Recombinan bo ine se um albumin domain II as bio ecep o o
och a oxin A cap u e
Ta iana Q. Aguia
a,b,*,1
, Tˆ
ania Leal
c,1
, Diana G. Rod igues
a
, Luís Ab unhosa
a,b
, Ca la Oli ei a
c
,
Lucília Domingues
a,b,**
a
CEB –Cen e o Biological Enginee ing, Uni e si y o Minho, 4710-057, B aga, Po ugal
b
LABBELS –Associa e Labo a o y, B aga/Guima ˜
aes, Po ugal
c
Uni e sidade Ca ´
olica Po uguesa, CBQF - Cen o de Bio ecnologia e Química Fina –Labo a ´
o io Associado, Escola Supe io de Bio ecnologia, Rua Diogo Bo elho 1327,
4169-005, Po o, Po ugal
ARTICLE INFO
Handling edi o : Kin-ichi Tsunoda
Keywo ds:
Och a oxin A
BSA domain II
Bio ecep o
Solid-phase ex ac ion
Recombinan p o ein
ABSTRACT
Es ablished ch oma og aphic echniques o myco oxin con ol in oods u s equi e p io sample en ichmen and
clean-up, ypically achie ed using immunoa ini y columns (IACs). Bo ine se um albumin (BSA) has ecen ly
eme ged as a cos -e ec i e al e na i e o an ibodies used in IACs. This s udy aimed a explo ing he BSA domain
II (BDII), which houses he p ima y binding si e o och a oxin A (OTA), as a bio ecep o o OTA cap u e.
Recombinan BDII ( BDII) was p oduced in soluble o m by Esche ichia coli O igami 2(DE3), used o a His6
(HisBDII) o hio edoxin-His6 (T xBDII) ag, wi h yields up o 19 ±4.3 mg/L
cul u e
in shake- lask. Fluo escence
and ci cula dich oism (CD) spec oscopy e ealed in e ac ion o OTA wi h bo h BDII a ian s, wi h es ima ed
binding cons an s o OTA-HisBDII/T xBDII complexes in he ange o 5.7–9.3 ×10
4
M
−1
. CD also showed an
α
/β
s uc u e o BDII a ian s, in opposi ion o he p edominan
α
-helical s uc u e o whole BSA, and sligh inc ease
in hei
α
-helical con en upon binding o OTA. T xBDII immobilized on Ni-NTA esin success ully cap u ed OTA
om spiked samples a he op imum pH ange o 6.5–7.0, allowing OTA ex ac ion, clean-up, and en ichmen
om spiked whi e g ape juice, wi h up o 84 ±7.4 % eco e y.
1. In oduc ion
Myco oxins a e oxic seconda y me aboli es na u ally p oduced by
ce ain ungal species ha a e ubiqui ously p esen in na u e and
con amina e a ious ag icul u al commodi ies. Och a oxin A (OTA) is
one o he mos common and conce ning ood and eed con amina ing
myco oxins, being linked o se e e heal h isks o humans and animals
due mainly o i s neph o oxici y and ca cinogenic po en ial (classi ied as
possibly ca cinogenic o humans - G oup 2B acco ding o he IARC
classi ica ion) [1]. Wi h high bioaccumula ion cha ac e is ics, OTA can
be ound in a wide a ie y o eed and ood s u s o plan o igin, namely
in ce eals, g apes, co ee beans, cacao, d ied spices, he bs, and hei
de i ed p oduc s, which aises addi ional conce ns [1,2]. To mi iga e
he heal h isks associa ed wi h i s consump ion, maximum limi s ha e
been es ablished in se e al coun ies and egions o a ious oods u s
[3]. In he Eu opean Union, he legisla ion o OTA was ecen ly
ex ended o 30 ood ca ego ies, wi h maximum limi s gene ally anging
om 2 o 20
μ
g/kg. Excep ions include liquo ice, which has limi s se
be ween 10 and 80
μ
g/kg, and child en’s oods, which ha e a limi o
0.5
μ
g/kg [4].
Se e al analy ical me hods ha e been success ully de eloped o
de ec ing and quan i ying OTA in ood ma ices [5]. Howe e , con-
en ional ch oma og aphic-based me hods emain he gold s anda d in
mos labo a o ies due o hei sensi i i y, accu acy, and p ecision [6,7].
High- o ul a-high-pe o mance liquid ch oma og aphy coupled wi h
luo escence de ec ion (HPLC-FD o UPLC-FD) a e he mos commonly
used echniques o ou ine OTA analyses [3,8,9]. Be o e analysis, an
e ec i e pu i ica ion and en ichmen is equi ed o elease OTA om
he ma ix, ypically ollowed by a clean-up s ep o educe ma ix in-
e e ences. Solid phase ex ac ion (SPE) using immunoso ben s immo-
bilized on solid suppo s such as aga ose o silica is he mos commonly
used me hod o his pu pose, enabling he selec i e ex ac ion, clean-up
* Co esponding au ho . CEB –Cen e o Biological Enginee ing, Uni e si y o Minho, 4710-057, B aga, Po ugal.
** Co esponding au ho . CEB –Cen e o Biological Enginee ing, Uni e si y o Minho, 4710-057, B aga, Po ugal.
E-mail add esses: [email p o ec ed] (T.Q. Aguia ), [email p o ec ed] (L. Domingues).
1
Equal con ibu o s.
Con en s lis s a ailable a ScienceDi ec
Talan a
jou nal homepage: www.else ie .com/loca e/ alan a
h ps://doi.o g/10.1016/j. alan a.2024.127126
Recei ed 5 July 2024; Recei ed in e ised o m 19 Oc obe 2024; Accep ed 29 Oc obe 2024
Talan a 283 (2025) 127126
A ailable online 30 Oc obe 2024
0039-9140/© 2024 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (
h p://c ea i ecommons.o g/licenses/by/4.0/ ).
and en ichmen o OTA in a ew s eps [2]. Mo e con en ional so ben s,
such as e e se o no mal silica, ion exchange, o mixed-mode phases
can also be used, bu hese o en equi e mo e complex and
ime-consuming op imiza ions [2,5]. Rega dless o hei ype, SPE so -
ben s a e usually packed in ca idges o columns, condi ioned, and
loaded wi h samples. A e insing wi h an adequa e washing solu ion o
emo e in e e en s, OTA can be elu ed om he so ben wi h pu i y and
concen a ion le els sui able o subsequen analy ical de ec ion and
quan i ica ion [2].
While e y selec i e, eliable, and sensi i e, he commonly used SPE
immunoa ini y columns (IACs) based on an ibodies speci ic o OTA
ha e limi a ions, p ima ily hei high cos and single-use na u e, which
es ic hei b oade use [5,8]. Hence, esea ch has ocused on de el-
oping al e na i e bio ecogni ion molecules and ma e ials, such as
ap ame s, sho pep ides, ecombinan p o eins and molecula ly
imp in ed polyme s, which ha e been used in myco oxin ex ac ion,
pu i ica ion, and biosenso pla o ms [2,3,6]. Among hese, bo ine and
human se um albumin (BSA and HSA) ha e eme ged as in e es ing al-
e na i es o an ibodies o use in OTA SPE de ices due o hei
easonably high a ini y o his myco oxin (10
5
-10
6
M
−1
o BSA [10,11]
and 10
6
-10
7
M
−1
o HSA [11,12]), wide a ailabili y a low cos , and
compa able pe o mance o IACs [7,8].
Se um albumins a e p ima ily ob ained om animal o human
plasma, bu la ge-scale ecombinan p oduc ion is likely he solu ion o
mee he inc easing demand o BSA and HSA in a ious pha macolog-
ical, biochemical, and bio echnological applica ions [13]. Addi ionally,
ecombinan p oduc ion allows o he enginee ing o se um albumins
wi h imp o ed s abili y [13], enhanced binding p ope ies, o speci ic
modi ica ions ha may be use ul o ce ain applica ions, such as sub-
sequen immobiliza ion on SPE suppo s [14]. In his con ex , signi ican
e o s ha e been made o de elop cos -e ec i e biop ocesses o e-
combinan albumin p oduc ion. Mic obial hos s, such as bac e ia and
yeas , a e he mos explo ed due o hei ad an ages in p oduc ion
speed, cos and yield [13,15,16]. Among hese, he bes p oduc ion
yields ha e been ob ained wi h yeas [16], as he olding o
disul ide- ich mul idomain albumins is challenging o bac e ial hos s
[13,15,17].
