Intein-Mediated Biotinylation of Proteins and its Application in Protein Microarray Lue Yee Peng Rina (B.Sc (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCES DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2004 Acknowledgements First, I would like to thank the National University of Singapore (NUS) and the Agency for Science, Technology and Research (A*STAR) of Singapore for the funding support I also thank the Department of Biological Sciences (NUS) for granting me the Research Scholarship that financially supported me through my post-graduate days I would also like to thank Joan and Reena for their advice on most of the administrative matters I really appreciate A/P Yao Shao Qin for his guidance and moral support As the supervisor for both my honors and master’s projects, he has always provided me with insightful discussion His continuing vision is the main key to the success of this project Special thanks to Grace for organizing the enjoyable lab outings Besides that, she has also given me lots of technical advice and assistance on the project Thanks also to Dr Zhu Qing for synthesizing the cysteine-biotin probe Last but not the least, I would like to thank all the people working in the Functional Genomic Laboratory (FGL) and my fellow lab mates in the Department of Chemistry for their valuable friendship i Table of Contents Acknowledgements i Table of Contents ii Summary iv List of Tables vi List of Figures vii Abbreviations ix Introduction Materials & Methods 11 2.1 Chemical synthesis of the cysteine-biotin 11 2.1.1 Using Boc-protected cysteine 11 2.1.2 Using Fmoc-protected cysteine 11 2.1.3 Purification and identification of cysteine-biotin 12 2.2 Cloning of target genes into pTYB1 & pTWIN expression vector 12 2.3 Site-directed mutagenesis of pTYB1-wtEGFP (Lys239)-intein 14 2.4 Expression of intein-fused proteins 14 2.5 Affinity purification & C-terminal biotinylation of recombinant proteins 15 2.6 SPR analysis 16 2.7 In vivo protein biotinylation in E coli 16 2.8 In vivo protein biotinylation of in mammalian cells 18 2.9 Generation of protein microarray 19 2.10 Cell free synthesis and biotinylation of MBP 20 Results & Discussion 21 ii 3.1 General features of pTYB expression vectors 21 3.2 Intein-Mediated Biotinylation of three model proteins 24 3.2.1 Cloning of target genes into pTYB1 expression vector 24 3.2.2 Expression and extraction of fusion proteins 25 3.2.3 Affinity purification and on-column biotinylation 26 3.3 Protein microarray application 29 3.4 Immobilization of biotinylated proteins onto self-assembled monolayers (SAM) in SPR analysis 34 3.5 Influence of C-terminal residues on biotinylation 37 3.6 High-throughput expression and biotinylation of yeast proteins 43 3.6.1 Cloning of yeast gene into pTYB1 expression vector 43 3.6.2 Expression, purification & biotinylation of yeast proteins 45 3.7 In vivo biotinylation of proteins 48 3.7.1 In bacterial cells 48 3.7.2 In mammalian cells 51 3.7.2.1 Construction of mammalian expression plasmid, pT-Rex-DEST30-EGFP-Sce VMA intein-CBD 51 3.7.2.2 Expression and in vivo biotinylation of EGFP in HEK 293 cells 52 3.7.3 Protein microarray generation using crude bacterial cell lysate 57 3.8 Protein biotinlyation using different inteins 59 3.9 Protein biotinylation in a cell-free system 63 Conclusion 66 References 68 iii Summary The post-genome era has led us to a new frontier of proteomics that requires us to gain information on the millions of proteins encoded by these identified genes The challenge ahead therefore lies in the development of protein microarray that would enable us to unravel the biological function of proteins in a massively parallel fashion This high-throughput screening technique would allow thousands of functional molecules to be analyzed simultaneously, possibly leading to a better understanding of how these molecules affect cellular functions It can be used for discovery of novel protein functions, screening of protein-protein interactions, detecting enzyme-substrate interactions and identifying protein targets of biologically active small molecules Beside basic protein expression studies, application of the protein microarray technology has also evolved to diagnostics, mutation analysis, and toxicology in recent years The idea of a protein microchip is to immobilize tens of thousands of protein molecules (e.g antibodies, receptors, enzymes) onto a solid surface such as glass slides Each of these proteins is geared towards identifying and binding of specific targets, thus it is