Introduction

Cell surface display is a technology that enables a desired protein to be targeted and fixed on the

outer surface of the cell thereby carrying out its assigned function in the exterior environment of

the cell. This technology overcomes several drawbacks of using whole-cell biocatalyst with

protein produced intracellularly or secreted outside the cell which face possible mass transfer

limitations due to the cell membrane, difficult and complex purification methods, and

unnecessary side reactions that occur inside the cell (Schüürmann et al., 2014; Smith et al.,

2015). Display of heterologous protein on the surface of bacteria was found to be useful for

several purposes such as peptide library screening, live vaccine development, bioadsorption and

whole-cell biocatalyst (van Bloois et al., 2011). Escherichia coli is the most commonly used

microorganism for bacterial surface display because of the wide variety of tools available for its

genetic manipulation and its ability to produce recombinant proteins in high yield. Cell surface

display requires the use of anchoring motifs to attach the desired protein to the cell surface.

Outer membrane proteins (Sousa et al., 1998), lipoproteins (Yang et al., 2008), autotransporters

(Yang et al., 2010), flagellum (Majander et al., 2005), and ice nucleation protein (INP) (Liu et

al., 2016) are among the various anchoring motifs that have been used to display proteins on the

surface of E. coli. The use of different anchoring motifs results in different performance for

surface display.

INP is one of the favorite choices of anchor for surface display. This is because it has

been proven to be a stable system with negligible effect on outer membrane integrity and cell

growth, and its ability to display not only large (up to 119 kDa) but also proteins containing

cofactors (Yim et al., 2010). INP is an outer membrane protein found in plant pathogenic

bacteria from the genus Pseudomonas, Xanthomonas, and Erwinia (Gurian-Sherman and
Lindow, 1993). It causes frost injury to plants as it is capable of nucleating ice in supercooled

water. The basic structure of INP consists of three domains; N-terminal domain, central

repeating domain (CRD), and C-terminal domain. The N-terminal domain makes up about 15%

of the entire structure, is hydrophobic and anchors the INP to the outer membrane (Liang et al.,

2011). The CRD is the longest section of the INP (about 81%), and consists of repeats with 8-,

16-, and 48- residue periodicity which provides a template for ice crystal formation in

supercooled water (Shimazu et al., 2001). The C-terminal domain is hydrophilic and is the site of

fusion to the desired heterologous protein for surface display. A study has also suggested that the

C-terminal domain is not prone to protease attack (Li et al., 2004). Despite its relatively wide use

in research, the mechanism of transport of INP to the cell surface is not clear and it lacks a signal

peptide in its sequence. It is most likely anchored to the outer membrane via a

glycosylphosphatidylinositol (GPI) anchor which is rarely found in prokaryotes but is common

in eukaryotes (Jung et al., 1998a; Kwak et al., 1999; Wu et al., 2006b).

Due to the many advantages of using INP, various researchers have chosen it as a display

anchor. The first INP to be used for surface display was InaK from P. syringae KCTC 1832 to

display levansucrase (Jung et al., 1998a) and it has since become the favorite INP among

researchers because of its proven performance. Other INPs that have been studied for its use as a

surface display anchor are InaQ (Li et al., 2012), InaV (Shimazu et al., 2001), InaPb (Liang et

al., 2011), and InaZ (Bao et al., 2015) and all are from the genus Pseudomonas. InaX from

Xanthomonas (Wu et al., 2006b) has also been shown to display transglucosidase successfully.

Despite the known advantages of INP, a drawback encountered by some researchers is that only

a portion of expressed INP were successfully transported to the outer membrane while the rest

remained in the cytoplasm where proteolysis occurred (Li et al., 2004; Li et al., 2009). This
suboptimal anchoring on the cell surface reduces the potential of the cell as a biocatalyst. As the

mechanism of transport of INP to the outer membrane is still unknown, the success and

efficiency of its surface display can only be known experimentally. A wider choice of INP

anchors may provide more variety for better surface display efficiency.

