rounds of magnetic screening and additional random mutagenesis, to isolate proteins that bind to iron (II, III) oxide nanopowder at pH 7.4 (Figure 1); the Sso7d library was previously generated by



mutagenesis of ten surface-exposed residues on three beta-strands (Figure S1)6. Briefly, in each round of magnetic screening, yeast cells expressing Sso7d mutants were incubated with iron oxide powder and magnetized cells were separated using a magnet. The colloidal stability of the iron oxide powder was not characterized; we noted that yeast could get magnetized by simply resuspending the powder regardless of its dispersion state. To ensure selection of proteins that can magnetize yeast in complex media, all screening was carried out in the presence of a large excess of competitor proteins. Additionally, to further increase stringency of selection, the amount of iron oxide nanopowder used in successive rounds was reduced, and screening was conducted in the presence of increasing concentrations of competitor yeast; cells expressing the wild-type Sso7d protein, or cells not expressing any Aga2-protein fusions, were used as competitors.

Figure 1: Overview of screening procedure used to isolate iron oxide binding proteins from a combinatorial library of Sso7d mutants.


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After six rounds of magnetic screening and an intermediate round of random mutagenesis, a pool of yeast cells that bound iron oxide nanopowder in the presence of 1% BSA and a 1000--fold excess of competitor yeast was obtained. Plasmid DNA was isolated from these cells and 10 clones were sequenced to identify the Sso7d mutants that bind iron oxide and enable magnetization of yeast when expressed as cell surface fusions (Table 1). Two clones, SsoFe2 and SsoFe10, were identical. Enrichment of mutations to basic residues was observed in the three beta-strands mutagenized to construct the Sso7d library; the mutagenized region is referred to as the 3B region hereafter. A greater prevalence of mutations to arginine was also observed (Table 1, Figure 2a). Protein/Position WT Sso7d SsoFe1 SsoFe2 SsoFe3 SsoFe4 SsoFe5 SsoFe6 SsoFe7 SsoFe8 SsoFe9 SsoFe10

20 K I R I V F Y L Y R R

21 K Q R R S K T R S L R

23 W L R I K R R A R I R

25 V S K K H R K S K K K

28 M C C C K C C G C C C

30 S R R R A K L K R F R

32 T W Y V R T Q V R C Y

34 D D D D D D D D D N D

40 T F Y S H C R R R L Y

42 R R R N S R R K N K R

44 A R I R C S K C F R I

Table 1: Sequences of iron oxide binders isolated from a library of Sso7d mutants. Positions mutagenized in the initial Sso7d library are shown in bold font. This result is consistent with previous studies wherein enrichment of basic residues was observed in proteins and peptides that bind iron oxide and other metal oxides, which are usually negatively charged at neutral pH 8–11. Interestingly, all ten clones sequenced contained at least one cysteine residue, most frequently at position 28 (Table 1, Figure 2a). Notably, it has been observed that cysteine residues commonly coordinate binding to iron (III) heme groups, and sulfur-containing 5 ACS Paragon Plus Environment


groups are known to coordinate with iron12,13. Cysteine residues are also frequently found in peptides that bind to type II-VI semiconductors14. One of the selected Sso7d mutants, referred to as SsoFe2 hereafter, was chosen for further characterization. SsoFe2 was chosen because it had the highest isoelectric point (pI) among the mutants, and the sequence was observed twice in the sequenced population (Table 1). Figure 2b shows the expected electrostatic potential map of the 3B region in SsoFe2 and the corresponding map for WT-Sso7d. The 3B region is slightly more positively charged and less hydrophobic in SsoFe2, suggesting that that basic residues in this region may mediate interaction with the negatively charged iron oxide. The estimated isoelectric point (pI) of SsoFe2 is 10.1; by comparison, WT-Sso7d has a pI of 9.9. It is important to note that screening of the yeast display library was conducted under high ionic strength conditions (137 mM NaCl), which is expected to shield long-range electrostatic interactions. Therefore, basic residues in the 3B region of the selected mutants likely mediate close-range interactions with negatively charged groups on the iron oxide surface.


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Figure 2: Analysis of mutations in Sso7d mutants that bind iron oxide. (a) Amino acid composition of the Sso7d mutants isolated from a combinatorial library. (b) Electrostatic potential of the 3B region of WT Sso7d and SsoFe2. Images were generated using the UCSF Chimera package 1.10.215.