necessary to immobilize them in its native conformation and correct orientation to preserves their functional sites There are several reported strategies of immobilizing proteins onto solid surfaces but many of these mode of attachments are unspecific, causing the molecules to be immobilized in the ‘wrong’ orientation In this report, we present an intein-mediated approach for efficient and site-specific immobilization of proteins The reactive Cterminal thioester generated from intein-assisted protein splicing, either in vitro or in live cells, served as an attractive, as well as exclusive site for attaching cysteine-containing biotin Using this novel biotinylation strategy, we were able to biotinylate many proteins from different biological sources in a potentially high-throughput fashion These proteins iv were subsequently immobilized onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy We highlighted the numerous advantages of using biotin over other tags (e.g GST, His tag etc) as the method of choice in protein purification/immobilization In addition, our intein-mediated strategies also provided critical advantages over other protein biotinylation strategies in a number of different ways We successfully demonstrated that, for the first time, intein-mediated protein biotinylation proceeded inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system Taken together, our results indicate the versatility of these inteinmediated strategies, which should provide invaluable tools for potential high-throughput proteomics applications They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo v List of Tables Table Page The influence of C-terminal residues on the in vivo cleavage of EGFP-intein and on-column cleavage/biotinylation of EGFP 39 vi List of Figures Figure Page Mechanism of protein splicing Biotin-tagging of protein via IPL reaction Chemical structure of cysteine-biotin derivative Three intein-mediated protein biotinylation strategies 10 Map and multiple cloning sites (MCS) of pTYB1 & pTYB2 expression vector 22 Cloning of gene fragment into pTYB1 & pTYB2 23 Affinity purification of MBP 28 On-column biotinylation of MBP 28 Site-specific immobilization of biotinylated, functionally active proteins onto avidin slides 29 10 Integrity of biotinylated proteins immobilized on avidin-functionalized glass surface 30 11 Chemical structure of glutathione, natural ligand of GST 30 12 Biotinylated GST on an avidin slide treated with different washing conditions 32 13 Overview of the on-column biotinylation strategy and site-specific immobilization procedure 33 14 SPR data showing immobilization of biotinylated MBP on avidin-functionalized sensor chip 35 15 SPR response of anti-MBP through the MBP-coated sensor chip 36 16 Influence of the C-terminal amino acid residue 40 17 Effect of an extra glycine residue on intein-mediated biotinylation 41 vii 18 DNA fragments obtained from PCR amplification of the yeast cDNA 44 19 DNA fragments obtained from NdeI and SapI digestion of the TA plasmid 44 20 Cloning of yeast gene fragment into pTYB1 45 21 High-throughput expression and biotinylation of yeast proteins 47 22 Purification and biotinylation of a yeast protein (YAL012W) 47 23 Optimizing in vivo biotinylation conditions in bacterial cells 50 24 In vivo biotinylation efficiency in bacterial cells 50 25 In vivo biotinylation of proteins in bacterial cells shown by anti-biotin blot 51 26 Construction of mammalian expression plasmid using GatewayTM Technology 54 27 Expression of EGFP-Sce VMA intein-CBD in different mammalian cell line 55 28 In vivo biotinylation of EGFP in mammalian cells shown by anti-biotin blot 55 29 In vivo biotinylation efficiency in mammalian cells 56 30 Site-specific immobilization of biotinylated proteins onto avidin slides using bacterial cell lysate 57 31 Schematic representation of pTWIN vectors 60 32 Recovery of the intein fusion proteins from the cell extract and its cleavage efficiency with MESNA and cysteine-biotin 61 33 Yield of EGFP from the different intein fusion 62 34 In vivo biotinylation of EGFP with different intein fusion 62 35 Protein biotinylation in a cell-free system 65 viii Abbreviations AA Amino acid Ala Alanine Ampr Ampicillin resistant Arg Arginine Asn Asparagine Asp Aspartic acid Boc t-Butoxycarbonyl CBD Chitin binding domain Cys Cysteine DCM Dichloromethane DMEM Dulbecco’s modified Eagle’s medium DMF Dimethylformamide DTT 1,4-dithiothreitol E.coli Escherichia coli ECL Enhanced ChemiLuminescent EDTA Ethylenediaminetetraacetic acid EGFP