In this study, INP from Erwinia ananas IN-10, InaA, truncated to contain only the N- and

C-terminal domain was studied for its use as a surface display anchor. InaA has been studied in

1989 for its property to nucleate ice and have been successfully cloned in E. coli (Abe et al.,

1989; Arai et al., 1989). Its sequence similarity to InaK and InaZ, both truncated to contain only

the N- and C-terminal domain, were compared. All the INPs were fused by C-terminal fusion to

endo-1,4-β-xylanase (EC 3.2.1.8) which was used as a reporter protein. Xylanase is the main

enzyme responsible for the hydrolysis of xylan, the most abundant component of hemicellulose,

into xylooligosaccharides. Xylooligosaccharides has a wide range of applications from

pharmaceuticals to agricultural industry (Vázquez et al., 2000). This study aims to discover the

suitability of using InaA from E. ananas as an anchor in E. coli by comparing it to two other

INPs and to the best of the author’s knowledge, no INP from any organism of this genus has

been studied for surface display.

Materials and Method

Bacterial strains, plasmid, and culture conditions

E. coli BL21 (DE3) was used as the cloning and expression host for all plasmid constructs. All E.

coli growth was done in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L sodium

chloride). E. coli harboring pET-21a(+) plasmid backbone was grown in LB medium

supplemented with ampicillin (100mg/ml). Expression was induced by 0.1 mM IPTG when the

culture reached an optical density at 600nm of 0.6. The induced cells were grown for 24 h with

shaking at 200 rpm and temperature 25 ˚C in LB medium supplemented with ampicillin.

Plasmid construction and cloning

InaA from E. ananas IN-10 (GenBank Ascension No. X17316.1), InaK from P. syringae KCTC

1832 (GenBank Ascension No. AF013159), and InaZ from P. syringae S203 (GenBank

Ascension No. X03035) were synthesized where all INPs were truncated to contain only the N-

and C-terminal domain. The N- and C-terminal domains were flanked by restriction enzymes

NdeI and BamHI respectively giving pInaA, pInaK, and pInaZ. The synthetic genes had pET-

21a(+) as their backbone vector thereby giving it ampicillin resistance and a His-Tag at the C-

terminal of the plasmid for detection. Endo-1,4-β-xylanase (GenBank Ascension No.

GQ458016.1) was taken from labstock. The gene was from Aspergillus fumigatus and has been

cloned into E. coli BL21 (DE3) using pET21-a(+) vector giving pXyl.

For cloning, PCR was carried out on pXyl to obtain the 663 bp of xylanase without the

stop codon. The forward primer used was F: 5'-ATATGGATCCACACCCGGCTCGGAGCAA-

3' and the reverse primer R: 5'-GCGCCTCGAGAGAAACAGTGATAGTAGC-3' where the

restrictions sites BamHI and XhoI are underlined respectively. The amplified xylanase and

synthetic INPs (InaA, InaK, and InaZ) were digested with BamHI and XhoI and ligated to obtain

pInaAXyl, pInaKXyl, and pInaZXyl. The products of ligation were transformed into E. coli

BL21 (DE3). The plasmid map of the constructed INP surface display system is shown in Figure

1.
Cell fractionation

Cell fractionation was performed according to the method described by Park et al. (2013) with

slight modification. After harvesting, cells were washed with 10 mM Na2HPO4 buffer and

disrupted by three cycles of sonication (20 s each at 15% maximum output) on ice. The cells

were then subjected to short centrifugation for 5 mins at 8000 rpm to remove cells that were

partially disrupted followed by centrifugation at 8000 rpm for 45 min at 4 ˚C. The pellet was the

total membrane protein. The pellet was then resuspended in 10 mM Na2HPO4 buffer with 0.5%

(w/v) sarcosyl solution. The mixture was incubated in a water bath at 37 ˚C for 30 min. After

centrifugation at 13 400 rpm for 5 min, the pellet obtained is the outer membrane. The pellet was

washed with 10 mM Na2HPO4 buffer and resuspended in TE buffer.

The soluble fraction which was made up of the cytoplasmic and periplasmic fractions

was fractioned by the following method. Harvested cells were washed with PBS buffer. The

pellet was resuspended with 300 μl of Bugbuster (Novagen, USA), 0.3 μl of 25 U/μl benzonase,

0.1 mM PMSF, and 0.3 μl of 10mg/ml lysozyme. The mixture was put on a shaking platform for

20 min and then centrifuged at 8000 rpm and 4 ˚C. The supernatant obtained was the soluble

fraction.