The binding affinity of a protein for a surface is commonly quantified using a Langmuir adsorption isotherm and soluble recombinant protein7. However, fitting the protein adsorption process to a Langmuir isotherm to calculate an equilibrium binding constant may result in erroneous estimates of affinity, since the underlying assumptions of reversible protein adsorption to a surface are often not met16. More importantly, such an equilibrium constant is less relevant 7 ACS Paragon Plus Environment


in the context of yeast magnetization, wherein the protein is tethered to the cell surface and not present in soluble form. Therefore, as an alternative, we developed a competition assay to assess the affinity of SsoFe2 for iron oxide in the desired context of cell surface expression, relative to similar proteins. Two other well-characterized Sso7d mutants – Sso7d-lysozyme (pI 8.9) and Sso7d-hFc (pI 9.9) – were chosen for comparison with SsoFe2 (Table S1)6,17. Sso7d-lysozyme was chosen because it contains four cysteine residues in the 3B region. Sso7d-hFc was chosen due to its high pI, similar to WT-Sso7d and SsoFe2. Yeast cells displaying SsoFe2 were combined with a 1000-fold excess of yeast cells displaying WT-Sso7d, and the mixture was incubated with a limiting amount of iron oxide nanopowder in the presence of 1% BSA. Here, WT-Sso7d acts as a competitor to SsoFe2 for binding to iron oxide. The presence of several surface exposed basic residues in the 3B region of WT-Sso7d, similar to SsoFe2, make WTSso7d a reasonable choice for use as a competitor. After incubation, cells binding to iron oxide were isolated with a magnet and the recovery of SsoFe2-displaying cells was quantified (Figure 3a). Nearly 70% of yeast cells displaying SsoFe2 were recovered. Strikingly however, in similar experiments, less than 10% of yeast cells displaying Sso7d-lysozyme or Sso7d-hFc were recovered. Importantly, the yeast surface expression levels of all proteins were found to be similar, as assessed by flow cytometry (Figure S2). These studies do not quantify the binding strength of SsoFe2. Nevertheless, we can conclude that yeast cell surface expressed SsoFe2 has higher affinity for iron oxide relative to other Sso7d mutants. Further, since SsoFe2 was selected under conditions with limiting iron oxide and wherein a 1000-fold excess of yeast displaying WT-Sso7d, and 0.1 mg/mL recombinant WT-Sso7d fused to streptavidin was present (Figure 1), it is reasonable to expect that SsoFe2 has higher affinity for iron oxide than WT Sso7d. It is also worthwhile to highlight that the ~ 7-fold difference in recoveries between cells displaying


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SsoFe2 and those displaying Sso7d-hFc is significantly lower than conventional cell separation methods such as flow cytometry wherein an antibody specific to a cell surface protein is typically used to discriminate between cell types. This is because of the promiscuous binding of iron oxide to many proteins, unlike antibodies18–21. Comparison of SsoFe2 with Sso7d-hFc and Sso7d-lysozyme also provides insight into the role that the specific amino acid composition of SsoFe2 plays in mediating binding to iron oxide. Sso7d-lysozyme has four surface-accessible cysteines, and Sso7d-hFc has similar pI as WT-Sso7d. Yet, unlike SsoFe2, Sso7d-lysozyme or Sso7d-hFc cannot compete with WT Sso7d for binding to iron oxide. Thus, the cysteine residue or overall positive charge alone does not mediate the binding of SsoFe2 to iron oxide; rather, SsoFe2’s specific amino acid sequence arrangement in the 3B region dictates binding.



Figure 3: Magnetic capture of yeast cells expressing SsoFe2 on the cell surface. (a) Capture of yeast cells expressing SsoFe2, Sso7d-hFc, or Sso7d-lysozyme, from a solution with high protein concentration, in the presence of 1000-fold excess competitor yeast expressing WT Sso7d. (b) Capture of yeast expressing (induced) or not expressing (uninduced) WT Sso7d or SsoFe2 in the presence of 1% BSA. (c) Capture of yeast cells expressing GOx, or both SsoFe2 and GOx from a solution containing high protein concentration. (d) Capture of yeast cells expressing GOx, both Sso7d-hFc and GOx, or both SsoFe2 and GOx from culture medium. Capture is quantified as the ratio of GOx activity of the iron oxide powder to GOx activity of cells in suspension. Error bars


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correspond to standard error of the mean (SEM) from at least three replicates (**p

An Engineered Sso7d Variant Enables Efficient Magnetization of

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