3 Biotech (2018) 8:254
Enhanced production and puriﬁcation of recombinant surface array
protein (Sap) for use in detection of Bacillus anthracis
· N. K. Tripathi
· V. Pal
· Ajay Kumar Goel
Received: 1 March 2018 / Accepted: 28 April 2018 / Published online: 11 May 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018
Surface array protein (Sap) can be an important biomarker for speciﬁc detection of Bacillus anthracis, which is released by
the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli.
The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap
in soluble form. The recombinant Sap was puriﬁed employing aﬃnity chromatography followed by diaﬁltration. The ﬁnal
yield of puriﬁed protein was 20 and 46 mg/l of culture during shake ﬂasks and batch fermentation, respectively. The protein
purity and its reactivity were conﬁrmed employing SDS–PAGE and Western blot, respectively. The antibodies raised against
puriﬁed Sap were evaluated by Western blotting for detection of Sap released by B. anthracis. Our results showed that the
Sap could be a novel marker for detection and conﬁrmation of B. anthracis.
Keywords Anthrax · Escherichia coli · Bioreactor · Puriﬁcation · Surface array protein
Anthrax is a potentially fatal zoonotic disease and humans
get infection on coming in contact with the aﬀected ani-
mals. Bacillus anthracis, the causative agent is recognized
as the most potential biological warfare agent (Goel 2015).
Besides, anthrax is also a public health problem in several
agrarian countries. Therefore, it is important to develop sim-
ple technologies for detection of B. anthracis.
Surface layers (S-layers) are generally the outermost com-
ponents of several bacteria. However, in B. anthracis S-lay-
ers are highly patterned ultra structure layers found beneath
the capsule made up of poly-D-glutamic acid (Mesnage et al.
1999). B. anthracis harbors two types of S-layer proteins,
viz., extracellular antigen-1 (EA1) and surface array protein
(Sap), which are encoded by the chromosomal genes eag or
sap, respectively (Kern et al. 2012; Mesnage et al. 1997).
B. anthracis bacteria are entrapped by Sap under exponen-
tial growth phase. Later on, this Sap is replaced by EA1
when the bacteria reaches stationary phase (Candela et al.
2005). Both, Sap as well as EA are highly antigenic and
immunogenic proteins (Walper et al. 2012). EA1 has been
used for species speciﬁc detection of B. anthrcais spores
(Lai et al. 2003). However, later on it was found that instead
of an integral component of spore, EA was a contaminant
during spore preparation (Williams and Turnbough 2004).
B. anthracis secretes Sap in growth medium under in vitro
conditions, whereas other bacteria do not. Thus, Sap may be
utilized as a biomarker for early detection of B. anthracis.
The present study was aimed at high level production of
recombinant Sap antigen in E. coli using bioreactor, and
its utility as an important biomarker for detection of B.
Materials and methods
Cloning of sap gene
B. anthracis Sterne grown in Luria Bertani (LB) broth was
used to extract the genomic DNA employing the method
described elsewhere (Jackson et al. 1997). The surface array
protein (sap) gene (GenBank accession no. Z36946.1) was
PCR ampliﬁed using the primers, sap-F (5′-CAT GGA TCC
GCA GGT AAA ACA TTC CCA G-3′, the BamH1 site under-
lined) and sap-R (5′-ATA CTC GAG TTT TGT TGC AGG TTT
* Ajay Kumar Goel
Bioprocess Technology Division, Defence Research
and Development Establishment, Jhansi Road,
Gwalior 474002, India