S uc u ally, se um albumins consis o h ee globula domains (I, II
and III), each con aining wo subdomains (A and B), and 17 conse ed
disul ide b idges. The p incipal OTA binding si e is loca ed wi hin
domain II, speci ically in Sudlow’s si e I (subdomain IIA), an impo an
d ug-binding si e [11,12]. S udies wi h ecombinan HSA p oduced by
yeas e ealed ha he binding a ini y o his domain o OTA (7.9 ×10
5
M
−1
) is only one o de o magni ude lowe han ha o he en i e HSA
(5.2×10
6
M
−1
) [12].
Gi en ha he in e ac ion o BSA wi h OTA had been explo ed p e-
iously o de elop BSA-based SPE columns o OTA de e mina ion in
wine, wi h pe o mances compa able o IACs [8], his wo k aimed a
explo ing he BSA domain II (BDII) as a bio ecep o o use in new OTA
sepa a ion pla o ms. To ensu e s able supply and acili a e immobili-
za ion on widely a ailable SPE suppo s, his wo k aimed o ob ain BDII
ecombinan ly om Esche ichia coli, used o a His6 pu i ica io-
n/immobiliza ion ag ( BDII). A e cha ac e iza ion o he in e ac ion o
BDII wi h OTA, BDII was immobilized on nickel-ni ilo iace ic acid
(Ni-NTA) c osslinked aga ose esin. Subsequen ly, he e ec i eness o
his BDII-based SPE sys em o OTA cap u e was assessed.
2. Ma e ials and me hods
2.1. Cloning, ecombinan p oduc ion and pu i ica ion o BDII usion
a ian s
The coding sequence o he domain II o BSA (amino acids 188–385)
wi h codons op imized o exp ession in E. coli was pu chased om
NZYTech, lanked by NcoI and XhoI ecogni ion si es a he 5
′
- and 3
′
-
end, espec i ely. Using hese es ic ion enzymes (New England
Biolabs), BDII was cloned in o he exp ession ec o pETM10 (EMBL), in
usion wi h an N- e minal His6 ag (pETM10_HisBDII), and pETM20
(EMBL), in usion wi h an N- e minal ag comp ising he E. coli hio-
edoxin 1 (T x), His6 and a obacco e ch i us (TEV) p o ease ecogni-
ion si e (pETM20_T xBDII) (Table 1), as con i med by Sange
sequencing (Eu o ins Genomics). Chemically compe en NZY5
α
E. coli
cells (NZYTech, #MB00402) we e used o plasmid cons uc ion and
p opaga ion, and E. coli O igami 2(DE3) (No agen), which con ains
mu a ions ha acili a e cy oplasmic disul ide bond o ma ion, was used
o p o ein exp ession.
E. coli s ains we e g own in LB medium (5 g/L yeas ex ac , 10 g/L
yp one, 10 g/L NaCl, pH 7.0) con aining 50
μ
g/mL kanamycin
(pETM10_HisBDII) o 100
μ
g/mL ampicillin (pETM20_T xBDII) o
plasmid selec ion and main enance. 12.5
μ
g/mL e acycline and 50
μ
g/
mL s ep omycin we e also used o ensu e he E. coli O igami 2(DE3)
geno ype. Fo ecombinan p o ein p oduc ion, 250 mL o medium we e
inocula ed wi h 5 mL o o e nigh cul u es and incuba ed a 37 ◦C and
200 pm un il eaching an op ical densi y (OD
600nm
) o ~0.5. Exp ession
o he usion p o eins was hen induced wi h 0.2 mM isop opyl-β-D-
hiogalac opy anoside (IPTG; NZYTech, #MB02603) o ~16 h a 18 ◦C
and 150 pm, a e which he cells we e ha es ed by cen i uga ion a
(10,000 pm, 10 min, 4 ◦C) and s o ed a -20 ◦C un il use.
Fo p o ein pu i ica ion, cell pelle s we e de os ed and esuspended
in NZY Bac e ial Cell Lysis Bu e (NZYTech, #MB17802) con aining
100
μ
g/mL lysozyme, 4
μ
g/mL DNaseI and 1 mM phenylme hylsul onyl
luo ide (PMSF; Sigma, #P7626) acco ding o he manu ac u e ’s in-
s uc ions. A e incuba ion a oom empe a u e o 25 min in a o a o
s i e (LBX Ins umen s, RD80 model) se a 30 pm, soluble cell- ee
ex ac s we e collec ed by cen i uga ion (10,000 pm, 10 min, 4 ◦C)
and il e ed h ough 0.22
μ
m PES sy inge il e o ob ain a clea ed lysa e.
HisBDII and T xBDII we e pu i ied om clea ed lysa es by immobilized
me al ion a ini y ch oma og aphy (IMAC), using a 5 mL HisT ap HP
p epacked column (Cy i a, #17524802) ope a ed wi h a pe is al ic
pump. Pu i ica ion was pe o med acco ding o he manu ac u e ’s in-
s uc ions, using 50 mM T is-HCl and 150 mM NaCl (pH 8.0) as he main
bu e solu ion, supplemen ed wi h 60 mM imidazole o equilib a ion
and washing, and wi h 300 mM imidazole o elu ion. Samples om
clea ed lysa es, binding, washing, and elu ion ac ions we e mixed 4:1
wi h 5×SDS-PAGE Sample Loading Bu e (NZYTech, #MB11701),
boiled o 10 min a 95 ◦C and analysed by SDS-PAGE using 15 % (w/ )
ac ylamide gels. PageRule Uns ained B oad Range P o ein Ladde
(The mo Scien i ic, #26630) was used as molecula weigh ma ke , and
BlueSa e (NZYTech, #MB15201) as p o ein s ain. PD10 desal ing col-
umns (Cy i a, #17085101) we e used o exchange he bu e o pu i ied
p o eins acco ding o he manu ac u e ’s ins uc ions. Pu i y was
assessed by densi ome y analysis o p o ein bands in BlueSa e s ained
SDS-PAGE gels using ImageJ so wa e e sion 1.53, as desc ibed in
Re . [18]. The concen a ion o pu i ied p o eins was es ima ed om he
abso bance a 280 nm using hei co esponding heo e ical mola
ex inc ion coe icien s (Table 1).
2.2. Fluo escence and ci cula dich oism (CD) spec oscopy s udies
The in e ac ion o pu i ied HisBDII and T xBDII wi h OTA (Supelco,
#34037-2 ML-R) was s udied by luo escence and CD spec oscopy in
10 mM T is-HCl (pH 7.0). Comme cial BSA (NZYTech, #MB046) was
used as posi i e con ol unde he same expe imen al condi ions o
compa a i e pu poses. Fluo escence measu emen s we e pe o med in a
CYTATION 3 (Bio ek) mic opla e eade using black 96-well la bo om
pla es (The mo Scien i ic, #611F96BK). All measu emen s we e ca ied
ou a 25 ◦C a e incuba ion o 100
μ
L p o ein (0–40
μ
M) wi h 100
μ
L
OTA (0–10
μ
M) a oom empe a u e o 15 min. P o eins and OTA we e
exci ed a an exci a ion wa eleng h (λ
ex
) o 295 nm, wi h emission
eco ded in he 325–550 nm emission wa eleng h (λ
em
) ange. A e a
p elimina y assessmen o he exci a ion maxima o OTA-HisBDII, OTA-
T xBDII and OTA-BSA complexes, OTA was exci ed a λ
ex
380 nm, and
T.Q. Aguia e al. Talan a 283 (2025) 127126
2
luo escence emission spec a o OTA-p o ein complexes we e ob ained
in he λ
em
ange o 410–550 nm. Binding cons an s o OTA-p o ein
complexes we e de e mined by non-linea i ing o OTA (0.5
μ
M)
luo escence emission in ensi y a 446–454 nm (λ
ex
295 nm o 380 nm)
agains inc easing concen a ions o HisBDII (0, 0.5, 1.25, 2.5, 5, 7.5, 10,
12.5, 15, 17.5, 20
μ
M), T xBDII (0, 0.5, 1.25, 2.5, 5, 7.5, 10, 12.5, 15,
17.5, 20
μ
M) and na i e BSA (0, 0.05, 0.125, 0.25, 0.375, 0.5, 0.75, 1,
1.5, 2.5, 3.75
μ
M), using he G aphPad P ism 10.2.3 so wa e and he
equa ions desc ibed in Re . [20] as de ailed in Knowledgebase A icle
#1725 [21].
