This laboratory project report details the expression and purification of the Clocl_2077 protein from Clostridium clariflavum for the purposes of crystallization and structural determination. The Clocl_2077 protein is a putative fused anti-sigma factor and sigma factor protein that may be involved in cellulose degradation regulation. The project involved cloning the Clocl_2077 gene, expressing it in E. coli, and purifying the protein using nickel affinity and size exclusion chromatography. Additional experiments were conducted to express and purify the CBM3b domain of the Clocl_1053 protein to test its ability to bind carbohydrates. The successful purification of Clocl_2077 paved the way for future crystallization

1. GEORGE S. WISE FACULTY OF LIFE SCIENCES
The Department of Molecular Microbiology and Biotechnology
Expression and purification Clostridium
clariflavum Clocl_2077 Un-known Module
to a Further Crystallization for Molecular
Deciphering
Laboratory Project Final Report
Written by: Oded Mizrachi (ID 038168233)
Under the supervision of: Prof. Raphael Lamed
Instructor: Leeron Piechota
Date: February 2014

2. 1
Abstract
Life on Earth depends on photosynthesis, which results in production of plant biomass having
cellulose as the major component. The carbon cycle is closed primarily as a result of the action of
cellulose-utilizing microorganisms [1]. Thus, microbial cellulose utilization is responsible for one
of the largest material flows in the biosphere and is of interest in relation to analysis of carbon
flux at both local and global scales. Cellulosic materials are particularly attractive in this context
because of their relatively low cost and plentiful supply. Aloof, breaking cellulose is no easy task.
The central technological impediment to more widespread utilization of this important resource
is the general absence of low-cost technology for overcoming the recalcitrance of cellulosic
biomass. A promising strategy to overcome this impediment involves the production of
cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired
products in a single process step via a cellulolytic microorganism or consortium [1, 2]. Generally,
the degradation processes can be achieved by a macromolecular complex named Cellulosome- A
machine comprising different enzymes which can digest the biomass efficiently [3]. This project
will deal with the cellulosome-producing anaerobic bacteria- Clostridium clariflavum working of
expression, purifying, crystallization and diffraction parts of the Clocl_2077 protein.
The main results of this work are the obtaining of a pure protein solution of Clocl_2077 and the
construction of experiments for future crystallizations, in order to reach a further understanding
helping the main goal above.
A side job of this project was also to over expresses and purifying the CBM3b protein (part of
Clocl_1053) protein of C. clariflavum and to construct a carbohydrate-binding assay to the
purifying protein in order to examine the protein ability to bind carbohydrates.
Introduction
Cellulolytic clostridia are prominently represented among bacterial species. These
organisms are able to solubilize lignocellulose, and their high rates of cellulose
utilization make them candidates for consolidated bioprocessing applications [4]. In
particular, anaerobic cellulolytic clostridia which grow at thermophilic temperatures
is aclariflavumlostridiumCto break down lignocellulose very efficiently.ableare
within the family Clostridiaceae isolated fromClostridiumIIIClustershaperod
dalthough retarde-motile-type positive, nonGram,c sludgethermophilic anaerobi
st because of its. This species is of intere], 54[flagella are presentedperitrichos
Clostridiumorganismmodel cellulolyticthe well studysimilarity to
environmental isolates to break downity ofand for the abilthermocellum
-omecellulosis aC. clostridiumned,oAs mentihemicellulose in addition to cellulose.
designed for efficientcomplexellulosomecheT.producing anaerobic bacterium
degradation of plant cell-wall polysaccharides in general and cellulose in particular. It
consist of a central 'scaffoldin' subunit that incorporates the various enzymes into the
complex, anchors the complex onto the cell surface of the bacterium and targets the
complex to the substrate (figure1). A cellulosome's major component is the CBM

