Cullen Lecture 5: High-Throughput Protein Protein Interactions Test Capen 257 Wed 15th 8:30-11AM

On your own, please familiarize yourself with chromatin IP analysis

Two-Hybrid Analysis

Two-Hybrid Analysis

What is Phage Display?

Peptide libraries may be constructed by grafting, in vitro, random DNA sequences into a carrier gene and then introducing the degenerate hybrid coding sequence into an expression organism. Phage display is the first expression organism for peptide library expression to be described and which still maintains predominance in this area because of its simplicity, minimal cost, ease of manipulation, power and robustness. Using phage as the host, a repertoire of random peptides can be expressed that may be searched by a variety of screening or selection procedures. By physically associating each element of the peptide library with its coding sequence, selection for a property of a specific peptide results in the enrichment of the corresponding gene thus facilitating its cloning and amplification.

What is phage display?

Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose amino-terminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.

What is Phage Display?

2-20 µg of GST-SH3 fusion protein bound to glutathione-Sepharose 4B gel (Amersham Pharmacia Biotech) were incubated with 1010 infectious particles from a nonapeptide library. After washing 10 times with PBS, 0.5% Tween 20, the bound phage was eluted with 100 mM glycine HCl, pH 2.2. After three selection cycles, the binding of isolated clones was confirmed by ELISA. Microtiter wells were coated with 109 particles of a clonal phage stock and incubated with 0.2 µg of GST-SH3 fusion protein. The wells were then washed 10 times with PBS, 0.1 Tween 20, and bound protein was detected with anti-GST goat primary antibody (Amersham Pharmacia Biotech) and a secondary anti-goat monoclonal alkaline phosphatase-conjugated antibody (Sigma). Clones with strong SH3 binding activity were selected for further analysis. The sequence of the peptides displayed by positive clones were determined by manual and automatic (ABI PRISM 310 Perkin-Elmer) sequencing of phage single-stranded DNA using universal M13-40 primer.

What is an ELISA?

What is ELISA?

ELISA is an abbreviation for "enzyme-linked immunosorbent assay."

What is an ELISA test?

An ELISA test uses components of the immune system and chemicals to detect immune responses in the body (for example, to infectious microbes). The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules).

What is the use of an ELISA test?

ELISA tests are widely utilized to detect substances that have antigenic properties, primarily proteins (as opposed to small molecules and ions such as glucose and potassium). The substances detected by ELISA tests include hormones, bacterial antigens and antibodies.

How does an ELISA test work?

There are variations of the ELISA test, but the most basic type consists of an antibody attached to a solid surface. This antibody has affinity for (will latch on to) the substance of interest, for example, human chorionic gonadotropin (HCG), the commonly measured protein which indicates pregnancy. A mixture of purified HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only HCG with linked enzyme will bind. The more HCG which is present in the test sample, the less enzyme linked HCG will bind. The substance the enzyme acts on is then added, and the amount of product measured in some way, such as a change in color of the solution.

What are the advantages of ELISA?

ELISA tests are generally relatively accurate tests. They are considered highly sensitive and specific and compare favorably with other methods used to detect substances in the body, such as radioimmune assay (RIA) tests. They have the added advantages of not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus).

What is an ELISA?

What is ELISA?

ELISA is an abbreviation for "enzyme-linked immunosorbent assay."

What is an ELISA test?

An ELISA test uses components of the immune system and chemicals to detect immune responses in the body (for example, to infectious microbes). The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules).

What is the use of an ELISA test?

ELISA tests are widely utilized to detect substances that have antigenic properties, primarily proteins (as opposed to small molecules and ions such as glucose and potassium). The substances detected by ELISA tests include hormones, bacterial antigens and antibodies.

What is an ELISA?

How does an ELISA test work?

There are variations of the ELISA test, but the most basic type consists of an antibody attached to a solid surface. This antibody has affinity for (will latch on to) the substance of interest, for example, human chorionic gonadotropin (HCG), the commonly measured protein which indicates pregnancy. A mixture of purified HCG linked (coupled) to an enzyme and the test sample (blood, urine, etc) are added to the test system. If no HCG is present in the test sample, then only HCG with linked enzyme will bind. The more HCG which is present in the test sample, the less enzyme linked HCG will bind. The substance the enzyme acts on is then added, and the amount of product measured in some way, such as a change in color of the solution.

What are the advantages of ELISA?

ELISA tests are generally relatively accurate tests. They are considered highly sensitive and specific and compare favorably with other methods used to detect substances in the body, such as radioimmune assay (RIA) tests. They have the added advantages of not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus).

