a sequence of nh2-aaaagaaacp - cooh was used as the switch - tag ( st ) ( munoz and serrano 1994 ) . we have previously observed that c - terminal cysteines , which have thiol and carboxylic acid groups in close proximity , can be self - proteolytic and thus added a protecting c - terminal proline residue which does not hydrogen bond to the helix and , in in silico modeling , orientates the  sh group of the cysteine towards the target binding surface ( figure 1a ) .
a synthetic st with additional n - terminal residues ( kydd ) to aid solubility and concentration measurements showed a coil to helix transition in increasing concentrations of the hydrophobic solvent tri - fluoroethanol ( tfe ) ( figure 1b ) .
specific binding of the peptide to gold from aqueous solution was assessed by spr ( biacore - x ) with bare gold ( au - chip ) surfaces and a synthetic switch tag peptide incorporating the flag antibody - binding motif ( dykddddkgg ) at its n - terminus ( flag - st ) . to reduce nonspecific adhesion
specific binding was determined by reference to peptides in which the cys  sh group was blocked by iodoacetamide .
this showed that 75% of surface mass is specifically bound via the cys after washing .
after infilling the remaining surface by injection of peg - thiol solution , to inhibit nonspecific binding ( nsb ) ( seigel et al 1997 ) , anti - flag antibody was injected . following a wash with 0.1% sodium dodecyl sulfate ( sds ) solution to remove nonspecifically bound antibody , 145 resonance units ( r.u .
= 0.0001 degree shift in spr minimum position ) remained bound to the iodoacetamide - blocked peptide - treated surface , whereas 1250 r.u .
were bound to the nonblocked peptide surface , clearly demonstrating the thiol - directed assembly of flag - peptides ( figure 2a ) .
the background is reduced still further if a longer ex situ assembly of peg - thiol is performed ( data not shown ) .
the st sequence was genetically engineered onto the c - terminus of the bacterial protein tolaiii , fusions to which are known to be well expressed and readily purified from escherichia coli .
the two naturally occurring cysteine residues in the protein were mutated to serine , leaving the st peptide cysteine as the only thiol group .
the complete plasmid allows for insertion of proteins between the tolaiii and st modules and subsequent proteolytic cleavage to remove the stabilizing tola fusion partner .
this was achieved with a multiple cloning site ( mcs ) to allow the simple insertion of any chosen protein sequence between the tola - iii and st coding regions .
a thrombin protease cut site was also included between the tola - iii and mcs sequences to allow the tola - iii protein to be cleaved from the tagged fusion protein and a flag epitope inserted in tolaiii .
the tolaiii- flag - st fusion protein was expressed in e. coli bl-21 cells and purified by ni affinity chromatography as described ( anderluh et al 2003 ) .
far uv - cd analysis showed that it was soluble and correctly folded and spr revealed that 510 r.u
the surfaces were briefly in - filled in situ with peg - thiol , and probed with anti - flag antibody , as described previously ; 1580 r.u . of antibody remained bound after washing to the normal surface whereas only 265 r.u .
a longer peg - thiol incubation would decrease this still further since errors in the sam are sites for nonspecific binding .
next , green fluorescent protein ( gfp ) was used as a model for an inserted protein domain as it is easily visualized on surfaces .
the coding dna sequence of gfp was cloned into the plasmid and protein expressed in bl-21 e. coli .
microcontact printing ( xia and whitesides 1998 ) was used to pattern arrays of 5-m diameter spots of a tolaiii - gfp - switch - tag fusion protein later in filled with 1 mm peg - thiol solution in 100% ethanol overnight to assemble a peg - terminated sam both in and around the spots .
the protein was easily visualized by fluorescence microscopy and thus remains folded ( figure 3a ) .
rat primary osteoblast cells incubated on the surface adhered to the islands of protein but were unable to bind to the surrounding peg - thiol coated surface , as shown in figure 3b .
the cells are much larger than the pattern and adjust their contact points to avoid the peg in - filled areas .
gfp has no known specific cell adhesion properties but provides clear anchor points within the peg - layer .
this illustrates that the st is able to specifically immobilize recombinant proteins as defined patterns within an otherwise homogeneous sam .
the stamped surface was backfilled with peg - thiol and incubated with primary osteoblast cells which avoided the protein - free region ( figure 3c ) .
the ability of phospholipid layers to limit nonspecific binding has been exploited in combination with patterned arrays of soluble proteins ( kung et al 2000 ) , whilst other approaches have combined the peptide with the lipid itself ( svedhem et al 2003 ) or printed proteins onto a reactive sam ( rozkiewicz et al 2006 ) .