3. 2
(Carbohydrate Binding Module). CBM is a family of proteins that share a discrete
fold which enables them to bind different carbohydrates. So far, 64 CBM families
have been identified, based on amino acid sequence similarity [6]. These families
feature a great diversity in ligand specificity. there are characterized CBMs that
recognize crystalline cellulose, non-crystalline cellulose, chitin, xylan, starch and ex.
[7]. An example that will later go through a carbohydrate binding assay- as a 'side job'
of this study- is CBM3b1
(family-3b CBM) which characterized by a module of
approximately 150 amino acids organized in a β-sandwich fold and has the ability to
glycosyl hydrolasesofvarietyaare known to organizeriflavumclaC.bind cellulose.
and other catalytic subunits outside of the cell by means of the cellulosome. In this
bacterium there are few different examples of a cellulosome architecture structural
proteins such as typeI and typeII cohesin-dockerin interaction (fighre1), in addition to
a changeable number of the subunits comprising the different macromolecules. In
terms of the anchoring proteins, 4 different structures have been identified containing
SLH2
(S-layer homology). In addition, in contrast to others cellulosomal
4haveclariflavumC.,modules are not very commonCBM2ganisms wheremicroor
(!) of these domains that also associated with variable modules. This specific and
C.wideness of theorder to show theelaborated description is important in
can becellulosome components. As a result, a numerous possibilitiesclariflavum
taken in account, in order to seek for the perfect combination to manipulate and
accomplish the best efficient carbohydrate degradation complex.
Figure13
.Simplified model of a typical cellulosome, based on the C. thermocellum paradigm. This figure represent diverse
interactions inside the complex. C. clariflavum also have different and wide possibilities installing the varied relationships
between the different sub-units. Note that not all the interactions described in the 'Introduction' section are presented.
1
Historically, the division of the family into subgroups (a, b and c) was based on minor sequence differences combined with the
fact that the known scaffoldin-borne CBM3s (subgroup a) from four different clostridia could be differentiated by the existence
of a distinctive Trp-containing. Subfamilies b and c lacked this loop and were further differentiated by the lack of the standard
aromatic binding residues in subfamily c [8].
It.archaea, as well as amongbacteriacommonly found incell envelopeis a part of the(SLH)(surface layer)homologylayer-S
2
.glycoproteinsorproteinsconsists of a monomolecular layer composed of identical
3
This figure is given due to a lack of such relevant data of this work's bacteria in order to convey a graphic clue of the different
interaction described.

4. 3
Former studies of other bacteria revealed the presence of putative genes for proteins,
homologous to the anti-σ factor RsgI protein of Bacillus subtilis [9]. Some of these
genes contain upstream to the rsgI an open reading frame (ORF) encoding for σ
factor, sigI, that are expected to be co-transcribed, as in B. subtilis [10]. Some of the
RsgI containing proteins are different in their modular structure from those of B.
subtilis RsgIs. These proteins have additional domains at the C-terminal that are
predicted to be located and act outside the cell membrane as CBMs. The proposed
regulatory mechanism of the cellulosomal genes, states that alternative σ factors are
activated in response to the polysaccharides in the extracellular surroundings (Fig. 2)
[9]. Bioinformatic analyses have shown a presence of such putative protein which
comprises anti-σ and σ factors in the same ORF also in C. clariflavum [11]. In
addition, an exclusive new sequence was discovered during examination the C.
clariflavum genome, indicating a new fused type of protein which comprises both
anti-σ and σ factors. The C-terminus putative module of these fused protein
designated Early set (E_set) and the complete protein is RsgI-like E_Set
(Clocl_2077). This protein contains an N-terminal sigI-like region (Region (Re). A)
fused to RsgI-like region with a trans-membrane domain (Re. B) that has a putative
sensing module (Re. C) which consist the E_set module at its C-terminus (fig. 2, ii).
The whole general function of Clocl_2077 is still unknown but can be predicted,
hence is the relevant of this study. The origin of the most basic hypothesis must lays
on a solid ground. That's why the initial work, carried out in this project, is to over-
express, purifying and figure the partial 'long' protein's structure of Clocl_2077, in
order to give a profound knowledge of the interactions between the subunits relating
to the cellulose utilization that will hopefully shed some light on the process. By
doing that I'm hoping to achieve some basis for further research of the cellulose
degradation by C. clariflavum. In addition, in this work I'm dealing with the CBM3b
protein part of Clocl_1053 in order to test its carbohydrates binding ability.