Fa -UV CD spec a (190–250 nm) we e eco ded on a Jasco J-1500
CD spec opho ome e (Jasco) a 25 ◦C, using a 0.1 cm pa h leng h
qua z cu e e and he ollowing expe imen al pa ame e s: bandwid h
o 1 nm; da a pi ch o 1 nm; scanning speed o 50 nm/min, esponse D.I.
T. o 1 s, 5 accumula ions. The CD spec um o he bu e was sub ac ed
om each eco ded spec um. The seconda y s uc u e elemen s pe -
cen age was calcula ed om he CD spec a using he BeS Sel web se e
[22].
2.3. OTA cap u ing assays
2.3.1. Cons uc ion o T xBDII-based SPE columns
To assess BDII as a bio ecep o o OTA cap u e, g a i y low SPE
columns we e p epa ed wi h he T xBDII a ian immobilized on His-
Pu ™Ni-NTA esin (The mo Scien i ic, #88222) and packed in o 2 mL
columns (The mo Scien i ic, #89896). A e he ini ial assessmen o he
maximum T xBDII-binding capaci y o he Ni-NTA esin (1.7–2.1 mg o
pu i ied T xBDII/0.125 mL o se led esin), an excess o ~2.5 mg o
pu i ied T xBDII in 1.5–3 mL o 50 mM T is-HCl and 150 mM NaCl (pH
8.0) we e incuba ed o 30 min a oom empe a u e and 30 pm (RD80
o a o s i e , LBX Ins umen s) wi h 0.125 mL o se led esin p e i-
ously cha ged wi h 0.1 M NiSO
4
⋅6H
2
O and equilib a ed wi h he same
T is-HCl bu e . A e incuba ion, he binding slu y was ans e ed o
he packing column, ollowed by wo washing s eps o emo e loosely
bound T xBDII: i s wi h 1 mL o 50 mM T is-HCl and 150 mM NaCl
supplemen ed wi h 20 mM imidazole (pH 8.0) and hen wi h 1 mL o he
same bu e wi hou imidazole. Samples we e collec ed a e each s ep
o SDS-PAGE analysis and p o ein quan i ica ion, as desc ibed in 2.1.
Columns packed wi h 0.125 mL o se led esin wi hou immobilized
T xBDII we e used as nega i e con ols in OTA cap u ing assays.
2.3.2. E alua ion o OTA ex ac ion om chemically de ined bu e ed
solu ions
Fo he ini ial assessmen o he cons uc ed columns, chemically
de ined bu e ed solu ions con aining 10
μ
g/L OTA we e p epa ed by
dilu ing a 10
μ
g/mL OTA s ock solu ion (Supelco, #34037-2 ML-R) in
10 mM T is-HCl (TB) o 10 mM Na–K phospha e bu e (PB) a pH
6.0–8.0. F eshly p epa ed g a i y low SPE columns we e equilib a ed
wi h 2 mL o he bu e unde s udy and hen loaded wi h 0.5 mL o 10
ng/mL OTA solu ion p epa ed in he same bu e . A e collec ion o he
low- h ough, he column was washed wi h 3 ×0.5 mL o he same
bu e , and hen elu ion was pe o med wi h 3 ×0.5 mL o me hanol
con aining 1 % ( / ) o ace ic acid, as desc ibed in Leal e al. [8], wi h an
incuba ion pe iod o 2 min a oom empe a u e p eceding he collec ion
o each elu ion ac ion. The samples collec ed a e each s ep o his
p ocedu e we e s o ed sepa a ely a -20 ◦C and dilu ed 1:1 in HPLC
mobile phase be o e OTA quan i ica ion.
Table 1
Amino-acid sequence and heo e ical physicochemical p ope ies o na i e BSA (UniP o : P02769) and ecombinan a ian s o i s domain II p oduced in E. coli
O igami 2(DE3). Molecula weigh (MW), isoelec ic poin (pI) and mola ex inc ion coe icien (
ε
) we e calcula ed using he P o Pa am ool [19], assuming all pai s o
cys eine esidues o m cys ines. In bold, i alics and unde lined a e indica ed he His6 ag, he E. coli hio edoxin 1 (T x) and he obacco e ch i us (TEV) p o ease
ecogni ion si es, espec i ely.
P o ein Amino-acid sequence MW (kDa) pI
ε
(M
−1
cm
−1
)
Ma u e BSA DTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQ
QCPFDEHVKLVNELTEFAKTCVADESHAGCEKSL
HTLFGDELCKVASLRETYGDMADCCEKQEPERNE
CFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFW
GKYLYEIARRHPYFYAPELLYYANKYNGVFQECC
QAEDKGACLLPKIETMREKVLASSARQRLRCASIQ
KFGERALKAWSVARLSQKFPKAEFVEVTKLVTD
LTKVHKECCHGDLLECADDRADLAKYICDNQDTIS
SKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTAD
FAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEY
AVSVLLRLAKEYEATLEECCAKDDPHACYSTVF
DKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNAL
IVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTK
PESERMPCTEDYLSLILNRLCVLHEKTPVSEKV
TKCCTESLVNRRPCFSALTPDETYVPKAFDEK
LFTFHADICTLPDTEKQIKKQTALVELLKHKPKAT
EEQLKTVMENFVAFVDKCCAADDKEACFAV
EGPKLVVSTQTALA
66.4 5.60 42925
HisBDII MKHHHHHHPMVLASSARQRLRCASIQKFGERA
LKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHG
DLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKS
HCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKD
AFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLE
ECCAKDDPHACYSTVFDKLKHLVDEPQN
23.8 6.05 16680
T xBDII MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCG
PCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPK
YGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLD
ANLAGSGSGHMHHHHHHSSGENLYFQGAMVLA
SSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEF
VEVTKLVTDLTKVHKECCHGDLLE
CADDRADLAKYICDNQDTISSKLKECCDKPLLEK
SHCIAEVEKDAIPENLPPLTADFAEDKDVCKNY
QEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKE
YEATLEECCAKDDPHACYSTVFDKLKHLVDEPQN
37.0 5.54 32275
T.Q. Aguia e al. Talan a 283 (2025) 127126
3
2.3.3. E alua ion o OTA ex ac ion om spiked g ape juice
To e alua e he capaci y o he columns o cap u e, clean-up and
concen a e OTA om complex ood samples, bo led whi e g ape juice
andomly pu chased om a local supe ma ke was il e ed h ough a
0.2
μ
m polyamide memb ane il e (Sa o ius, #2500747 N) and spiked
wi h 2 o 0.5
μ
g/L OTA. Then, wi hou dilu ing spiked juice samples,
hei pH was adjus ed o 6.8 by adding T is base powde o a inal
mola i y o ~50 mM. Fo hese cap u ing assays, eshly p epa ed
g a i y low SPE columns we e equilib a ed wi h 2 mL o 10 mM PB (pH
6.5) and hen loaded wi h 2 mL o g ape juice (pH 6.8) spiked wi h 2 ng/
mL OTA (assay A) o 8 mL o g ape juice (pH 6.8) spiked wi h 0.5 ng/mL
OTA (assay B). A e collec ion o he low- h ough, he column was
washed wi h 2 ×0.5 mL o 10 mM PB (pH 6.5), and elu ion was pe -
o med as desc ibed in 2.3.2. The samples collec ed a e each s ep we e
s o ed sepa a ely a -20 ◦C and dilu ed 1:1 in HPLC mobile phase be o e
OTA quan i ica ion.
2.4. HPLC analysis
OTA was quan i ied by HPLC-FD using a ch oma og aphic sys em
Shimadzu Nexe a 40 Se ies coupled o an RF-20 Axs luo escence de-
ec o (se a λ
ex
: 333 nm, λ
em
: 460 nm, gain: 16x-High). The column was
a Syne gy 2.5
μ
m Hyd o-RP 100 A (100 ×3 mm). The eluen s we e
il e ed wi h a 0.2
μ
m memb ane (Sa o ius, #2500747 N) and degassed.