incorporation of functional synthetic peptides into sam is straightforward and patterning has been achieved by micro - contact printing ethylene glycol - terminated sam in patterns leaving exposed gold for subsequent peptide assembly ( zhang et al 1999 ) .
the system described here provides a generic method to co - assemble proteins with classical sam .
we have designed a plasmid which attaches a short peptide sequence which can insert into an 11 carbon chain thick sam ( figure 3d ) .
it was previously shown that hydrophobic helical peptides can be assembled into dense sam and retain a helical structure ( kitagawa et al 2004 ) .
the current system avoided altering the protein s hydrophobicity by allowing the peptide to be random coil in solution and helical in the sam .
although the behavior of the peptide and modified proteins confirmed the first property of water solubility , it was unfortunate that the relatively low immobilization density prevented ftir confirmation of helix formation in the sam ( kitagawa et al 2004 ) .
nevertheless the tolaiii - st expression system is able to produce engineered proteins that show specific adhesion without compromising water solubility or expression levels in e. coli .
we have previously shown that by pre - treating the surface with -mercapto - ethanol surface interactions are reduced and proteins keep their 3d structure ( terrettazet al 2002 ; keegan et al 2005 ) the subsequent assembly with lipids stabilizes the array and further contributes to retaining protein structure ( terrettaz et al 2002 ; holt et al 2005 ; cisneros et al 2006 ) .
this expression system has the potential to increase the range and complexity of proteins that can be specifically immobilized on surfaces designed to modify cellular growth ( shah et al 2007 ) or the development of new detector elements for spr , saw , or qcm .
the st sequence was developed using agadir ( munoz and serrano 1994 ) ( http://www.embl-heidelberg.de/services/serrano/agadir/agadir-start.html ) .
the helix - disrupting glycine residue was added to a poly - alanine sequence at different positions and the resulting helix propensities calculated .
a sequence with a central glycine retained sufficient helical propensity that an increase in environmental hydrophobicity would cause this sequence to rearrange from random coil to -helix .
molecules based on this sequence , with the addition of a c - terminal cysteine and proline residues and n - terminal sequences of either kydd to aid solubility and concentration measurements or dykddddkgg , a flag epitope , were synthesised by f - moc chemistry and purified to > 95% as assessed by high - pressure liquid chromatography and maldi - tof mass spectrometry ( invitrogen , evotrack , paisley , uk ) .
helical structure and thio alkane monolayer were modeled and energy minimized using the quanta v4.0 package ( accelrys , orsay cedex , france ) .
solutions of 0.2 mg / ml peptide in 50 mm nah2po4 , ph 7.0 were made with increasing concentrations of the hydrophobic solvent tfe ( sigma - aldrich , gillingham , uk ) up to 80% tfe , the limit of peptide solubility .
all measurements were made with 0.2 mm path length quartz suprasil cuvettes ( hellma uk , southend on sea , uk ) using a jasco j-810 spectropolarimeter , over a wavelength range of 250195 nm at a scan speed of 20 nm min .
the mean spectrum derived from 10 scans was corrected by subtraction of a background buffer blank .
surface plasmon resonance measurements of sam formation used a biacore x system with  au  gold sensor chips .
the gold surface was cleaned with piranha solution ( kitagawa et al 2004 ) for 30 minutes , rinsed with h2o , 1% sds and finally extensively with h2o before drying with air and immediate docking into the biacore system .
a buffer of 200 mm nacl , 0.5 mm edta , and 20 mm tris ph 8.0 was used for all solutions and also as the running buffer .
peptide and protein solutions at a concentration of 0.1 mg / ml were used in all experiments . the peg - thiol ( 11-mercapto-1-triethyleneglycol - undecane , hsc11-eg3 )
( prochimia surfaces , sopot , poland ) was used at a concentration of 0.5 mm .
the solutions used for thiol binding steps were supplemented with 0.1 mm tcep ( tris(2-carboxyethyl)phosphine hydrochloride ) for 1 h before use .
anti - flag monoclonal antibodies were obtained from sigma - aldrich and used at a dilution concentration of 0.1 mg / ml prepared daily in running buffer .
tolaiii - switch tag fusion protein expression constructs were based on the ptol - t vector ( anderluh et al 2003 ) .
they were expressed in e. coli bl-21 cells and purified as detailed previously ( anderluh et al 2003 ) .