5. 4
Fig2: i. Proposed mechanism for the activation of alternate σ factors by extracellular polysaccharides. The RsgI trans-
membrane proteins (red) contain an extracellular carbohydrate-active module (CBM) and an intracellular anti-σ peptide domain.
In the OFF state, the anti-σ domain interacts strongly with the alternative σ factor (blue), thereby inactivating it. In the ON state,
extracellular polysaccharides (green) interact with the CBM, which, in turn, induces a conformational change on the intracellular
anti-σ domain, resulting in the release of the alternative σ factor. The σ factor is now free to interact with RNA polymerase
(RNAP) and promote transcription of the σ-dependent promoters. Note that the σ factor also promotes transcription of its own
bicistronic operon. The deviation of the Clocl_2077 protein is mark in blue bracket and also the E_set protein is given (orange).
ii. Modular structure of the RsgI-like E_Set protein in C. clariflavum Putative rsgI-E_set gene. (A) SigI domain is
represented in green; (B) RsgI domain represented in grey and the transmembrane region in black; (C) C-terminus module
including E_set. The whole Clocl_2077 protein was divided and defined as followed: region (Re.) C only ("short" protein),
region C together with a part of region B (Re. B') ("long" protein) and the whole protein containing regions A, B and C ("Whole"
protein).
and methodssMaterial
:C. clariflavumof2077_Cloning Clocl:STAGE1
A DNA fragment encoding Clocl_2077 was amplified by PCR from C. clariflavum
DSM119732 genomic DNA isolated as described by Murray & Thompson (1980)
using two specific primers4
: 5'-ATCGCATATGAATCGTACACCGGCGT-TTTC-3' (forward) and
5'-TGCCTCGAG TTATTCGACAACCGTAACGGTAAC-3' (reverse). The PCR products were purified and
both the DNA fragment and the expression vector pET-28a(+) (Novagen, Madison,
Wisconsin, USA) were cleaved with the restriction enzymes NdeI and XhoI. The
cleaved fragments were incubated with DNA ligase. The obtained construct contained
an N-terminal hexahistidine (His) tag and thrombin cleavage site. Transformation of
the ligated vector into competent Escherichia coli BL21 (DE3) was done. LB
4
The italics 6 nucleotides in each primer refer to the restriction enzymes sites (Forward primer: Nde1, reverse primer: Xho1).
Re. C-
'short'Re. C+B'-
'long'
Re. C+B+A-
'whole'
E-set
i ii

6. 5
(Lysogeny Broth) + Kanamycin plates were used in order to identify the
transformants colonies.
Fig3. The predicted pET-28a(+) vector. Note that just the Clocl_2077 insert is present. Clocl_1053 was cloned at the same way
and place and using of the same vector.
***Transformated E. coli bacteria which adopted the vector containing Clocl_1053
were provided as a gift from Dr. Oren Yaniv The cloning was done using the primers:
forward (cut by NdeI) 5'-GCACATATGTCTGTTAAGCTCGGTATGTACAA-3', reverse (cut by XhoI)
5'-GCACTCGAGTTAGGGTTCAGTACCCCATAC-3'.
:2077 only)_(CloclssayAdetection by colony PCRectorV:STAGE2
4 colonies were chosen for colony PCR using the original primers described earlier. A
colony that received the vector will yield a ~735bp bend after electrophoresis in
Agarose gel, which refers to the Clocl_2077 gene's length (including the primers).
:1053 separately)_2077 & Clocl_locl(Cein expressionProt:STAGE3
4 different pre-cultures (starters) of E. coli harboring the expression plasmid were
cultivated. A gentle touch of a chosen colony (which showed a positive figure at the
colony-PCR assay) was mix with 5ml LB and 5µl kanamycin 50µg/ml for expression
at 310ºK, 270rpm rotation, O.N. Day afterwards each starter was grown under aerobic
conditions at 310ºK, 270rpm rotation 24h in TB (Terrific Broth) medium The
specifics growing conditions were chosen due to some results of previously studies [6,
7, 8].

