Microfluidics and microarray based approaches to biological analysis 4

CHAPTER 4 INTEIN-MEDIATED BIOTINYLATION OF PROTEINS AND ITS APPLICATION IN A PROTEIN MICROARRAY 4.1 Introduction 4.1.1 Developing Microarrays of Functionally Active Proteins DNA micro

CHAPTER 4 INTEIN-MEDIATED BIOTINYLATION OF PROTEINS AND

ITS APPLICATION IN A PROTEIN MICROARRAY

4.1 Introduction

4.1.1 Developing Microarrays of Functionally Active Proteins

DNA microarray is currently the method of choice for high-throughput analysis of nucleic acids at their transcriptional level However, it has been shown that the mRNA expression level in a cell does not correlate well with the abundance of proteins.1 To gain more insights into protein functions, a number of techniques2 and binding chemistry have been developed to immobilize small molecules,3,4 peptides,5,6,7 and proteins 8,9,10 in a microarray for high-throughput protein studies.11

As mentioned in chapter 3, various strategies have been developed for the site-specific immobilization of kinase substrates onto functionalized slides However, proteins are more difficult to handle than peptides Indeed, they are delicate, denature in ‘harsh environments’ and protein arrays are stable and useful only for a very short period of time Consequently, different approaches have been developed to ensure they retain

their activity Schreiber et al dissolved proteins in a 60 % solution in order to keep

them hydrated;8 whereas Zhu et al attached them to the surface of PDMS micro wells.9

However, in most cases, protein immobilization was achieved via their nucleophilic residues, resulting in random orientations of proteins on the glass surface, whereas an oriented immobilization on the slide surface is desired for optimal protein activity Indeed, in order for arrayed proteins to retain their full biological activity, they need to

be arrayed in a proper orientation to ensure accessibility of their active sites with interacting molecules

Thus far, there has only been one report of site-specific attachment of proteins on glass slides.12 Approximately 6000 yeast proteins were expressed as His-tag fusions, spotted onto Ni-NTA functionalized slides, and >80 % were found to retain their full biological activities, presumably as a result of site-specific immobilization which ensures most proteins on the slide to be oriented correctly However, the binding between Ni-NTA and His-tag proteins is neither very strong, nor very stable and susceptible to interference by many commonly used chemicals,13 making this immobilization method incompatible with many protein screening assays

Besides the site-specific immobilization, another problem when working with protein microarrays is the high throughput expression and purification of proteins So far, all protein arrays involve commercially available proteins or recombinant proteins.9 In the second case, proteins need to be purified before spotting, implying long, and

labor-intensive protocols The in vitro expression of proteins appears as a promising solution and a protein array of in situ synthesized proteins has been reported.14 He at al developed a new protein array production procedure termed PISA (protein in situ

array) PISA is designed to generate protein arrays directly from PCR generated DNA via cell-free protein synthesis and simultaneous in situ immobilization of the generated proteins on a surface However, this array consists of microwells rather than a real array and this strategy has not been applied to the array format yet In addition, post

translation modifications may be lacking for in vitro synthesized proteins, and new strategies are required for the in vivo synthesis, purification and functionalization of

proteins for site-specific immobilization

4.1.2 Site-Specific Protein Biotinylation

On the contrary to the interaction between His tag and Ni-NTA previously used for protein microarrays, the biotin-avidin interaction is one of the strongest known non-covalent interaction.15 It is very stable toward a variety of harsh conditions,16 and has

been widely used in standard biochemical assays for immobilization purposes The in vivo and in vitro biotinylation of proteins have previously been reported,17,18 but with limited success due to low yields and non-specific nature of the biotinylation reaction.19 In some cases biotinylation resulted in the addition of a long peptide to the target protein, which may interfere with proper folding of the protein.20 Furthermore, avidin is known to be toxic to cells, making the expression of avidin-fused proteins a difficult task.21

4.1.3 Intein-Mediated Protein Engineering

Inteins are naturally occurring proteins that are involved in the precise cleavage and formation of peptide bonds in a process known as protein splicing The mechanism of protein splicing (Figure 4.1) was elucidated using a combination of site directed mutagenesis and chemical analysis and allowed the rational engineering of inteins for use in protein chemistry The process of protein splicing involves the excision of an intervening protein sequence (the intein) from a precursor protein with the concomitant fusion of the two flanking protein regions through a native peptide bond Controlled cleavage at single intein splice junctions led to the development of fusion protein purification on chitin columns.22

In so-called “native chemical ligation”23 described in chapter 3, an N-terminal cysteine containing peptide is chemically ligated to a second peptide possessing a thioester group with the resultant formation of a native peptide bond at the ligation junction