Enhanced green fluorescent protein FITC Fluorescein Isothiocyanate Fomc 9-Fluorenylmethoxycarbonyl GFP Green fluorescent protein Gln Glutamine Glu Glutamic acid Gly Glycine ix expressing EGFP-Sce intein-CBD, EGFP-Mxe intein-CBD and EGFP-Mth-intein-CBD before further incubation at °C for 24 hrs Cells were harvested, lysed and subjected to SDS-PAGE Anti-biotin was used to confirm the presence of biotinylated EGFP in the cell lysate (Figure 34) Improved in vivo biotinylation of EGFP was observed with EGFP-Mxe intein-CBD fusion EGFP-Mth-intein-CBD, on the other hand, does not produce any significant amount of the biotin-tagged EGFP, suggesting that in vivo biotinylation is greatly reduced by the pre-matured cleavage activity of the fusion protein Herein, we showed that the yield of biotin-tagged EGFP is highly affected by the choice of intein in both in vitro and in vivo system These experimental results further emphasize the importance of selecting the best intein for our biotinylation strategy EGFP-Sce intein kDa 82 55 Fusion protein Sce intein-CBD B A EGFP-Mxe intein EGFP-Mth intein kDa 55 Fusion protein 28 Mxe intein-CBD 15 Mth intein-CBD B A B A Figure 32 Recovery of the intein fusion proteins from the cell extract and its cleavage efficiency with MESNA and cysteine-biotin Fusion proteins, EGFP-Sce intein-CBD, EGFP-Mxe intein-CBD & EGFP-Mth intein-CBD, were expressed, extracted and incubated with chitin beads After washing, bound proteins were incubated with MESNA and cysteine-biotin B: Proteins bound on chitin beads before cysteine-biotin/MESNA elution, A: proteins remaining on chitin beads after cysteine-biotin/MESNA elution Coomassie blue staining of the SDS gels are presented 61 Coomassie Stain MESNA + cysteine-biotin EGFP Anti-biotin blot MESNA + cysteine-biotin EGFP Figure 33 Yield of EGFP from the different intein fusion EGFP eluted from the chitin column were analyzed by SDS-PAGE and immunoblot Lane 1: EGFP-Sce intein-CBD fusion; lane 2: EGFP-Mxe intein-CBD fusion; lane 3: EGFP-Mth intein-CBD kDa Coomassie Stain 27 EGFP Anti-biotin blot 27 EGFP Figure 34 In vivo biotinylation of EGFP with different intein fusion Anti-biotin blot was used to confirm the presence of biotin-tagged EGFP within the cell lysate Lane 1: EGFPSce intein-CBD fusion; lane 2: EGFP-Mxe intein-CBD fusion; lane 3: EGFP-Mth inteinCBD 62 3.9.Protein biotinylation in a cell-free system We have thus far successfully demonstrated the utilities of intein-mediated biotinylation strategies in both in vitro and in vivo settings In both cases, intein-fused proteins need to be successfully expressed in soluble forms in the host cell before biotinylation (either in vitro or in vivo) could take place However, numerous problems may arise during protein expression in a host cell The formation of inclusion bodies is one This is especially true when one attempts to express eukaryotic proteins in prokaryotic hosts Other problems include potential proteolytic degradation of the protein by endogenous proteases, as well as expression of proteins toxic to the host cell Cell-free protein synthesis provides an attractive alternative for protein expression which may potentially overcome many of these problems (method C, Figure 4), and is well-suited for protein microarray applications because (1) minute quantities of proteins generated in cell-free system are sufficient for spotting in a protein array, and (2) the method could be easily adopted in 96and 384-well formats with a conventional PCR machine for potential high-throughput protein synthesis.89-91 To assess whether our intein-mediated strategy is suitable for biotinylation of proteins synthesized in a cell-free system, pMYB5, the plasmid expressing MBP-intein-CBD fusion under the transcription control of T7 promoter, was used as the DNA template in a Rapid Translation System (RTS) 100 E coli HY kit Optimal temperature for most protein synthesis is at 30 °C, however, lower temperatures may be used for synthesis of proteins that tend to aggregate at that temperature Protein synthesis can proceed for up to h but the synthesis reaction is usually 90% complete after h After cell-free protein 63 synthesis, the reaction was incubated with cysteine-biotin/MESNA, followed by analysis with SDS-PAGE and western blots (Figure 35A) The presence of a 42 kDa band on the anti-biotin immunoblot, and not any other band (Figure 35A, lane 2), indicated successful and exclusive biotinylation of the MBP protein synthesized in the cell-free system It should be noted that, among three