SDS-PAGE and Western blot

Equal volume of loading buffer was mixed with each cell fraction (total membrane protein, outer

membrane and soluble fraction) and boiled for 10 min. E. coli BL21 (DE3) harboring pET-

21a(+) was used as a control. The samples were resolved with 15% SDS-polyacrylamide gel. For
Western blot analysis, the samples were electroblotted onto PVDF membranes. Incubation with

primary antibody, chicken anti-polyhistidine tag polyclonal antibody (Merck Millipore, USA)

with dilution of 1:1000 was done followed by secondary antibody, rabbit anti-chicken IgY (IgG)

alkaline phosphatase conjugate (Merck Millipore, USA) with dilution of 1:5000. Both antibodies

were diluted in TBST/3% BSA. After repeated washings, a developing solution BCIP/TNBT

(Merck Millipore, USA) was added to the membrane until a strong signal appeared.

Reducing sugar assay

Enzyme activity was determined by detection of reducing sugar using the 3,5-dinitrosalicylic

acid (DNS) method (Miller, 1959). 300 μl of cells (final concentration OD600nm 3) was added to

100 μl of PBS buffer and 1% beechwood xylan (Sigma-Aldrich, St. Louis, MO) dissolved in

PBS. The mixture was incubated at 37 ⁰C for 15 min. Then, the mixture was centrifuged, the

supernatant taken and 500 μl of DNS was added. The mixture was boiled for 10 mins and the

reducing sugar was determined at 540 nm. One unit of enzyme activity was defined as the

amount of enzyme releasing 1 μmol of reducing sugar (xylose equivalent) per minute under the

assay condition.

Immunofluorescence microscopy and flow cytometry

Recombinant E. coli with surface displayed INP were expressed for 24 h and harvested. The cell

pellet was washed twice with PBS buffer. The optical density of the cells was then standardized

to OD600nm 5.0 in 500 µl. Then it was blocked with PBS buffer containing 2% BSA at 4˚C for 2 h
on a rocking platform. After that washing was done twice with PBS buffer. The cells were then

stained with primary antibody, His-probe mouse monoclonal antibody (Santa Cruz

Biotechnology, USA) for His-Tag detection with a dilution of 1:500. Staining was done

overnight at 4 ˚C on a rocking platform. The cells were washed thrice with PBS buffer before

being incubated with the secondary antibody goat anti-mouse IgG-FITC (Santa Cruz

Biotechnology, USA) at a dilution of 1:100 in PBS/2% BSA. The incubation was done at 4˚C on

a rocking platform for 2 h in the dark. Finally, the cells were washed thrice with PBS buffer and

resuspended in a final volume of 300 µl before being examined under an inverted fluorescence

microscope (Nikon Eclipse Ti-S/L 100). For flow cytometry analysis using BD FACS CANTO

II, final resuspension volume was 500 µl.

Multiple sequence alignment, signal peptide and transmembrane prediction

Protein sequences were aligned using BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and

Clustal Omega. The presence of signal peptide was predicted using SignalP 4.1

(http://www.cbs.dtu.dk/services/SignalP/) while the prediction for transmembrane spans was

done using HMMTOP (http://www.enzim.hu/hmmtop/index.php).

Results and Discussion

Molecular characterization of InaA

Although INP has long been an established mode of transport and anchor of enzymes to the

surface of bacteria, most of the INPs used in research originated from the genus Pseudomonas
(Bao et al., 2015; Kwak et al., 1999; Wu et al., 2006a) and Xanthomonas (Wu et al., 2006b).

However, INPs are found in plant pathogenic bacteria causing frost injuries to plants and these

include those from the genus Erwinia. In an effort to diversify the INP anchors available for

surface display and discover better INPs for display of enzymes, InaA, was explored for its

abilities as a surface display anchor in E. coli. Full length InaA is a protein that contains 1322

amino acid residues and has a typical INP structure of an N-, CRD, and C-terminal domain.

