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A generic expression system to produce proteins that co-assemble with alkane thiol SAM.

Chaffey BT, Mitchell E, Birch MA, Lakey JH - Int J Nanomedicine (2008)

Bottom Line: 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 thio-alkanes The expression plasmid is constructed so that any stable protein domain can be easily cloned, expressed, purified and immobilized.

View Article: PubMed Central - PubMed

Affiliation: The Institute for Cell and Molecular Biosciences, The Medical School, Framlington Place, The University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, Great Britain.

ABSTRACT
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 thio-alkanes The expression plasmid is constructed so that any stable protein domain can be easily cloned, expressed, purified and immobilized.

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(A) Image of the predicted structure of the switch tag peptide (ST) in its helical conformation. (B) Far UV CD spectra of ST. A 0.2 mg/ml solution of peptide in 50 mM NaH2PO4 buffer, pH 7.0 was used throughout. Increasing the quantity of the hydrophobic solvent TFE promoted the transition of peptide secondary structure from random coil to α-helix.Abbreviation: TFE, tri-fluoro ethanol.
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f1-ijn-3-287: (A) Image of the predicted structure of the switch tag peptide (ST) in its helical conformation. (B) Far UV CD spectra of ST. A 0.2 mg/ml solution of peptide in 50 mM NaH2PO4 buffer, pH 7.0 was used throughout. Increasing the quantity of the hydrophobic solvent TFE promoted the transition of peptide secondary structure from random coil to α-helix.Abbreviation: TFE, tri-fluoro ethanol.

Mentions: 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 the gold was pretreated with 2-mercaptoethanol (Keegan et al 2005). 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. (Anderluh et al 2003). 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 Ni2+ 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. of fusion protein remained bound after washing versus only 25 R.U. of the iodoacetamide-blocked species. 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. remained on the blocked protein surface (Figure 2b). 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-μm 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 GFP version was also printed using a stamp which contains holes. The stamped surface was backfilled with PEG-thiol and incubated with primary osteoblast cells which avoided the protein-free region (Figure 3c).


A generic expression system to produce proteins that co-assemble with alkane thiol SAM.

Chaffey BT, Mitchell E, Birch MA, Lakey JH - Int J Nanomedicine (2008)

(A) Image of the predicted structure of the switch tag peptide (ST) in its helical conformation. (B) Far UV CD spectra of ST. A 0.2 mg/ml solution of peptide in 50 mM NaH2PO4 buffer, pH 7.0 was used throughout. Increasing the quantity of the hydrophobic solvent TFE promoted the transition of peptide secondary structure from random coil to α-helix.Abbreviation: TFE, tri-fluoro ethanol.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2626940&req=5

f1-ijn-3-287: (A) Image of the predicted structure of the switch tag peptide (ST) in its helical conformation. (B) Far UV CD spectra of ST. A 0.2 mg/ml solution of peptide in 50 mM NaH2PO4 buffer, pH 7.0 was used throughout. Increasing the quantity of the hydrophobic solvent TFE promoted the transition of peptide secondary structure from random coil to α-helix.Abbreviation: TFE, tri-fluoro ethanol.
Mentions: 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 the gold was pretreated with 2-mercaptoethanol (Keegan et al 2005). 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. (Anderluh et al 2003). 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 Ni2+ 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. of fusion protein remained bound after washing versus only 25 R.U. of the iodoacetamide-blocked species. 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. remained on the blocked protein surface (Figure 2b). 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-μm 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 GFP version was also printed using a stamp which contains holes. The stamped surface was backfilled with PEG-thiol and incubated with primary osteoblast cells which avoided the protein-free region (Figure 3c).

Bottom Line: 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 thio-alkanes The expression plasmid is constructed so that any stable protein domain can be easily cloned, expressed, purified and immobilized.

View Article: PubMed Central - PubMed

Affiliation: The Institute for Cell and Molecular Biosciences, The Medical School, Framlington Place, The University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, Great Britain.

ABSTRACT
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 thio-alkanes The expression plasmid is constructed so that any stable protein domain can be easily cloned, expressed, purified and immobilized.

Show MeSH