Eluen A was ace oni ile/ace ic acid (99:1, / ) and eluen B was
H
2
O
dd
/ace ic acid (99:1, / ). The isoc a ic sepa a ion was achie ed
wi h 45 % A and 55 % B a 30 ◦C and cons an low a e o 0.4 mL/min
o e a 10 min un. The injec ion olume was 10
μ
L. An 8-poin cali-
b a ion cu e (10–0.04 ng/mL) was p epa ed by se ial dilu ion o he
p ima y s ock (Supelco, #34037-2 ML-R) in mobile phase. OTA e en-
ion ime was 8.4 min. The quan i ica ion was done by compa ing he
peak a ea o each compound wi h he espec i e calib a ion cu e. A se
o calib an s p ese ed a -20 ◦C was injec ed be o e each daily analysis.
The eg ession deli e ed a linea i : Y =2.22 ×10
6
X wi h an R
2
=
0.9994, RFRSD =5.9 %, n=4, wi h LOD =0.03 ng/mL and LOQ =0.09
ng/mL (calcula ed om he calib a ion cu e by 3.3
σ
/s and 10
σ
/s,
espec i ely).
3. Resul s and discussion
3.1. Recombinan p oduc ion and pu i ica ion o HisBDII and T xBDII
E. coli exp ession s ains wi h mu a ions in bo h he hio edoxin
educ ase ( xB) and glu a hione educ ase (go ) genes, which enhance
disul ide bond o ma ion, ha e been used o exp ess soluble and unc-
ional HSA and BSA [13,15,17]. Likewise, in his wo k, BDII was p o-
duced by E. coli O igami 2(DE3) in soluble o m using wo
IPTG-inducible exp ession ec o s, gi ing ise o wo di e en usion
p o eins, HisBDII and T xBDII (Fig. 1). Bo h HisBDII and T xBDII con ain
an N- e minal His6 ag o acili a e pu i ica ion/immobiliza ion [18,23].
Since he E. coli hio edoxin 1 (T x) is epo ed o imp o e he soluble
p oduc ion yield and s abili y o usion pa ne s [24,25], T xBDII was
designed wi h an N- e minal T x pa ne , which can be emo ed
oge he wi h he His6 ag h ough he TEV p o ease clea age si e
posi ioned immedia ely be o e BDII (Fig. 1). Al hough nei he BDII
usion was easily de ec ed in he soluble ac ions o cellula lysa es
(Fig. 1, lane 1), bands co esponding o he p edic ed molecula weigh
o monome ic HisBDII (24 kDa) and T xBDII (37 kDa) we e isible in he
elu ion ac ions a e IMAC pu i ica ion (Fig. 1, lanes 3 and 4). Addi-
ionally, bands wi h appa en molecula weigh ≥150 kDa we e isible
in bo h HisBDII and T xBDII pu i ied ac ions. This indica es he p es-
ence o s able BDII oligome ic o ms, as al eady epo ed o pu i ied
HSA ecombinan ly ob ained om E. coli [15,17]. A con aminan band
o 40–50 kDa was also isible in bo h HisBDII and T xBDII pu i ied
ac ions, which co esponds o a na i e p o ein p oduced by he hos
ha a aches igh ly o he IMAC column, and which has been obse ed
a e IMAC pu i ica ion o o he His6- agged p o eins, namely HSA [15].
Pu i ied HisBDII and T xBDII could be ob ained wi h 60–80 % pu i y.
Despi e BDII cons i u ing only ~60 % o he molecula weigh o he
T xBDII usion p o ein compa ed o ~95 % o HisBDII, he p oduc ion
yields o pu i ied BDII wi h ei he His6 ag alone (14 ±4.0 mg/L
cul u e
)
o he T x-His6-TEV pa ne (19 ±4.3 mg/L
cul u e
) we e iden ical. This
indica es ha he T x pa ne did no imp o e he soluble p oduc ion
yield o BDII. Howe e , T xBDII exhibi ed signi ican ly be e s abili y
han HisBDII, as he la e displayed loss o soluble p o ein concen a-
ion a e o e nigh s o age a 4 ◦C. Un agged BDII ob ained om
T xBDII ollowing clea age wi h TEV p o ease also displayed low s a-
bili y, p ecipi a ing a e o e nigh s o age a 4 ◦C. The e o e, he T x
pa ne appa en ly enhanced he s abili y o BDII, as al eady epo ed
o o he a ge p o eins [24,25] and u he discussed in subsec ion 3.2.
The p oduc ion yields ob ained he e o BDII a e 16 h o
IPTG-induc ion all wi hin he ange o he bes yields epo ed o e-
combinan HSA and BSA p oduced in E. coli, which a e 10–19 mg/L
cul u e
[13,17]. S ill, hese yields a e below he 65–300 mg/L
cul u e
epo ed o
ecombinan HSA domains (speci ically domain II) p oduced by he
yeas Pichia pas o is a e 96–120 h o me hanol-induc ion [26].
3.2. In e ac ion o OTA wi h ecombinan HisBDII and T xBDII
The spec al p ope ies o OTA a e known o change subs an ially in
he p esence o BSA o o he albumins, and he in insic luo escence o
hese p o eins is quenched by OTA [11,27]. Thus, o examine he
unc ionali y o he p oduced BDII usion a ian s, he abili y o pu i ied
HisBDII and T xBDII o in e ac wi h OTA was assessed by luo escence
spec oscopy.
Despi e e aining he yp ophan (W) 213 esidue o BSA highly
sensi i e o luo escence quenching by OTA and o he myco oxins [11,
Fig. 1. BSA s uc u e and scheme o he BDII usion a ian s p oduced by
E. coli O igami 2(DE3), wi h BlueSa e s ained SDS-PAGE gels ep esen a i e o
hei soluble p oduc ion and IMAC pu i ica ion. In he scheme, he g een and
blue colo s ep esen he subdomains IIA and IIB highligh ed in he BSA
s uc u e wi h he same colo . Legend o he gels: lane 1, soluble lysa es om
p oduc ion cul u es (5×dilu ed); lane 2, low- h ough collec ed a e passing
he clea ed lysa es h ough he IMAC column (5×dilu ed); lanes 3 and 4, pu-
i ied ac ions ob ained a e elu ion; *, unknown p o ein om he hos .
T.Q. Aguia e al. Talan a 283 (2025) 127126
4
28,29], he in insic luo escence o HisBDII was no a ec ed by he
p esence o OTA. This is e iden om i s iden ical emission a a ound
340 nm a e exci a ion a 295 nm, alone (Fig. 2A, blue lines) o in he
p esence o OTA (Fig. 2A, yellow/b own lines). Fo T xBDII, OTA
induced only a sligh dec ease in i s in insic luo escence (Fig. 2B, blue
lines s yellow/b own lines), sugges ing weak OTA in e ac ion wi h
T xBDII. Compa ed o BSA (Fig. 2C), he quenching e ec o OTA o e
T xBDII was minimal and unsui able o de e mining S e n-Volme
quenching cons an s. Howe e , as obse ed o BSA (Fig. 2C&F), he
p esence o inc easing concen a ions o HisBDII (Fig. 2A&D) and
T xBDII (Fig. 2B&E) esul ed in inc easingly highe luo escence
emission in ensi y o OTA a a ound 450 nm, sugges ing he o ma ion o
OTA complexes no only wi h T xBDII, bu also wi h HisBDII. This in-
dica es ha bo h BDII usion a ian s p oduced by E. coli a e unc ional.
OTA displayed a luo escence emission maximum a ound 450 nm
bo h in he absence and p esence o HisBDII, T xBDII o BSA (Fig. 2),
whe eas i s exci a ion maximum shi ed om ~374 nm o ~381 nm
upon binding o HisBDII and T xBDII, and o ~390 nm upon binding o
BSA. This ed shi in he luo escence exci a ion maximum o OTA in he
p esence o albumins has been widely epo ed [10,12], and he absence
o he domain I has been shown o lead o he educ ion o his shi [12].
The e o e, in ag eemen wi h he al eady epo ed o ecombinan HSA
domain II [12], HisBDII and T xBDII did no a ec he luo escence
exci a ion maximum o OTA as much as he en i e BSA.