Comparatively, InaA is longer than its full length counterparts InaK and InaZ which are made up

of 1148 and 1200 amino acids respectively. However, when truncated and only the N- and C-

terminal domains are used for surface display, the protein sequence of InaA is shorter than both

the protein sequences of InaK and InaZ. A comparison was done by protein sequence alignment

of InaA to InaK and InaZ (Fig. 2). BLASTP showed that the sequences have a similarity of only

about 59% and 62% respectively where the majority of the difference in sequence lies in the N-

terminal domain, the region that is important for surface anchoring. Although the N-terminal

domain alone is sufficient for effective surface display (Liang et al., 2013; Song et al., 2015) , the

C-terminal domain was included in this study because one study has shown that this domain is

structurally stable while the N-terminal domain is not (Li et al., 2004). The instability of the N-

terminal domain may lead to free enzyme being detected on the outer membrane without its

anchor. In this study, computational analysis using SignalP 4.1 and HMMTOP found that InaA

does not contain a signal peptide or transmembrane spans, causing its mechanism of anchorage

to the outer membrane to be uncertain. The same situation was observed for InaK and InaZ. This

is in agreement to Jung et al. (1998b) who discussed that the crossing of INP through the

cytoplasmic membrane and anchorage to the outer membrane could be achieved without

additional gene sequences or signal peptides. By far the only known INP with transmembrane
segments and a weak secretion signal in the N-terminal domain is InaQ from P. syringae MB03

(Li et al., 2012).

Comparison of whole-cell xylanase activity between different INPs

To determine if the sequence difference in the N-terminal domain between InaA and that of InaK

and InaZ would result in better surface expression for improved enzyme activity, expression of

all the INPs fused to xylanase was carried out and the whole-cell xylanase activity is shown in

Figure 3. Interestingly, the activity produced by cells with InaAxyl was 92.2 U/g dry cell weight

which was about two-fold higher than InaKxyl (50.7 U/g dry cell weight) and more than three-

fold when compared to InaZxyl (25.9 U/g dry cell weight). A possible explanation for this might

be that the difference in the N-terminal domain of InaA allowed more efficient surface transport

and interaction with the phospholipid component of the outer membrane compared to the other

two INP constructs hence, providing a better anchoring system and more abundant surface

display. This is consistent with the findings of Shimazu et al. (2001) who compared the surface

display efficiency of InaV and InaK where the differences of amino acid sequences were mainly

in the N-terminal domain. Other studies that have documented the display of xylanase on the

surface of E. coli used other anchors such as Lpp-OmpA, a chimera from E. coli (Qu et al., 2015)

and PgsA from Bacillus subtilis (Chen et al., 2012). The display of both anchors resulted in 72

U/g dry cell weight and 49 U/g dry cell weight xylanase activities respectively, both of which are

still lower than the xylanase activity from anchorage using InaA. Taken together, this study

shows that the surface display of xylanase using InaA as anchor produced a superior whole-cell

biocatalyst for the degradation of xylan.

Surface display of INPs and xylanase fusion proteins

Further analysis using SDS-PAGE and Western blot was carried out to confirm that the INPs

were indeed localized on the surface of E. coli. Cell fractionation was performed to obtain the

outer membrane of cells and the soluble fractions which were the combination of the cytoplasm

and periplasm. From Figure 4 (a), no fusion proteins were found in the soluble fraction of the

cell while in Figure 4 (b), there were clear expression of the fusion proteins InaKxyl, InaAxyl,

and InaZxyl with molecular weights of approximately 55, 50, and 55 kDa respectively in both

the total membrane and outer membrane fractions. The predicted molecular weights for InaKxyl,

InaAxyl, and InaZxyl were 47, 45, and 49 kDa respectively. Although the reason for the higher

molecular weights on the SDS-PAGE gel is unknown, this occurrence is not an isolated one as

other researchers have reported similar findings when using INPs (Fan et al., 2011; Jung et al.,

1998b). Besides that, Western blot was also done for specific detection of the proteins and the

results are shown in Figure 4 (c). It confirms the results shown in SDS-PAGE that the protein

bands found at molecular weights of 55, 50, and 55 kDa were indeed InaKxyl, InaAxyl, and

InaZxyl as the antibody used for detection was specific to the His-Tag marker found at the end of

the xylanase sequence. These prove that the constructs were successfully targeted to the cell

outer membrane and displayed on the cell surface. It is a confirmation that InaAxyl was anchored

to the cell surface resulting in the highest whole cell activity. This was the most suitable

construct for the display of xylanase.