protein biotinylation strategies presented herein (e.g Figure 4), the cell-free method seems to be the simplest of all In our hands, however, it is also the least reliable: the efficiency of protein expression as well as the subsequent protein biotinylation depends greatly on a number of different factors, including the nature of the protein itself, the amount and quality of the DNA template used (Figure 35 B) and the kind of cell lysates used for protein expression, etc For optimized performance of this biotin-tagging strategy in cell-free system, more experiments have to be done to further assess some of these issues 64 A kDa 42 MBP B kDa 42 MBP Figure 35 Protein biotinylation in a cell-free system MBP fusion encoded by pTYB1MBP-intein was first synthesized using the RTS cell-free system, followed by incubation with MESNA and cysteine-biotin Proteins were precipitated and analyzed by a 12% SDS-PAGE gel (A) Lane 1: coomassie stained gel; lane 2: western blots of lane with anti-biotin antibody (B) Different volume of DNA templates were used in the 25 µl RTS reaction mix Lane 1: No DNA template added; lane - 5: µl, µl, µl & µl of DNA template added, respectively 65 Conclusions Our intein-mediated biotinylation strategies have several advantages over other traditional methods in which biotin ligase is used First, the precise splicing mechanism of intein allows coupling of biotin moiety to the C-terminus of proteins without introduction of additional amino acids sequences that otherwise may compromise the native protein activity Second, most commonly used biochemical reagents not inhibit the inteinmediated ligation reaction, thus enabling purification/biotinylation of the desired protein to be done efficiently in a single step Third, cell toxicity due to over-expression of fusion proteins (unless the target protein is toxic to the host strain itself) is unlikely, since there is no competition of endogenous biotin consumption Finally, since protein biotinylation is solely dependent on the cleavage of the fusion protein from the intein tag, use of expensive enzyme is not required and co-expression of biotin ligase is not necessary for in vivo biotinylation of proteins Our findings herein indicate that the intein-mediated biotinylation approaches are sufficiently general and versatile, enabling proteins from different biological sources to be site-specifically biotinylated under different conditions, and subsequently used in a wide range of avidin/biotin technologies Expressed proteins fused to an intein tag could be efficiently purified and biotinlyated, in vitro, in a single purification step We also showed that the strategy proceed in both live bacterial and mammalian cells We further showed intein-fused proteins synthesized from a cell-free system could undergo inteinmediated biotinylation reaction as well We emphasized the unique utilities of our strategies in the area of protein microarray, as this technology may be one of the most 66 powerful tools for high-throughput analysis of protein functions Several essential aspects of a protein microarray were addressed using our intein-mediated biotinylation strategies Firstly, protein biotinylation/immobilization was site-specific, leading to uniform orientation, and more importantly retention of the functional integrity of proteins immobilized on the array Secondly, no extra macromolecular tag was introduced in the immobilized protein, further ensuring the biological activity of proteins was minimally perturbed Thirdly, avidin/biotin interaction was extremely stable, enabling immobilized proteins to be thoroughly washed to remove cellular contaminants, and subsequently screened under even the most stringent conditions Finally, the strategy upon modifications, does not involve tedious protein purification/elution steps and allows the facile generation of biotinylated proteins, making it possible for proteins in crude lysates to be spotted directly onto a protein array (e.g in live cells or in a cell-free system) This enables expression, without further processing (e.g 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10953-10964 94 Yeo, S.Y.D., Srinivasan, R., Uttamchandani, M., Chen, G.Y.J., Zhu, Q., Yao, S.Q (2003) Cell-permeable small molecule probes for site-specific labeling of proteins Chem Commun., 2870-2871 75 [...]