Based on he i ing cu es p esen ed in Supplemen a y Fig. S1,
equilib ium cons an s o OTA-HisBDII, OTA-T xBDII and OTA-BSA
complexes we e de e mined by non-linea i ing o OTA (0.5
μ
M)
luo escence emission in ensi y a 446–454 nm agains inc easing con-
cen a ions o HisBDII (0–20
μ
M), T xBDII (0–20
μ
M) and na i e BSA
(0–3.75
μ
M) (Table 2). These measu emen s we e eco ded using an λ
ex
o 295 nm o 380 nm, as p e iously done by o he s [27]. The cons an s
de e mined using bo h exci a ion wa eleng hs show good co ela ion
and sugges he o ma ion o s able OTA-HisBDII/T xBDII complexes. As
epo ed o he ecombinan domain II o HSA [12], he binding con-
s an s de e mined he e o OTA-HisBDII and OTA-T xBDII complexes
we e lowe (~2 o de s o magni ude) han ha de e mined o
OTA-BSA. In e es ingly, no subs an ial di e ences we e obse ed be-
ween he binding cons an s de e mined o HisBDII and T xBDII, indi-
ca ing ha he p esence o he T x usion pa ne does no hinde he
OTA- BDII in e ac ion. O no e, gi en he di e en o igin and pu i y
le els o he HisBDII and T xBDII used in hese assays (70–80 %)
compa ed o he na i e BSA used (>98 %), hese cons an s should be
used cau iously o b oade compa a i e pu poses. S ill, hey p o ide a
ealis ic unde s anding o he cap u ing sys em we a e de eloping and
s udying he e.
To gain u he insigh on he in e ac ion o OTA wi h pu i ied
HisBDII and T xBDII, CD spec oscopy s udies we e pe o med o e al-
ua e hei seconda y s uc u e and he occu ence o con o ma ional
changes induced by OTA binding. The a -UV CD spec a ob ained o
BDII in usion wi h he ~1 kDa His6 ag (HisBDII) o wi h he ~14 kDa
T x-His6 ag (T xBDII) a he N- e minal (Fig. 3A and B) indica e
s uc u es esembling hose o an
α
/βp o ein a he han hose o an
α
-helix ich p o ein like BSA (Fig. 3C). Simila esul s we e epo ed o
he ecombinan domain II o HSA, which we e asc ibed o he inc eased
ins abili y o he newly exposed long helical egions a he N and C
e mini o his domain [26]. While he T x pa ne does no seem o al e
he seconda y s uc u e o he BDII signi ican ly (Fig. 3D), i may
enhance he s abili y o i s N- e minal. T x i sel is a small globula
Fig. 2. Fluo escence emission spec a o HisBDII (A), T xBDII (B), na i e BSA (C) and OTA (A–F), alone o in complex, a he indica ed inal concen a ion o OTA:
p o ein a io. All measu emen s we e pe o med in 10 mM T is-HCl (TB) a pH 7.0, using λ
ex
295 nm (A–C) o 380 nm (D–F). The da a p esen ed in A-B was
sub ac ed om he esidual luo escence o he bu e and show ha OTA induced a concen a ion-dependen dec ease in he luo escence emission signal o T xBDII
and BSA (~340 nm). The da a p esen ed in D-E was sub ac ed om he esidual luo escence o he bu e ( o OTA) o p o ein a he co esponding concen a ion
( o OTA-p o ein complexes) and show ha HisBDII, T xBDII and BSA induced a concen a ion-dependen inc ease in he luo escence emission signal o OTA (~450
nm). (a.u) a bi a y uni .
T.Q. Aguia e al. Talan a 283 (2025) 127126
5
p o ein wi h a cha ac e is ic
α
/β opology [30], being composed o 31 %
α
-Helix, 27 % β-Shee , 22 % Tu n and 20 % O he s, as de e mined om
i s PDB:2TRX esol ed s uc u e using he BeS Sel web se e [22].
Gi en i s inhe en p ope ies, his usion pa ne is used o imp o e he
p oduc ion yield, solubili y and s abili y o ecombinan p o eins [24,
25]; and indeed, an imp o ed s abili y o T xBDII o e HisBDII was
obse ed h oughou his wo k. On he o he hand, he a -UV CD
spec um ob ained o he na i e BSA unde simila condi ions (Fig. 3C)
is in good ag eemen wi h published da a [29], showing he wo nega-
i e ellip ici ies o na i e BSA a 209 and 222 nm ha a e he hallma k o
i s ich
α
-helical con en .
As p e iously epo ed o HSA [27], he addi ion o OTA o BSA
induced changes in i s seconda y s uc u e ha led o dec eased
α
-hel-
ici y (Fig. 3D). The p esence o OTA also induced sligh changes in he
miscellaneous
α
/βs uc u e o HisBDII and T xBDII, bu an inc ease in
α
-helical con en was obse ed ins ead (Fig. 3D). The occu ence o
OTA-induced con o ma ional changes in bo h BDII a ian s indica e
ha hey conse e he abili y o he na i e BSA o bind o OTA, namely
in he p esence o he T x pa ne . Howe e , while OTA binding induces
a dec ease in he
α
-helical con en o BSA, in BDII a ian s i induces a
sligh inc ease in
α
-helical con en . These di e en con o ma ional
changes may be asc ibed o s uc u al and binding di e ences be ween
he en i e BSA, which exhibi s a
α
-helix ich s uc u e wi h a leas wo
binding si es o OTA [12], and BDII, whose miscellaneous
α
/βs uc-
u e con ains only one binding si e o OTA.
3.3. OTA cap u e by T xBDII-based SPE columns om chemically de ined
bu e ed solu ions
Gi en he simila OTA-binding pe o mance displayed by HisBDII
and T xBDII and he supe io s abili y displayed by T xBDII upon pu-
i ica ion, namely in p elimina y OTA cap u ing assays (da a no
shown), subsequen s udies we e pe o med only wi h T xBDII. The
sui abili y o T xBDII as a bio ecep o o OTA cap u e was e alua ed
ollowing he SPE me hod p e iously de eloped by ou eam [8], bu
gi ing a s ep o wa d in using a mo e cos -e ec i e and expedi ious
immobiliza ion s a egy, which akes ad an age o he His6 ag ha was
gene ically used o BDII N- e minal o apid si e-di ec ed immobili-
za ion on Ni-NTA esin [14]. Using his immobiliza ion s a egy, g a i y
low SPE columns we e p epa ed using 1.7–2.1 mg o pu i ied T xBDII
immobilized on 0.125 mL o se led Ni-NTA.
In p elimina y OTA cap u ing assays, he in e ac ion o OTA wi h
T xBDII was assessed by incuba ing 5 ng OTA in 0.5 mL 10 mM TB pH
7.0 wi h immobilized T xBDII o up o 18 h a oom empe a u e. The
pe cen age o unbound OTA a e less han 1 min o incuba ion
dec eased o 3.2 %, and no signi ican changes in his pe cen age we e
seen wi h inc easing incuba ion imes (da a no shown). These esul s
con i med he abili y o immobilized T xBDII o cap u e OTA and
indica ed ha he in e ac ion o OTA wi h his bio ecep o is almos
ins an aneous, no equi ing long con ac imes. Subsequen g a i y low
Table 2
Equilib ium cons an s o OTA-HisBDII, OTA-T xBDII and OTA-BSA complexes,
de e mined by non-linea i ing o OTA (0.5
μ
M) luo escence emission in-
ensi y a 446–454 nm agains inc easing concen a ions o HisBDII, T xBDII and
na i e BSA, using as λ
ex
295 nm o 380 nm. Da a ep esen s mean ±SE (n=2).
Complex λ
ex
(nm)
K
D
(M) K
A
(M
−1
) logK
A
R
2
OTA-BSA 295 1.18 (±0.12) x
10
−7
8.47 (±0.86) x
10
6
6.93 0.977
380 1.01 (±0.15) x
10
−7
9.91 (±1.51) x
10
6
7.00 0.950
OTA-
HisBDII
295 1.76 (±0.09) x
10
−5
5.67 (±0.29) x
10
4
4.75 0.996
380 1.32 (±0.11) x
10
−5
7.60 (±0.63) x
10
4
4.88 0.990
OTA-
T xBDII
295 1.08 (±0.04) x
10
−5
9.28 (±0.39) x
10
4
4.97 0.995
380 1.38 (±0.07) x
10
−5
7.39 (±0.39) x
10
4
4.86 0.992
Fig. 3. Fa -UV CD spec a o ecombinan HisBDII (A), T xBDII (B) and na i e BSA (C) in he absence and p esence o OTA, wi h indica ion o co esponding
seconda y s uc u e elemen s pe cen age calcula ed om he CD spec a (D). A ows indica e a dec ease (down) o inc ease (up) in
α
-helici y in he p esence o OTA.