micro - contact printing using pdms stamps was used to transfer patterns of st protein onto gold substrates ( xia and whitesides 1998 ) .
the stamps were inked using the relevant alkanethiol or protein and dried in a stream of compressed air , placed on the gold and left for 15 seconds .
the stamps were removed and the surface air dried for 10 minutes . if required the surface of the gold was then immersed in the relevant infill solution and left for a minimum of 4 hours to allow formation of a sam .
primary osteoblastic cells were isolated from the calvariae of five - day old rats ( bokhari et al 2005 ) they were maintained in a culture medium of dulbecco s modified eagle s medium ( dmem , sigma aldrich ) supplemented with 1% penicillin / streptomycin solution , 1% l - glutamine solution , and 10% fetal calf serum .
solutions of 0.2 mg / ml peptide in 50 mm nah2po4 , ph 7.0 were made with increasing concentrations of the hydrophobic solvent tfe ( sigma - aldrich , gillingham , uk ) up to 80% tfe , the limit of peptide solubility .
all measurements were made with 0.2 mm path length quartz suprasil cuvettes ( hellma uk , southend on sea , uk ) using a jasco j-810 spectropolarimeter , over a wavelength range of 250195 nm at a scan speed of 20 nm min .
the mean spectrum derived from 10 scans was corrected by subtraction of a background buffer blank .
surface plasmon resonance measurements of sam formation used a biacore x system with  au  gold sensor chips .
the gold surface was cleaned with piranha solution ( kitagawa et al 2004 ) for 30 minutes , rinsed with h2o , 1% sds and finally extensively with h2o before drying with air and immediate docking into the biacore system .
a buffer of 200 mm nacl , 0.5 mm edta , and 20 mm tris ph 8.0 was used for all solutions and also as the running buffer .
peptide and protein solutions at a concentration of 0.1 mg / ml were used in all experiments . the peg - thiol ( 11-mercapto-1-triethyleneglycol - undecane , hsc11-eg3 )
( prochimia surfaces , sopot , poland ) was used at a concentration of 0.5 mm .
the solutions used for thiol binding steps were supplemented with 0.1 mm tcep ( tris(2-carboxyethyl)phosphine hydrochloride ) for 1 h before use .
anti - flag monoclonal antibodies were obtained from sigma - aldrich and used at a dilution concentration of 0.1 mg / ml prepared daily in running buffer .
tolaiii - switch tag fusion protein expression constructs were based on the ptol - t vector ( anderluh et al 2003 ) .
they were expressed in e. coli bl-21 cells and purified as detailed previously ( anderluh et al 2003 ) .
micro - contact printing using pdms stamps was used to transfer patterns of st protein onto gold substrates ( xia and whitesides 1998 ) .
the stamps were inked using the relevant alkanethiol or protein and dried in a stream of compressed air , placed on the gold and left for 15 seconds .
if required the surface of the gold was then immersed in the relevant infill solution and left for a minimum of 4 hours to allow formation of a sam .
primary osteoblastic cells were isolated from the calvariae of five - day old rats ( bokhari et al 2005 ) they were maintained in a culture medium of dulbecco s modified eagle s medium ( dmem , sigma aldrich ) supplemented with 1% penicillin / streptomycin solution , 1% l - glutamine solution , and 10% fetal calf serum .
 surface biology aims to observe and control biological processes by combining bio- , surface , and physical chemistry . 
 self - assembled monolayers ( sam ) on gold surfaces have provided excellent methods for nanoscale surface preparation for such studies . however , 
 extension of this work requires the specific immobilization of whole protein domains and the direct incorporation of recombinant proteins into sam is still problematic . in this study 
 a short random coil peptide has been designed to insert into thioalkane layers by formation of a hydrophobic helix . 
 surface plasmon resonance ( spr ) studies show that specific immobilization via the internal cysteine is achieved . 
 addition of the peptide sequence to the terminus of a protein at the genetic level enables the production of a range of recombinant fusion - proteins with good yield . 
 spr shows that the proteins display the same gold - binding behavior as the peptide . 
 it is shown that cell growth control can be achieved by printing the proteins using soft lithography with subsequent infilling with thioalkanes the expression plasmid is constructed so that any stable protein domain can be easily cloned , expressed , purified and immobilized . 
Results and discussion
Materials and methods
Circular dichroism spectroscopy
Surface plasmon resonance measurements
Micro-contact printing
Eukaryotic cell culture