... thioester protein and the cysteine-containing biotin tag resulting in a native peptide bond formation In our work with 3 model proteins, namely MBP (Maltose Binding Protein) , EGFP (Enhanced Green Fluorescent Protein) and GST, we demonstrated that site-specific biotinylation of proteins could be efficiently carried out by applying a cysteine-containing biotin tag (Figure 3) to the intein- fused protein purified... (nucleotide sequences in pink) This results in an extra glycine residue added to the C-terminus of the target protein indicates the intein cleavage site 23 3.2 Intein- Mediated Biotinylation of three model proteins 3.2.1 Cloning of target genes into pTYB1 expression vector In the proof -of- concept experiment, the gene fragments of three model proteins, namely MBP, EGFP and GST were cloned into pTYB1 to generate... Three intein- mediated protein biotinylation strategies: (A) in vivo biotinylation in live cells; (B) in vitro biotinylation of column-bound proteins; & (C) cell-free biotinylation of proteins 10 2 Materials and Methods 2.1 Chemical synthesis of the cysteine-biotin Cysteine-biotin derivatives (Figure 3) can be synthesized with either with (1) Bocprotected, or (2) Fmoc-protected cysteine: 2.1.1 Using Boc-protected... determined by BioEvaluation software installed on the BIAcore X 2.7 In vivo protein biotinylation in E coli For in vivo biotinylation of proteins in E coli., pMYB5 and pTYB-1 constructs containing two yeast proteins (YAL012W & YGR152C) were used Liquid cultures of 16 ER2566 carrying the intein- fusion construct were grown to OD600 of ~0.6 in LB medium supplemented with 100 µg/ml of ampicillin Expression of. .. 3 Chemical structure of cysteine-biotin derivative 9 H2N SH Gene Gene Expression Protein A Protein Intein CBD Biotinylation in vivo Intein CBD Lysis Lysis H2N Column Purification & Biotinylation in vitro Chitin column HS B S Cell-free protein synthesis & Biotinylation N C Biotinylated protein H2N HS Gene C O O H2N HS H2N = HS N H Cysteine-biotin N H S N N O Applications Protein microarray, SPR analysis,... chemistry in protein engineering Intein- mediated protein ligation (IPL) is an extremely useful method for protein synthesis with a variety of peripheral applications.63-67 It has been used to incorporate noncoded amino acids into a protein sequences64, purify cytotoxic proteins6 5, study protein structure/function relationship by segmental isotopic labeling of proteins for NMR analysis66, and introduce... proteins were subsequently used to generate corresponding protein array in a single step without further downstream processing (Method A in Figure 4) Beside cell expressed recombinant proteins, intein- mediated biotinylation strategy may also be extended to biotinylate proteins synthesized in a cell-free synthesis system (Method C in Figure 4).31 Figure 4 summarizes the 3 intein- mediated protein biotinylation. .. techniques for protein chemistry and engineering For example, through identification of the residues directly participating in the breakage and peptide bond formation, Chong et al were able to engineer inteins with controllable cleavage at single splice junctions.59 By fusing the chitin binding domain (CBD, 5kD) of Bacillus circulans60 to one terminus of the intein, they developed an intein- mediated affinity... analogous to RNA splicing.41,42 These protein “introns”, known as intein, are found within genes of other proteins and translated as a single polypeptide chain After translation, the intein initiates an autocatalytic event to excise itself and join the flanking host segments with a new peptide bond to form the final protein product (Figure 1).43 To date, over 100 inteins have been discovered in unicellular... using biotin-containing chemicals This leads to random biotinylation of proteins and in many cases, the subsequent inactivation of some protein biological activities.33 Alternative techniques have been developed which allows for site-specific labeling of proteins with biotin 34,35 A stretch of amino acids sequences has been identified by Cronan for site-specific tagging of biotin to proteins. 35 The covalent ... Three intein-mediated protein biotinylation strategies: (A) in vivo biotinylation in live cells; (B) in vitro biotinylation of column-bound proteins; & (C) cell-free biotinylation of proteins 10... (Lys239)-intein 14 2.4 Expression of intein-fused proteins 14 2.5 Affinity purification & C-terminal biotinylation of recombinant proteins 15 2.6 SPR analysis 16 2.7 In vivo protein biotinylation in E... BioEvaluation software installed on the BIAcore X 2.7 In vivo protein biotinylation in E coli For in vivo biotinylation of proteins in E coli., pMYB5 and pTYB-1 constructs containing two yeast proteins