T.Q. Aguia e al. Talan a 283 (2025) 127126
6
SPE assays p e o med wi h 5 ng OTA in 0.5 mL 10 mM TB pH 7.0 u he
suppo ed hese esul s (Table 3). While he e was also non-speci ic
adso p ion o OTA o he Ni-NTA esin (nega i e con ol), 91 % o he
ini ially bound OTA was los du ing he column washing s eps (Table 3).
In con as , he column con aining immobilized T xBDII e ained a
g ea e ac ion o OTA du ing he wash (68 %). Simila non-speci ic
binding was epo ed by Leal e al. [8] using a cyanogen
b omide-ac i a ed aga ose esin blocked wi h 0.2 M glycine pH 8.0.
The e o e, we decided o skip his blocking s ep, as i would no
signi ican ly educe he non-speci ic binding o OTA. On he o he hand,
we obse ed a conside ably highe loss o OTA wi h T xBDII-based
columns du ing column wash wi h 10 mM TB pH 7.0 (31 %) han Leal
e al. [8] epo ed o BSA-based columns using he same bu e (0 %).
OTA possesses mono- and di-anionic o ms, he p e alence o which
is pH sensi i e. A pH 7.0, bo h mono- and di-anionic OTA o ms a e
p esen a a a io o ~1:1 [31], bu upon binding o se um albumins
(namely BSA, HSA and ecombinan HSA domain II), OTA su e s
comple e dep o ona ion o i s di-anionic o m [10,28]. Howe e , he
p e ailing OTA o m a pH 6.0 is he mono-anionic and a pH 8.0 is he
di-anionic [31]. Thus, o s udy he pH in luence on he in e ac ion o
OTA wi h immobilized T xBDII, u he cap u ing assays we e pe -
o med using TB a pH 6.5 and 8.0. As seen in Table 3, he amoun o
OTA los du ing he binding and washing s eps was highes a pH 8.0 (78
%) and lowes a pH 6.5 (18 %), sugges ing highe a ini y o immobi-
lized T xBDII o mono-anionic OTA han o di-anionic OTA, con a y o
wha is epo ed o se um albumins (namely ecombinan HSA domain
II) [12]. Howe e , he na u e o he bu e used (e.g., T is- o
phospha e-based) has been epo ed o po en ially in luence p o ein
in e ac ion s udies [23]. In his case, he exis ence o p o ona ed T is
amino g oups in TB a he pH ange es ed may ha e weakened he ionic
in e ac ions in ol ed in he binding o di-anionic OTA o posi i ely
cha ged amino acids in he Sudlow’s si e I [12,28]. Thus, u he assays
we e pe o med wi h a di e en bu e (10 mM PB, pH 6.0 o 7.0) o
assess i imp o emen s could be ob ained in e ms o OTA e en ion.
In e es ingly, using he PB bu e ing sys em, he esul s ob ained a he
di e en pHs es ed we e iden ical (Table 3). Compa ed o TB, highe
e en ion o OTA du ing column wash was gene ally achie ed wi h PB,
which enabled supe io eco e y upon elu ion. Al hough highe
non-speci ic binding o OTA o he Ni-NTA esin was obse ed wi h PB
han wi h TB, non-speci ically bound OTA was also almos en i ely los
du ing he column wash. Conside ing hese esul s, 10 mM PB (pH 6.5)
was selec ed as he washing bu e o subsequen p oo -o -concep as-
says. Using TB o PB (pH 6.5–7.0), he maximum binding capaci y o he
de eloped T xBDII-based SPE columns was ~15 ng OTA (i.e, ~7.5 ng
OTA pe mg immobilized T xBDII).
3.4. OTA ex ac ion, clean-up and concen a ion om spiked g ape juice
using T xBDII-based SPE columns
Gi en he in e es ing pe o mance o he de eloped T xBDII-based
SPE columns in de ined bu e ed solu ions, he applicabili y o hese
columns o eal complex samples was in es iga ed by p epa ing and
analysing samples o bo led whi e g ape juice spiked wi h OTA a wo
inal concen a ions - 2 ng/mL (assay A) o 0.5 ng/mL (assay B) – o
e alua e he e ec o OTA concen a ion and sample olume. As seen in
Fig. 4, he % o OTA bound, e ained, and eco e ed om he column in
he assay whe e 4 ng OTA/2 mL o juice we e used (assay A) was
Table 3
Adso p ion and deso p ion pe o mance o he de eloped g a i y low SPE columns based on ecombinan T xBDII (~2 mg) immobilized on 0.125 mL o se led Ni-
NTA esin owa d OTA in chemically de ined bu e solu ions. OTA-cap u ing assays we e pe o med a he indica ed pH by loading 0.5 mL o 10 mM T is-HCl (TB) o
10 mM Na–K phospha e bu e (PB) spiked wi h OTA a 10 ng/mL (equi alen o 4.7 ±1.1 ng OTA loaded, as de e mined by HPLC). A e h ee washing s eps wi h he
same bu e , OTA was elu ed om he column wi h 1 % ( / ) ace ic acid in me hanol. Nega i e con ol (NC) co esponds o 0.125 mL o se led Ni-NTA esin wi hou
immobilized T xBDII a pH 7.0. The pe cen age o OTA in he low- h ough, wash and elu ion ac ions was calcula ed om he a io be ween he amoun o OTA (in
ng) eco e ed in hese ac ions and he amoun o ini ially loaded OTA (which co esponds o 100 %). The indica ed alues (in %) ep esen he a e age (±SD) o wo
independen assays.
SPE s ep Sample
Volume
10 mM TB 10 mM PB
pH 6.5 pH 7.0 pH 8.0 NC pH 6.0 pH 6.5 pH 7.0 NC
Flow- h ough 0.5 mL 0.0 ±0.0 2.0 ±0.1 10 ±0.5 44 ±3.2 0.0 ±0.0 1.5 ±0.1 0.0 ±0.0 21 ±1.4
Column wash 1s 0.5 mL 4.1 8.0 33 30 2.2 3.0 1.9 40
2nd 0.5 mL 7.5 10 22 17 3.9 3.6 2.7 20
3 d 0.5 mL 6.8 13 13 4.1 4.7 4.1 3.1 9.1
To al 18 ±1.1 31 ±2.1 68 ±3.4 51 ±0.7 11 ±0.5 11 ±1.6 8.0 ±2.6 69 ±3.7
Elu ion 1s 0.5 mL 33 32 20 2.2 49 51 52 8.7
2nd 0.5 mL 17 22 2.1 1.1 22 16 16 0.0
3 d 0.5 mL 9.8 5.6 0.0 0.0 6.9 8.0 7.0 0.0
To al 60 ±3.5 60 ±3.9 22 ±3.3 3.3 ±1.0 78 ±1.4 75 ±4.5 75 ±3.2 8.7 ±0.4
Fig. 4. Adso p ion and deso p ion pe o mance o he de eloped g a i y low
SPE columns based on ecombinan T xBDII (~2 mg) immobilized on 0.125 mL
o se led Ni-NTA esin owa d OTA in a complex ma ix. OTA-cap u ing assays
we e pe o med using comme cial whi e g ape juice spiked wi h 4 ng OTA/2
mL juice (assay A) o 4 ng OTA/8 mL juice (assay B) and wi h pH adjus ed o
6.8 wi h T is base a a inal concen a ion o 50 mM (equi alen o 3.7 ±0.5 ng
OTA loaded, as de e mined by HPLC). The pe cen age o OTA binding o he
SPE column was de e mined om he di e ence be ween he amoun o OTA
(in ng) p esen in he liquid ma ix be o e (loaded ac ion) and a e passage
h ough he column (unbound ac ion). A e wo washing s eps wi h 0.5 mL o
10 mM PB (pH 6.5), OTA was elu ed om he column wi h 3 ×0.5 mL 1 % ( /
) ace ic acid in me hanol (E1-3). The pe cen age o OTA in he wash and
elu ion ac ions was calcula ed om he a io be ween he amoun o OTA (in
ng) eco e ed in he wash and elu ion ac ions and he amoun o OTA p esen
in he ini ially loaded ma ix (which co esponds o 100 %). Ba s ep esen he
a e age (±SD) o h ee independen assays.
