The lysin motif (LysM) is a ubiquitous motif across kingdoms, which in bacteria allows cell wall degrading enzymes to bind noncovalently to peptidoglycan. This property has been exploited for two decades to design mucosal vaccines consisting of LysM-tagged recombinant proteins anchored to bacterium like particles (BLP) as carriers. Surprisingly, less attention has been paid to apply the LysM motif to protein purification of recombinant proteins. Thus, our goal was to determine if the LysM motif is suitable for recombinant protein purification.

1. Affinity purification of recombinant proteins
using a LysM domain and bacterium like
particles
Carla Luciana Padilla Franzotti1, Maria Fernanda Raya Tonetti1, Lorena
Arce1, Maria Jose Rodriguez Vaquero2 and Maria Guadalupe Vizoso Pinto1,*
1Infection Biology Lab, INSIBIO (UNT-CONICET); Faculty of Medicine of the National University of Tucumán, Argentina.
2Chair of Microbiology, Faculty of Biochemistry, Chemistry and Pharmacy of the National University of Tucumán, Argentina. (UNT-CONICET)
CONCLUSIONS
RESULTS
METHODS
Binding
AssaysBLP
Acid/Heat
Treatment
Lactobacilli
Elution
Assays
E. coli
Rossetta
Cell Lysis
Recombinant
protein (RP)
5 LysM motifs
Venus
Fluorescence
Microscopy
Expression
(IPTG)
RP bound to
BLP (BP)
TEM
SDS-PAGE
Three washes
during 20 min
at RT in orbital
shaker
Mix during 1h at
RT in orbital
shaker
During 30 min
at RT in orbital
shaker
SDS-PAGE
Changing:
Ionic strength, pH and
Buffer composition
Cellular
Lysate (CL)
Binding and Washes in PBS pH 7.4
Changing Buffer composition
Changing ionic strengthChanging pH
Binding and Washes in PBS pH 7.4
Binding in Buffer 4M
urea pH 7.4
INTRODUCTION
The Lysin motif (LysM) is conserved and present across kingdoms. It specifically binds to peptidoglycan and chitin. This property has been used for surface display of
heterologous proteins on the surface of Gram positive bacteria for vaccine development and may also have multiple medical and industrial applications (1). The
possible use of this property for protein purification was proposed in reviews about the multiple applications of this motif but, to the best of our knowledge, there are
no reports on purification of proteins (1). Bacterium like particles (BLP) are Gram positive bacteria which expose the peptidoglycan of their cell walls after a heat and
acid treatment depletes them from other components such as proteins and teichoic acids. Furthermore, they loose membrane permeability, proteins and DNA content,
thus, are not viable. This particulate form is advantageous for handling, therefore, we have used BLP as matrix for developing an affinity chromatography purification
method of LysM-tagged recombinant proteins.
(1) Visweswaran GR, Leenhouts K, van Roosmalen M, Kok J, Buist G. Exploiting the peptidoglycan-binding motif, LysM, for medical and industrial applications. Applied microbiology and biotechnology. 2014;98(10):4331-45.
(2) de Haan A, Haijema BJ, Voorn P, Meijerhof T, van Roosmalen ML, Leenhouts K. Bacterium-like particles supplemented with inactivated influenza antigen induce cross-protective influenza-specific antibody responses
through intranasal administration. Vaccine. 2012;30(32):4884-9
We selected LysM domains from L. fermentum (results not shown) and constructed a destination vector (Gateway compatible) to tag with the LysM domain genes of
interest contained in Entry libraries. Using the fluorescent protein Venus as model, we developed a new purification method for proteins using a cheap and renewable
matrix and batch conditions. Proteins obtained under denaturing conditions are suitable for antigen production with diagnostic purposes. They may also be renatured
using standard dialysis methods. Nevertheless, we are working on purification of proteins under native conditions and on changing batch to column.
LysM-tagged proteins in cellular lysates and inclusion bodies lysates (after dilution) bound to the cell walls of BLPs as demonstrated by fluorescence microscopy. First we
tested 4 different species of lactobacilli and found that tagged proteins bound better to L. fermentum BLPs. Binding buffer added with 4M urea avoided unspecific
binding of an E. coli protein of ~30KDa. Changing pH (6-9) or ionic strength (1.5 M NaCl) were not suitable for eluting the protein. We tested different concentrations of
urea, and eluted the protein with urea 6 to 8 M.
Using Gateway recombinatorial cloning (Gateway), we obtained fusion proteins N-His-Venus-LysM5,
which were expressed in E. coli Rosetta induced with IPTG. Cells were lysed using standard buffers added
with lysozyme. BLPs were obtained by treating pellets from o.n. cultures of lactobacilli with HCl 1N for
30 min in a boiling water bath. Cellular and inclusion bodies lysates were diluted in PBS or Buffer Tris-Cl
and mixed with BLPs for an hour at RT in microcentrifuge tubes. Pellets were washed with 3 volumes of
washing buffer incubating for 20 min in orbital shaker 3 times. Different buffers were tested to try to
elute the bound protein and to reduce unspecific binding of E.coli proteins. All steps were followed by
SDS –PAGE.
REFERENCES
We acknowledge
FoNCyT and CONICET, Argentina,
for financial support.
*Email: mgvizoso@fm.unt.edu.ar