7. 6
:1053 separately)_2077 & Clocl_(Cloclprotein purificationSTAGE4:
After over-expression of the protein, the bacteria were harvest by centrifuge (5000g,
30min, 4ºC). In order to have accesses to the proteins the cells were broke down by
sonication. Therefore after the first centrifuge the pellet of the processes was re-
suspended in sonication buffer (1:4 volume ratio of wet pellet (g) and sonication
buffer (ml) respectively). In order to eliminate unwanted nucleic-acids and preventing
degradation of the desirable protein 2µl of DNase and 100µl protease inhibitors
mixture were added. The whole suspension was kept on ice during sonication (to
prevent denaturation of the proteins). Finally, the broken cells mixture was
centrifuged (18000g, 45min, 4°C), and the supernatant fluids were collected for
further protein purification.
:1053 separately)_2077 & Clocl_(CloclIDA chromatography-NiSTAGE5:
The recombinant Clocl_2077/Clocl_1053 containing an N-terminal His-tag was first
isolated by metal-chelate affinity chromatography using Ni packed colona according
to the manufacturer’s recommended protocol. For separation of the un-bound
proteins the supernatant was loaded to a 16/10 HR (High Resolution) glass column
and washed by 40ml washing buffer (flow rate of 1.5ml/min). The column was then
connected to Fast Protein Liquid Chromatography (FPLC) ÄKTA-prime System (GE
healthcare, USA). Elution of the bound His-tagged protein was done with 50ml
elution buffer (flow rate of 2ml/min). 2ml fractions were collected and later subjected
to SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamid Gel Electrophoresis) to
estimate the amount and the purification level of the expressed protein.
2077 &_(Cloclchromatographysize exclusion(GF)filtration-GelSTAGE6:
:1053 separately)_Clocl
After analysis of the purification made by the Ni-IDA processes the fractions refers to
the best absorption noted over the assay were combined. The gathered protein was
concentrated using Centriprep YM-3 10kD centrifugal filter devices (Amicon
Bioseparation, Millipore, Billerica, Massachusetts, USA) and the protein
concentration was determined by measuring the absorbance at 280nm. Then, third
purification step was done- the protein was separated by neat exit gradient (large to
small size order) using FPLC size exclusion chromatography ÄKTA-prime System