Peptide thioesters for use in native chemical ligation are generated solely through chemical synthesis The intein-based purification protocol has found wide applications

in protein engineering where the expressed protein ligation (EPL) strategy is utilized to incorporate non coded amino acid sequences into a protein sequence (Figure 4.2 a).24,25 This strategy has been extended to modifications of proteins at their C-termini with a number of chemical tags.26 Recently, Tolbert et al reported the use of TEV protease to

generate N-terminal cysteines from affinity-tagged fusion proteins (Figure 4.2),27 but unfortunately, the simultaneous protease cleavage and thioester labeling of the affinity-tagged fusion proteins was unsuccessful since the thioester labels are inhibitors of the TEV protease, possibly because the TEV protease is a cysteine protease with an active site cysteine that can be acylated by the thioesters

HN

H2N

O

N

O S

NH2 O

O

Intein

Intein

O O

N

O S

NH2 O O

H2N

S

N

HS O

Figure 4.1 Principle of intein-mediated protein splicing

R

Protein

R

His6 ENLYFQ His6 ENLYFQ Protein

Protein

R

N

OHS

O

+ NH3

O

O-NH 3

O

O-S

O

NH3+ O

HS

+NH3 O

O HS

O

HS

O

+ H 3 N HS

O

N HS

O O

O

O-O

O-O

O-a

b

SR O

Figure 4.2 Generation of N-terminal cysteine proteins using TEV protease for (a)

expressed protein ligation, (b) Generation of N-terminal

4.2 Results and Discussion

4.2.1 Intein-Mediated Biotinylation of Proteins at their C-Terminal 1

As described in chapter 3, the native chemical ligation developed by Kent et al allows

for the site-specific reaction between the N-terminal cysteine and the thioester function

of any other compound EPL represents a novel semisynthetic approach that has greatly expanded the utility of native chemical ligation chemistry and allows for the site-specific incorporation of non-coded amino acids into the protein of interest By expressing a protein of interest as fusion to an intein, which also contains a chitin-binding domain for purification on chitin column, one can both purify and label at its C-terminal by flushing the column with the cysteine containing labeling agent

1 The expression and biotinylation of proteins was performed by Lue Yee Peng Rina, and cysteine biotin was synthesized by Dr Zhu Qing

Therefore, by reacting cysteine biotin with intein fused protein, one can in a single step, purify and site-specifically biotinylate proteins at their C-terminal (Figure 4.3)

S

HS

S

O

O

O

S O

Target protein Intein ta g

N

Chitin column

N

N

a) In vivo

expression

c) Biotinylation

Spontaneous rearrangement

DNA

b) Purification

N

Intein tag

S

HS

S

O

O

O

S O

Target protein Intein ta g

N

Chitin column

N

N

a) In vivo

expression

c) Biotinylation

Spontaneous rearrangement

DNA

b) Purification

N

Intein tag

H 2 N N H

S H

O

H N

N H O

H 2 N H N O

O

S

H N N H O S

H N O

O

S

H N N H O N

H S

H 2 N N H

S H

O

H N

N H O

H 2 N H N O

O

S

H N N H O S

H N O

O

S

H N N H O N

H S

Figure 4.3 Intein-mediated site-specific biotinylation of proteins

Three proteins of interest, namely MBP (Maltose Binding Protein), EGFP (Enhanced Green Fluorescent Protein) and GST (Glutathione S-Transferase) were chosen as

models and expressed in vivo as fusion proteins with an intein tag (intein fused to

chitin binding domain) at their C-termini The proteins were purified and biotinylated,

in a single step (Figure 4.3), by first loading the crude cell lysate onto a column packed with chitin beads, then flushing the column with biotinylated cysteine (Figure 4.4), to obtain the C-terminally biotinylated proteins

Figure 4.4 Cysteine Biotin used for Intein-Mediated Site-Specific Biotinylation

The site-specific biotinylation of the proteins was unambiguously confirmed by SDS-PAGE (Figure 4.5, a) and western blotting (Figure 4.5, b) Based on SDS-SDS-PAGE, the biotinylation reaction took place with 90-95 % efficiency, generating proteins in sufficient purity (> 95 %) Labeling of proteins expressed as intein fusion is very simple since the ligation reaction can be combined with affinity purification, allowing C-terminally modified proteins to be obtained from crude bacterial lysates in a single step

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

(b)

NH2

H N N

H

HS

O

O S

NH HN O

(a)