Immunofluorescence microscopy and flow cytometry

For an anchor to be effective, it should be located on the outer membrane of the cell and

displaying the enzyme towards the medium for reaction to take place. Fluorescence microscopy

and flow cytometry test was done to prove this. For fluorescence microscopy, only InaAxyl

showed fluorescence bright enough to be detected for viewing (Fig. 6). For quantitative analysis

of surface display efficiency, flow cytometry data was obtained. All three genes showed surface

expression however, InaAxyl gave a significant peak compared to InaKxyl and InaZxyl (Fig. 7).

The controls used in the flow cytometry experiment were unstained cells harboring pET-21a (+)

where none of the population displayed fluorescence and stained pET-21a (+) which showed 0.1

% of population displaying fluorescence. A total of 5.5 % of cells expressing InaAxyl exhibited

strong fluorescence in comparison to InaKxyl and InaZxyl which showed 1.4 % and 1.5 %

respectively. It can be deduced that InaAxyl had higher surface expression of about 4 times

compared to the other two genes. Therefore, it can also be concluded that xylan hydrolysis was

most efficient using InaAxyl construct because of its higher surface localization. This makes it a

good surface anchor for the display of xylanase and its application in the degradation of

hemicellulose.

Effects of surface display on cell growth

Overexpression of cell surface protein could result in growth defects and membrane

destabilization (Lee et al., 2003). Cells expressing InaAxyl were also studied to determine if the

surface display caused a negative effect on cell growth. Cells harboring plasmid pET-21a(+) was

used as a control. Figure 8 shows that the growth curve of cells displaying InaAxyl was normal

and did not affect cell growth. It has a similar growth profile with InaKxyl and InaZxyl. All three
constructs had a higher growth rate than the control cell. One of the reasons INPs have been

shown to be good anchors for surface display is its ability to be expressed without hampering cell

growth (Jung et al., 1998b; Khodi et al., 2012). Therefore, InaA fits this characteristic perfectly

as well. Based on all the results obtained, it can be said conclusively that InaAxyl was displayed

well on the surface of E. coli BL21 (DE3) with no inhibition to cell growth.

Screening expression conditions of surface displayed InaAxyl

Due to the promising results of InaA as an INP for surface display, the optimum cultural

conditions for expression were studied using one-factor-at-a-time (OFAT). For post-induction

harvest time or duration of induction, it was found that the expression of InaAxyl increased till it

reached the maximum at 8 h before decreasing. Duration of induction is important because it

affects the folding, accumulation and productivity of expressed proteins in E. coli (Malik et al.,

2016). Prolonged duration of induction results in increased secretion of periplasmic proteins

which weakens the outer membrane and increases its permeability (Donovan et al., 1996). In this

study, LB medium was found to be the optimum medium for expression. The medium of

expression plays an important role in regulating cell growth as the composition of different

mediums either accelerate or pace cell growth according to its function. Although surface

expression has a tendency to drop at lower growth rates, it has been suggested that surface

expression is inversely proportional to growth rate (Gustavsson et al., 2011). It may be the case

therefore that, richer media such as TB and SOB gave high cell densities but did not support

optimal surface expression while minimal media, M9 yielded cell densities which were too low.

Besides that, the optimum inducer concentration was found to be 0.3 mM IPTG. The optimum
inducer concentration ensures efficient membrane transport and localization while preventing

cellular burden (Li et al., 2012). High IPTG concentration may also cause formation of inclusion

bodies which reduces protein display on the surface (Fan et al., 2011). The agitation rate of 200

rpm in this study gives the most suitable aeration for cell expression while 25 ˚C was found to be

the optimum temperature for expression. This temperature is the ideal condition for protein

translation compared to higher temperatures which may promote the formation of inclusion

bodies (Golotin et al., 2016).

Conclusion

We have proven in this first comprehensive study of InaA as a surface display anchor that it was

successfully anchored to the outer membrane of E. coli and was able to display active xylanase

(~22kDa). Compared to InaK and InaZ, InaA was the better INP for the display of xylanase.

InaA had about 4 times higher surface expression based on flow cytometry data and resulted in

highest whole-cell xylanase activity. It also does not hamper cell growth. Therefore, InaA is a

good anchor for xylanase display and its application as a whole-cell biocatalyst for the hydrolysis

of xylan to xylooligosaccharides. It is an attractive alternative to the current INPs in use.

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