8. 7
(GE healthcare, USA). 4ml of protein solution was administrated into 4ml injection
loop at a rate of 2 ml/min. 2ml fractions were collected and analyzed on SDS-PAGE.
**Note: few more GF chromatography procedures were made before continuing the
next stages in order to reach a satisfactory level of purified protein.
:2077 only)_(CloclThrombin cleavage:STAGE7
In order to prevent possible folding interruptions during crystallization The His tag of
Clocl_2077 was cleaved off by thrombin protease (Novagen, EMD Chemicals Inc.,
San Diego, California, USA) following the manufacturer’s instructions. Small volume
of purified protein was kept aside and wasn't cleaved by thrombin in order to execute
a further comparison to the cut-protein. After thrombin cleavage, the cleaved His tag
was removed from the preparation by metal-chelate affinity chromatography. For this
purpose, the solution was incubated with Ni–IDA resin and the suspension was
decanted into a column. The Flow-Through fluid (containing the cleaved protein and
thrombin) was collected and purified by FPLC using a Superdex 75 16/60 column
(GE Healthcare). Finally, also the cut protein and the uncut protein (which saved
earlier) were concentrated using Centriprep YM-3 10kD centrifugal filter devices and
the proteins concentration was determined by measuring the absorbance at 280nm.
The purified proteins solution consisted of 15.8mg/ml for the cut protein and
18.5mg/ml for the uncut protein.
:)2077 only_(CloclCrystallizationSTAGE8:
The protein sample was screened for crystallization using the hanging-drop vapor
diffusion Method using a 24-well VDX plate (Hampton Research). The size of the
droplets, which consisted of equal volumes of protein (cut & uncut separately) and
reservoir solution that placed on inversed siliconizes microscope slip, were 1.5µl
each. 400µl of the matching solution were added into each well. 192 conditions were
screened of the Crystal Screen I and II, PEG/ION I and II kits (Hampton Research,
CA, USA). In addition, Samples of the purified uncut protein were dispensed using
an Oryx-6 crystallization robot from Douglas Instruments (www.douglas.co.uk). A
1µl sample of the protein solution together with a 1µl aliquot of the crystallization
condition was dispensed into each well. 4 kits of 96 wells each were carried out a
mixture of silicone and paraffin oils combined in a 1:1 volume ratio was used to cover

9. 8
the crystallization wells (the 4 kits were Crystal screen, Index, wizard 1-2 and Seat-
Rx). Crystallization was performed at 293ºK in a temperature-controlled room.
Diffraction:E9:STAG
As for this writing, except emerging of few salt crystals, none relevant crystals had
appeared, therefore this stage wasn't carried out.
1053 only):_binding assay (CloclsCarbohydrateQualitativeSTAGE10:
The binding of Clocl_1053 to the insoluble Carbohydrates was determined as follows.
Clocl_1053 (50µg) was mixed with insoluble Carbohydrates (5mg) in a 50mM Tris-
HCl GF buffer (pH 7.5) in a final volume of 0.2ml and mix by rotation for 1h at R.T.
After centrifugation (3min, 10,000g), the supernatant (unbound) and the pellet
(bound) were treated differently. The pellets of each tested carbohydrate+Clocl_1053
were washed 3 times with 1ml of 50mM GF buffer. Afterwards, 65µl of S.B (Sample
Buffer) and 135µl GF buffer added to a final volume of 0.2ml. The supernatant
(20µl) were re-suspended in 10µl of S.B. Boiling of the samples for 10min eluted the
bound protein. Finally, the binding of the protein to the carbohydrate was evaluated of
both the bound and unbound samples by SDS–PAGE (12% SDS-PAGE which run at
70v for 20 min and 120v for 1.25h). The polysaccharides tested were cellulose,
amorphous cellulose, banana stem, switch grass, xylan (oat spelt), xylan (brich wood),
starch, chitin, lichenan and pectin. In addition, P.C (Positive Control) sample
containing cipA CBM3a+cellulose and N.C (Negative Control) sample containing
2506 CBM3b+cellulose were generated.
Results
i. Colony-PCR assay:
Plasmid transformation of pET-28a(+) into the bacteria's cell was examined over
LB+Kan plate. E. coli are sensitive bacteria to the Kan antibiotic, the vector consist
the gene 𝐾𝑎𝑛 𝑅
, therefore every growing colony suspected to receive the vector.
According to the results (fig4), All 4 chosen colonies had received the vector and are
ready for proteins translation. The N.C example gave no amplification as expected, so
we can assume that the reaction reagents used were specific and suitable.