Figure 4.5 MBP purification and biotinylation (a) SDS-PAGE (1) protein marker,

(2) uninduced cell extract, (3) induced cell extract, (4) flow-through from column loading, (5) flow-through from column wash, (6) proteins bound to chitin column before cleavage, (7) flow-through from quick flush of cleavage agent, (8-9) first two elution fractions after overnight incubation at 4 °C with cysteine biotin (10) remaining proteins bound to chitin column after cleavage (b) Western blotting of biotinylated MBP Biotinylated MBP was run on SDS PAGE and after transfer was detected using

Streptavidin-HRP

4.2.2 Microarrays of Site-Specific Immobilized Active Proteins

After biotinylation, without any further purification, the three biotinylated proteins were spotted directly, without any further treatment, onto an avidin-functionalized slide to obtain the corresponding protein array (Figure 4.6)

Immobilization

STA slide

O

Immobilization

STA slide

O

Immobilization

STA slide

O

Immobilization

STA slide

H N O

O

S

H N N H O N

H S

H N O

O

S

H N N H O N

H S

H N O

O

S

H N N H O N

H S

H N O

O

S

H N N H O N

H S

H N O

O

S

H N N H O N

H S

H N O

O

S

H N N H O N

H S

H N O

O

S

H N N H O N

H S

Figure 4.6 Site specific immobilization of functionally active proteins

A protein array was generated with the biotinylated EGFP, MBP and GST, and probed with Cy3-anti-EGFP, Cy5-anti-MBP and FITC-anti-GST, respectively Three corresponding non-biotinylated proteins were also spotted onto the same slide, as controls, and the array was incubated with either individual antibodies (Figure 4.7), or

a mixture of all three antibodies (Figure 4.8) Only specific binding between the biotinylated proteins and their corresponding antibodies were observed, regardless of the presence of other proteins (Figure 4.7) and antibodies (Figure 4.8), indicating the specific immobilization and versatility of this new protein array Furthermore, no fluorescence signal was observed with the non-biotinylated control proteins (data not shown), confirming the essence of biotinylation for protein immobilization

(a) (b) (c)

Figure 4.7 Site-specific immobilization of proteins via avidin-biotin interaction

Three biotinylated proteins : (a) EGFP, (b) MBP and (c) GST were arrayed onto avidin functionalized slides and individually detected with Cy3-anti-EGFP (green), Cy5-anti-MBP (red) and FITC-anti-GST (blue), respectively

(a) (b) (c)

Figure 4.8 Site-specific immobilization of proteins via avidin-biotin interaction

Three biotinylated proteins : (a) EGFP, (b) MBP and (c) GST were arrayed onto avidin functionalized slides and detected with a mixture of Cy3-anti-EGFP (green), Cy5-anti-MBP (red) and FITC-anti-GST (blue)

4.2.3 Arrays of Functionally Active Proteins

The most critical issue in generating a protein array is to ensure that proteins maintain their native activity, as it is previously known that proteins tend to denature on glass surfaces In order to confirm that biotinylated proteins immobilized on the avidin slide retain their proper folding, the native fluorescence of EGFP on the slide was monitored

(Figure 4.9) No loss of fluorescence intensity was observed after prolonged incubation

at 4 0C, suggesting that folding of the protein was properly maintained on the slide

Figure 4.9 Fluorescence from the native EGFP

In a separate experiment, a slide immobilized with EGFP, MBP and GST was incubated with Cy3-labeled glutathione (Figure 4.10), a known natural ligand of GST The result showed exclusive binding between GST and glutathione (Figure 4.11), further indicating full retention of the native GST activity

N H

H N

OH

O

H2N

OH

O

SH

O

O

Figure 4.10 Structure of glutathione

Figure 4.11 Functional activity of arrayed biotinylated GST Biotinylated GST was

arrayed onto avidin functionalized slides and incubated with its specific natural ligand, glutathione, labeled with Cy3

Furthermore, all data gathered thus far indicates that the presence of avidin as a molecular layer between the immobilized proteins and the glass surface also serves to minimize nonspecific absorption of proteins.Future improvement may be readily made

by using streptavidin as the immobilization agent on the slide in place of avidin, which

is a glycoprotein and known to have higher nonspecific binding characteristics.28

4.2.4 Comparison of Avidin-Biotin Interaction Stability with Other Existing Site-Specific Immobilization Strategies

Thus far, the only reported method for site-specific attachment of proteins in a microarray has been the immobilization of His-tag proteins on slides functionalized with Ni-NTA.12 However, the binding between His-tag proteins and Ni-NTA complex

is not very strong, and incompatible with many commonly used chemicals such as

DTT, SDS, EDTA, etc The binding is also depleted outside the 4 to 10 pH range, or

when the buffer contains high concentrations of common salts

On the contrary, as mentioned in chapter 3, the binding between biotin and avidin is one of the strongest known non covalent interaction Avidin is also extremely stable,16