T.Q. Aguia e al. Talan a 283 (2025) 127126
7
iden ical o ha ob ained in p e ious OTA-cap u ing assays pe o med
wi h 5 ng OTA/0.5 mL PB (Table 3). Mo eo e , 68 % o he loaded OTA
was eco e ed in he i s elu ion s ep, wi h a 2.7 ±0.2 concen a ion
ac o ela i e o he loaded sample, and wi h ewe ma ix in e e en s
(Supplemen a y Fig. S2A). In assay B, whe e 4 ng OTA/8 mL o juice
we e used, 56 % o he ini ially loaded OTA was los du ing he binding
and column wash s eps (Fig. 4). Ne e heless, 39 % o he loaded OTA
could s ill be eco e ed in he i s elu ion s ep wi h a 6.3 ±0.3 con-
cen a ion ac o (and also highe pu i y) ela i e o he loaded sample,
g ea ly imp o ing OTA de ec ion (Supplemen a y Fig. S2B). Gi en ha
in his assay he amoun o OTA loaded in he column was he same as in
assay A, he lowe OTA e en ion obse ed in assay B indica e ha o
highe juice sample olumes he e may be mo e molecules compe ing
wi h OTA binding, as se e al compounds ound in plan -based oods and
be e ages can bind o he domain II o se um albumins [32].
Conside ing hese esul s, he de eloped T xBDII-based SPE columns
pe o med be e wi h lowe sample olumes (assay A), allowing he
eco e y o up o 84 ±7.4 % o OTA om spiked g ape juice. This e-
co e y yield alls sho behind he 91–107 % OTA eco e y yields ob-
ained wi h immobilized BSA o HSA om spiked wine samples [7,8] o
wi h immobilized HSA om spiked bee samples [7]. Conside ing he
compa a i ely lowe binding a ini y o T xBDII han na i e BSA
(Table 2), and ha he amoun o immobilized T xBDII used he e (~2
mg) was lowe han ha p e iously used by Leal e al. [8] wi h BSA (~6
mg), addi ional imp o emen s may be o eseen by inc easing he
amoun o esin used wi h immobilized BDII (which would p o ide
mo e binding si es o OTA) and by u he op imizing binding, washing,
and elu ion condi ions. These p oo -o -concep assays hus demon-
s a ed he sui abili y o immobilized T xBDII o di ec OTA ex ac ion,
clean-up and en ichmen om complex whi e g ape juice samples and
he e ec i eness o BDII ob ained om E. coli as bio ecep o o OTA
cap u e, se ing he basis o u u e s udies.
4. Conclusion
This wo k demons a ed, o he i s ime, ha His6- agged BDII
p oduced and pu i ied om E. coli e ains he abili y o na i e BSA o
bind o OTA, albei wi h lowe a ini y, and holds g ea po en ial as a
low-cos bio ecep o o OTA cap u e. In solu ion, bo h BDII e sions
(HisBDII and T xBDII) unde go sligh con o ma ional changes in hei
seconda y s uc u e upon binding o OTA. A e apid si e-di ec ed
immobiliza ion on Ni-NTA esin h ough he His6 ag used o he
BDII N- e minal, BDII main ained i s OTA-binding capaci y. U ilizing
his s aigh o wa d and cos -e ec i e immobiliza ion s a egy, we
de eloped new g a i y low SPE columns based on immobilized
T xBDII, enabling di ec ex ac ion, clean-up, and en ichmen o OTA
om spiked whi e g ape juice samples, wi h up o 84 ±7.4 % eco e y.
While we pe o med he pu i ica ion and immobiliza ion o His6- agged
BDII in wo s eps o be e con ol o e he amoun o immobilized
p o ein, u u e s udies may conside combining hese s eps in o a single
p ocess o simpli y and speed up he p oduc ion o BDII-based SPE
columns [14]. This s eamlined app oach has he po en ial o u he
enhance he p ac ical applica ion and comme cial iabili y o
BDII-based sys ems o OTA de ec ion and analysis.
CRediT au ho ship con ibu ion s a emen
Ta iana Q. Aguia : W i ing – e iew &edi ing, W i ing –o iginal
d a , Me hodology, In es iga ion, Fo mal analysis, Concep ualiza ion.
Tˆ
ania Leal: W i ing –o iginal d a , Me hodology, In es iga ion,
Fo mal analysis. Diana G. Rod igues: Me hodology, In es iga ion,
Fo mal analysis. Luís Ab unhosa: W i ing – e iew &edi ing, Supe -
ision, Me hodology, Funding acquisi ion, Fo mal analysis, Concep u-
aliza ion. Ca la Oli ei a: W i ing – e iew &edi ing, Supe ision,
Me hodology, Fo mal analysis, Concep ualiza ion. Lucília Domingues:
W i ing – e iew &edi ing, Supe ision, Funding acquisi ion,
Concep ualiza ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Acknowledgemen s
This s udy was suppo ed by he Po uguese Founda ion o Science
and Technology (FCT) unde he scope o he s a egic unding o UIDB/
04469/2020 uni (h ps://doi.o g/10.54499/UIDB/04469/2020) and
P ojec MycoP oA ini y (h ps://doi.o g/10.54499/2022.03438.
PTDC). Luís Ab unhosa acknowledges FCT o he assis an esea ch
con ac CEECIND/00728/2017. The au ho s would also like o
acknowledge Heiða Aðals einsson, La ínia Pin o and Rui M. Rod igues
o hei assis ance in CD spec oscopy analyses.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j. alan a.2024.127126.
Da a a ailabili y
Da a will be made a ailable on eques .
Re e ences
[1] W.-C. Liu, K. Pushpa aj, A. Meyyazhagan, V.A. A umugam, M. Pappuswamy, H.
K. Bho la, R. Baska an, U. Issa a, B. Balasub amanian, A. Mousa i Khaneghah,
Och a oxin A as an ala ming heal h h ea o li es ock and human: a e iew on
molecula in e ac ions, mechanism o oxici y, de ec ion, de oxi ica ion, and
die a y p ophylaxis, Toxicon 213 (2022) 59–75, h ps://doi.o g/10.1016/j.
oxicon.2022.04.012.
[2] V. Pichon, A. Comb`
es, Selec i e ools o he solid-phase ex ac ion o Och a oxin A
om a ious complex samples: immunoso ben s, oligoso ben s, and molecula ly
imp in ed polyme s, Anal. Bioanal. Chem. 408 (2016) 6983–6999, h ps://doi.o g/
10.1007/s00216-016-9886-0.
[3] Y. Wang, C. Zhang, J. Wang, D. Knopp, Recen p og ess in apid de e mina ion o
myco oxins based on eme ging bio ecogni ion molecules: a e iew, Toxins 14
(2022) 73, h ps://doi.o g/10.3390/ oxins14020073.
[4] Eu opean Commission, Commission Regula ion (EC) 2023/915 o 25 Ap il 2023 on
maximum le els o ce ain con aminan s in ood and epealing Regula ion (EC) No
1881/2006, O . J. Eu . Union L119 (2023) 103–157.
[5] E. Janik, M. Niemcewicz, M. Podog ocki, M. Ce emuga, L. Go niak, M. S ela,
M. Bijak, The exis ing me hods and no el app oaches in myco oxins’de ec ion,
Molecules 26 (2021) 3981, h ps://doi.o g/10.3390/molecules26133981.
[6] Y. Alhamoud, D. Yang, S.S. Fia i Kens on, G. Liu, L. Liu, H. Zhou, F. Ahmed,
J. Zhao, Ad ances in biosenso s o he de ec ion o och a oxin A: bio- ecep o s,
nanoma e ials, and hei applica ions, Biosens. Bioelec on. 141 (2019) 111418,
h ps://doi.o g/10.1016/j.bios.2019.111418.
[7] J. Ye, H. Bao, M. Zheng, H. Liu, J. Chen, S. Wang, H. Ma, Y. Zhang, De elopmen o
a no el magne ic-bead-based au oma ed s a egy o e icien and low-cos sample
p epa a ion o och a oxin A de ec ion using myco oxin–albumin in e ac ion,
Toxins 15 (2023) 270, h ps://doi.o g/10.3390/ oxins15040270.
[8] T. Leal, L. Ab unhosa, L. Domingues, A. Venˆ
ancio, C. Oli ei a, BSA-based sample
clean-up columns o och a oxin A de e mina ion in wine: me hod de elopmen
and alida ion, Food Chem. 300 (2019) 125204, h ps://doi.o g/10.1016/j.
oodchem.2019.125204.