10. 9
1000kb
750kb
500kb
250kb
DNA 1 2 3 4 N.C
ladder
1 2 3 marker 4 5 6 7 8 9 10 11 FT 12 12 13 14 15 16 marker 17 18 19 20 21 22 23
Marker 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fig4. The chosen colonies had adopted the pET-28a(+) vactor. Gel-electrophoresis analysis of colony PCR reaction using the
primers described earlier. All 4 colonies had positive PCR amplification which suitable for the Clocl_2077 length. Samples 1-4
were the one that continued the growth and purification process. N.C sample of DDW as template for the PCR reaction was
executed. (2% Agarose gel which run at 110v for 25min, the DNA ladder is 1kb ng/0.5µgr).
ii. Purification by Ni-IDA chromatography:
Fig5. 15% SDS-PAGE referring to Clocl_2077 Ni-IDA purification. As infer from this results the protein samples from tubes
1-11 and 17-19 indicate to a good relatively purification level. Notice that the FlowThrough (FT) samples contain a mixture of
proteins as expected (run at 70v for 20 min, and afterwards at 120v for 1.25h).
Fig6. 12% SDS-PAGE referring to Clocl_1053 Ni-IDA purification. As infer from this results the protein samples from tubes
13-19 indicate to a good relatively purification level (run at 70v for 20 min, and afterwards at 120v for 1.25h).
iii. Purification by GF size-exclusion chromatography:
iii-a. According to fig5, samples 1-11 and 17-19 showed satisfactory amount of
cleaned and sized relevant protein. Therefore those samples will function as good
candidates for further purification procedure.
37kD
25kD
20kD
15kD
37kD
25kD
20kD
15kD
37kD
25kD
20kD
15kD

11. 10
21 22 marker 23 24 25 26 27 30 31 32 33 34 35
36 37 38 39 40 41 marker 42 43 44 45 46 47 48
31 marker 32 33 34 35 36 37 38 39 40 41 42 43 marker 44 44 45 46 47 48 49
Fig7. 15% SDS-PAGE referring to Clocl_2077 GF#1 purification. As infer in this results samples 30-35 shows good amounts
of suitable size protein, the purification level though is not very high. Hence, another GF procedure was executed. (run at 70v for
20 min, and afterwards at 120v for 1.25h).
iii-b. In order to reach a high purification level another GF size-exclusion procedure
was executed injecting the 30-35 samples from fig7. The protocols of this action are
similar to the earlier one.
Fig8. 15% SDS-PAGE referring to Clocl_2077 GF#2 purification. As infer in this results samples 37-42 shows good amounts
of suitable size protein and a better purification level than the one reached in GF#1 (run at 70v for 20 min, and afterwards at 120v
for 1.25h).
iii-c. Good crystallization demand threshold concentration of the protein. Therefore an
appropriate volume of protein solution is required. Hence, another GF procedure was
executed using protein solution 'leftovers' (from samples 1-11, 17-19, fig5) after
purified by Ni-IDA chromatography and concentration by Centriprep YM-3 10kD
centrifugal filter device.
Fig9. 15% SDS-PAGE referring to Clocl_2077 GF#3 purification. As infer in this results samples 36-43 shows good amounts
of suitable size protein in a good purification level (run at 70v for 20 min, and afterwards at 120v for 1.25h).
37kD
25kD
20kD
15kD
37kD
25kD
20kD
15kD
37kD
25kD
20kD
15kD
37kD
25kD
20kD
15kD

12. 11
marker 20 21 22 23 24 25 26 27 28 29 30 31
iii-d. According to fig6, samples 17-19 had showed the biggest amount of sized
relevant Clocl_1053 protein (~20kD). The purification level though was poor so the
samples were gathered, concentrated by Centriprep YM-3 10kD centrifugal filter
devices and accomplished GF size-exclusion procedure.
Fig10. 12% SDS-PAGE referring to Clocl_1053 GF purification. As infer from this results the protein samples from tubes 22-
26 indicate to a good relatively protein amount in a poor purification level (run at 70v for 20 min, and afterwards at 120v for
1.25h).
iv. Qualitative Carbohydrates binding assay:
Purified Clocl_1053 protein at concentration of 0.26 mg/ml plus different insoluble
cellulostic polymers were used in this assay (the protein solution gathered from the
relevant samples after the GF procedure and a Centriprep YM-3 10kD treatment).
Results are present below.
Protein substrate
cellulose Amorphous
cellulose
Banana stem Switch
grass
Xylan
(oat spelt)
Xylan
(brich wood)
starch chitin lichenan pectin
+ - + - + - + - + - + - + - + - + - + -
Clocl_1053 n.d n.d
2506 CBM3b
(N.C)
cipA CBM3a
(P.C)
*n.d= not determined.
Fig10. Interactions of family-3 CBMb (Clocl_1053) with cellulosic substrates. The partition of the Clocl_1053 CBM3b bands
between the bound (+) and unbound (-) is shown. The N.C show none relevant Clocl_1053 protein as expected (upper rectangle),
but respectively showed protein presence in both bound & unbound samples of the 2506 CBM3b protein (lower rectangle). The
P.C show relevant protein at the bound sample but none at the unbound sample, as expected.
37kD
25kD
20kD
15kD

13. 12
Discussion
Based on the concentration value of Clocl_2077 reached in this project, I can assume
that the purification methods which had been chosen are suitable and the expression
and purification stages were executed satisfactory. As for this writing, though, no
crystals were observed. Therefore some explanation must be provided. First, the 'long'
protein of Clocl_2077 is an unknown module and perhaps not enough time for
crystallization may be the cause for getting no crystals. Second, and inevitably, there
is a good chance of different possible options existence for handling the whole
procedure which can be involves in successful crystallization. Therefore this section
will deal with possible improvements or alternatives of the different stages of
expression, purification and crystallization procedures.
The 'long protein' composes of two different modules: one partially trans-membrane
protein (anti-σ) and the other is outer cell protein (E-set). Therefore, when dealing
with membrane protein (MP) (or pseudo-membrane protein) analysis special acts
must be execute to achieve reliability result. E. coli is a popular host for over
expression due to, among others, its well understood genetics and rapid growth [12].
However, as with other expression systems, high-level MP production is typically
toxic to the cell and the yields of biologically active material are generally poor.
Based on the observation that the over expression of MPs in E. coli leads to their
aggregation and to reduce levels of host membrane and secretory proteins [12], it has
been suggested that special E. coli strain which will aid in properly MP expression is
needed. Previously studies shows that when expression of a pure membrane protein
was induced in BL21(DE3) E. coli strain (just as used in this project), most of the
BL21(DE3) host cells died. Similar effects were also observed with expression
vectors for 10 globular proteins (GP). Therefore, protein over-production in this
expression system is either limited or prevented by bacterial cell death. Out of the few
survivors of BL21(DE3) a mutant host C41(DE3) was selected that grew to high
saturation cell density, and produced the protein as inclusion bodies at an elevated
level without toxic effect. Some proteins that were expressed poorly in BL21(DE3),
and others where the toxicity of the expression plasmids prevented transformation
into this host, were also over-produced successfully in C41(DE3). The examples
include GPs as well as MPs, and therefore, strain C41(DE3) is generally superior to
BL21(DE3) as a host for MP over-expression [13]. The final concentration of the