[9] L.J.G. Sil a, A.C. Teixei a, A.M.P.T. Pe ei a, A. Pena, C.M. Lino, Och a oxin A in
bee s ma ke ed in Po ugal: occu ence and human isk assessmen , Toxins 12
(2020) 249, h ps://doi.o g/10.3390/ oxins12040249.
[10] F.S. Chu, In e ac ion o och a oxin A wi h bo ine se um albumin, A ch. Biochem.
Biophys. 147 (1971) 359–366, h ps://doi.o g/10.1016/0003-9861(71)90391-2.
[11] M. Po´
o , Y. Li, G. Ma isz, L. Kiss, S. Kuns´
agi-M´
a ´
e, T. K˝
oszegi, Quan i a ion o
species di e ences in albumin–ligand in e ac ions o bo ine, human and a se um
albumins using luo escence spec oscopy: a es case wi h some Sudlow’s si e I
ligands, J. Lumin. 145 (2014) 767–773, h ps://doi.o g/10.1016/j.
jlumin.2013.08.059.
[12] Y.V. Il’iche , J.L. Pe y, F. Rüke , M. Dockal, J.D. Simon, In e ac ion o och a oxin
A wi h human se um albumin. Binding si es localized by compe i i e in e ac ions
wi h he na i e p o ein and i s ecombinan agmen s, Chem. Biol. In e ac . 141
(2002) 275–293, h ps://doi.o g/10.1016/S0009-2797(02)00078-9.
[13] O. Khe sonsky, M. Goldsmi h, I. Za e sky, S. Hame -Rogo ne , O. Dym, T. Unge ,
M. Yona, Y. F idmann-Si kis, S.J. Fleishman, S able mammalian se um albumins
T.Q. Aguia e al. Talan a 283 (2025) 127126
8
designed o bac e ial exp ession, J. Mol. Biol. 435 (2023) 168191, h ps://doi.
o g/10.1016/j.jmb.2023.168191.
[14] A.I. F ei as, L. Domingues, T.Q. Aguia , Tag-media ed single-s ep pu i ica ion and
immobiliza ion o ecombinan p o eins owa d p o ein-enginee ed ad anced
ma e ials, J. Ad . Res. 36 (2022) 249–264, h ps://doi.o g/10.1016/j.
ja e.2021.06.010.
[15] A. Sha ma, T.K. Chaudhu i, Re isi ing Esche ichia coli as mic obial ac o y o
enhanced p oduc ion o human se um albumin, Mic ob. Cell Fac o ies 16 (2017)
173, h ps://doi.o g/10.1186/s12934-017-0784-8.
[16] W. Zhu, R. Xu, G. Gong, L. Xu, Y. Hu, L. Xie, Medium op imiza ion o high yield
p oduc ion o human se um albumin in Pichia pas o is and i s e icien pu i ica ion,
P o ein Exp . Pu i . 181 (2021) 105831, h ps://doi.o g/10.1016/j.
pep.2021.105831.
[17] M.T. Nguyen, Y. Heo, B.H. Do, S. Baek, C.J. Kim, Y.J. Jang, W. Lee, H. Choe,
Bac e ial o e exp ession and pu i ica ion o soluble ecombinan human se um
albumin using mal ose-binding p o ein and p o ein disulphide isome ase, P o ein
Exp . Pu i . 167 (2020) 105530, h ps://doi.o g/10.1016/j.pep.2019.105530.
[18] T.Q. Aguia , L. Domingues, Recombinan p o ein pu i ica ion and immobiliza ion
s a egies based on pep ides wi h dual a ini y o i on oxide and silica, Bio echnol.
J. 18 (2023) 2300152, h ps://doi.o g/10.1002/bio .202300152.
[19] E. Gas eige , C. Hoogland, A. Ga ike , S. Du aud, M.R. Wilkins, R.D. Appel,
A. Bai och, P o ein Iden i ica ion and analysis ools on he ExPASy se e , in: J.
M. Walke (Ed.), P o eomics P o oc. Handb., Humana P ess, To owa, NJ, 2005,
pp. 571–607, h ps://doi.o g/10.1385/1-59259-890-0:571.
[20] H.J. Mo ulsky, R.R. Neubig, Analyzing binding da a, Cu . P o oc. Neu osci. 52
(7.5.1–7.5.65) (2010), h ps://doi.o g/10.1002/0471142301.ns0705s52.
[21] G aphPad, Fi ing binding o luo escen ligands, Knowledgebase A icle #1725,
h ps://www.g aphpad.com/suppo / aq/ i ing-binding-o - luo escen -ligands/,
2011. (Accessed 19 Janua y 2024).
[22] A. Micsonai, ´
E. Moussong, F. Wien, E. Bo os, H. Vad´
aszi, N. Mu ai, Y.-H. Lee,
T. Moln´
a , M. R´
e ´
egie s, Y. Go o, ´
A. Tan os, J. Ka dos, BeS Sel: webse e o
seconda y s uc u e and old p edic ion o p o ein CD spec oscopy, Nucleic Acids
Res. 50 (2022) W90–W98, h ps://doi.o g/10.1093/na /gkac345.
[23] A.I. F ei as, L. Domingues, T.Q. Aguia , Ba e silica as an al e na i e ma ix o
a ini y pu i ica ion/immobiliza ion o His- agged p o eins, Sep. Pu i . Technol.
286 (2022) 120448, h ps://doi.o g/10.1016/j.seppu .2022.120448.
[24] S. Cos a, A. Almeida, A. Cas o, L. Domingues, Fusion ags o p o ein solubili y,
pu i ica ion and immunogenici y in Esche ichia coli: he no el Fh8 sys em, F on .
Mic obiol. 5 (2014) 63, h ps://doi.o g/10.3389/ micb.2014.00063.
[25] M. Schenkel, A. T e , C.M. Debe , G. K aine , M. Schlie , Hea ea men o
hio edoxin usions inc eases he pu i y o
α
-helical ansmemb ane p o ein
cons uc s, P o ein Sci. 30 (2021) 1974–1982, h ps://doi.o g/10.1002/p o.4150.
[26] M. Dockal, D.C. Ca e , F. Rüke , The h ee ecombinan domains o human se um
albumin, J. Biol. Chem. 274 (1999) 29303–29310, h ps://doi.o g/10.1074/
jbc.274.41.29303.
[27] F. Sueck, M. Po´
o , Z. Faisal, C.G.W. Ge zen, B. C ame , B. Lemli, S. Kuns´
agi-M´
a ´
e,
H. Gohlke, H.-U. Hump , In e ac ion o och a oxin A and i s he mal deg ada ion
p oduc 2
′
R-och a oxin a wi h human se um albumin, Toxins 10 (2018) 256,
h ps://doi.o g/10.3390/ oxins10070256.
[28] Y.V. Il’iche , J.L. Pe y, J.D. Simon, In e ac ion o och a oxin A wi h human se um
albumin. P e e en ial binding o he dianion and pH e ec s, J. Phys. Chem. B 106
(2002) 452–459, h ps://doi.o g/10.1021/jp012314u.
[29] M.A. Qu eshi, S. Ja ed, A la oxin B
1
induced s uc u al and con o ma ional
changes in bo ine se um albumin: a mul ispec oscopic and ci cula dich oism-
based s udy, ACS Omega 6 (2021) 18054–18064, h ps://doi.o g/10.1021/
acsomega.1c01799.
[30] D.S. Vazquez, I.E. S´
anchez, A. Ga o e, M.P. Sica, J. San os, The E. coli hio edoxin
olding mechanism: he key ole o he C- e minal helix, Biochim. Biophys. Ac a
BBA - P o eins P o eomics 1854 (2015) 127–137, h ps://doi.o g/10.1016/j.
bbapap.2014.11.004.
[31] I. Cagnasso, G. Tonachini, S. Be o, A. Giacomino, L. Mand ile, A. Ma anzana,
F. Du biano, Comp ehensi e s udy on he deg ada ion o och a oxin A in wa e by
spec oscopic echniques and DFT calcula ions, RSC Ad . 9 (2019) 19844–19854,
h ps://doi.o g/10.1039/C9RA02086A.
[32] A. L´
opez-Ye ena, M. Pe ez, A. Vall e dú-Que al , E. Esc ibano-Fe e , Insigh s in o
he binding o die a y phenolic compounds o human se um albumin and ood-
d ug in e ac ions, Pha maceu ics 12 (2020) 1123, h ps://doi.o g/10.3390/
pha maceu ics12111123.
T.Q. Aguia e al. Talan a 283 (2025) 127126
9