14. 13
protein used for crystallization was relatively poor (in spite of what was written at the
beginning of this section) and this special strain perhaps may grant a better one.
In addition, the pET E. coli expression vector that was used in this project which is a
T7 RNA polymerase promoter driven and IsoPropyl-b-D-ThioGalactopyranoside
(IPTG) inducible are useful tool for the generation of expression constructs.
Alternatively, the pBAD vector system for E. coli expression which uses arabinose
induction has been implemented successfully for the production of MPs for X-ray
studies. Supporting studies to this assumption had found tight regulation, modulation,
and high-level expression of MPs by vectors containing the Arabinose PBAD
Promoter [14, 15].
Another issue to consider concerning the dis-crystallization is the vital isolation of
membrane fraction from MP during protein preparation. Diffraction quality crystals
are particularly difficult to prepare currently when a membrane source is used. The
reason for this is our limited ability to manipulate proteins bearing
hydrophobic/amphiphilic surfaces that are usually enveloped with membrane lipid.
More often than not, the protein gets trapped as an intractable aggregate in its watery
course from membrane to crystal. As a result, access to the structure, and thus
function is limited. Hence, for purification and crystallization, MPs need to be
extracted from the lipid membrane in which they were expressed using a special
detergent. For most expression systems, this extraction is performed on the isolated
membrane fraction but can be extracted from whole cells [16]. Whether solubilizing
from membranes or from whole cells, the goal is similar- to yield a water-soluble
Protein–Detergent–Lipid Complex (PDLC) (Fig11),Which will further lose the lipids
component and yield Protein-Detergent Complex (PDC). The identification and
desirable concentration of the detergent most suitable for a particular protein target is
an empirical process, when the ideal detergent extract all of the membrane protein
target from the membrane, maintains the native fold of the protein and forms a PDC
that is stable throughout purification and crystallization [16].

15. 14
Fig11. Detergent solubilization of membrane proteins- Schematic of the solubilization process. From left to right: free
detergent monomers (a) associate to form detergent micelles (b). When added to a membrane preparation (c), the micelles extract
membrane proteins from the lipid bilayer yielding a solution containing PDLC complexes, free lipid-detergent micelles and
detergent monomers (d).
Although the idea of using the PDC module when a MP crystallization is desired
seems to be logical, there are few adjustments in the general procedure that are
necessary to be execute after deciding going that road. For instance, since there is
evidence that the presence of certain detergents may inhibit the activity of various
proteases (thrombin for example), placing the His-Tag at the opposite terminus of the
protein or adding linker sequence to serve as a spacer between the cleavage site and
the protein can be good actions to overcome the problem. Additional issues require
original solutions can be pop up when using the PDC module.
When examination the carbohydrate binding assay results, I can first infer that the
controls are suitable (see explanations of fig10). I will describe each
binding/unbinding characters to Clocl_1053 of the cellulostic polymers that were
used, based on the assay results, and then try to phrase some conclusions. It is
important to mention that having a reliable statement as for binding interaction
between Clocl_1053 and a carbohydrate we must lay on significant results, this will
express in strong presence of the inspected protein at the bound sample together with
an absence of the protein at the unbound sample. And of course the other way around
for the un-binding interaction statement.
Fig12. Carbohydrates binding assay results analysis. Key: - Tight Specific Binding, - Tight Specific Un-Binding,
- Week Un-Binding, MRN- More replicates are Needed, n.d- not determined.
cellulostic polymer Interaction with Clocl_1053 cellulostic polymer Interaction with Clocl_1053
Cellulose Xylan (brich wood)
Amorphous cellulose MRN starch
Banana stem Chitin
Switch grass n.d Lichenan MRN
Xylan (oat spelt) MRN pectin n.d

16. 15
This assay results can infer that the Clocl_1053 CBM3b protein doesn't bind chitin
and xylan (brich wood), and does bind -as previously studies confirms- cellulose. For
further data about the protein relationship with the others cellulostic polymers
additional repeated (at least 3 times) and more precise experiment must be execute. In
spite of the un-satisfactory dis-accurate results of this assay the main issue of the
specific CBM3b does correlates to the presence knowledge- the protein bind
cellulose. The whole (yet small) date according to this assay is an configuration
knowledge which can be used in further studies in order to assist the main goal
mentioned in this work- to create the best efficient degradation system that can serve
as a green energy solution for the next generation.
Acknowledgments. This project was supported by Prof. Rafael Lamed laboratory at the department of
molecular microbiology and biotechnology of George S. Wise faculty of life science, TAU. I want to
thank all the members of Lamed group especially to Dr. Oren Yaniv, Yehuda Halfon and Dr. Harish
Kumar Reddy Y. A special gratitude goes to Leeron Piechota for all the scientific guidance, endless
patience and professional teaching attitude.
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