Supplemental Data

S4. Profile Matrix Used to Score the Yeast Peptides Containing Ligand Consensus Sequences.

The position-specific scoring matrix (PSSM) used to search for potential target peptides is based on 20 rows by 9 columns matrices. For a specific SH3 domain, each element of the profile matrix contains a position-specific score that is derived from the frequency of each of the 20 amino acid (rows) at each of the nine positions (columns) in the list of the aligned ligand peptides. When scanning the yeast proteome the PSSM calculates a total score representing the likelihood that the query peptide binds to the SH3 domain under consideration. This is obtained by summing, over the nine peptide positions, the elements of the PSSM corresponding to the specific amino acid found at that position in the query peptide. We tried several scoring matrices whose elements were calculated in different ways. In the simplest approach the matrix contains the frequency of occurrence of a specific amino acid at any given position of the peptide ligand. However, the number of ligands selected for each SH3 domain (10-20) is not sufficient to provide statistically significant frequencies in the positions that are not conserved. We improved the method by incorporating, in the PSSM, complementary information extracted from the peptides that do not bind a specific SH3 domain. This list of "non-binder" peptides was obtained from ELISA assays in which 40 different peptides (see S3), two representative clones from the list that were successful in the selection experiment, were tested against each of the 25 soluble SH3 domains. As expected, a PSSM obtained simply by subtracting the "negative" from the "positive" frequencies did not perform well because the list of non-binders was obtained by screening a biased peptide collection (most of the peptides tested contain PxxP). Instead, positive and negative frequencies were combined, according to an empirical function designed to lessen the contribution of the non-binder peptides in the positions that were conserved in the binding ligands. We examined several functions whose prediction results only differed slightly, thereby moving the score of some potential target proteins above or below the empirically set threshold of 20% of the maximum score. The interaction network displayed in Fig. 2A was obtained with the function Sij= pij-nij(1-pij)2 where Sij is the score at the position ij of the matrix and pij and nij are the frequencies of occurrence of amino acid i at position j in the peptides that do and do not bind the SH3 domain respectively. This method scores proline-rich peptides relatively high, even if they do not contain some of the ligand consensus residues. To overcome this problem, we included a step in which all the peptides that do not contain a close match to the consensus sequence were filtered out. Because the paralogs Myo3 and Myo5 selected peptides with the same consensus residues, the peptide data were pooled and the SH3 domains were analyzed together.

S2. Phage Display Analysis

DNA fragments corresponding to each of the 28 yeast SH3 domains were PCR amplified from yeast genomic DNA and ligated into one of the pGEX expression vectors (Pharmacia) such that they were fused to the glutathione S-transferase gene, creating pGex-Abp1(535-591), which codes for an Abp1 SH3 domain spanning residues 535 to 591 within the Abp1 protein; pGex Bbc1(8-67); pGex-Bem1-1(75-130);

…

and pGex-Ysc84(411-468). After expression and affinity purification, the 25 SH3 domains that could be prepared in a soluble form (all but Bem1-2, Cdc25, Sla1-1, and Sla1-2) were used to screen a random nonapeptide library displayed at high density by fusion to the bacteriophage lambda fd pVIII gene [F. Felici, L. Castagnoli, A. Musacchio, R. Jappelli, G. Cesareni, J Mol Biol 222 , 301 (1991); G. Cestra

et al.

, J Biol Chem 274 , 32001. (1999)]. Phage clones were selected by binding to the GST fusion protein through three cycles of binding and washing. Binding to the appropriate SH3 domain was confirmed by ELISA assay and the sequence of the displayed peptides deduced from the DNA sequence of the hybrid pVIII gene. We also tested, by ELISA assay, 40 different peptides, two representative clones from the list that were successful in the selection experiment, against each of the 25 soluble SH3 domains (see S3). Some SH3 domains (Abp1, Bbc1, Bem1-1, Bzz1-2, Sdc25, Hof, Myo3, Myo5, Nbp2, Rvs167, Sla1-3, Yfr024c, Ygr136w,Yhl002w, Ypr154w, Ysc84) were also screened with a library [A. B. Sparks, L. A. Quilliam, J. M. Thorn, C. J. Der, B. K. Kay, J Biol Chem 269 , 23853 (1994)] that invariantly had proline at specific residues 1064987xPxxP1064987x (x = any amino acid, P = proline). To map the SH3 domain target sites within Las17, five different fragments of the

LAS17

gene, encoding proline rich peptides, were fused to the D gene of bacteriophage lambda, between the Spe I and Not I sites of the lambda display vector Dsplay1 [A. Zucconi, L. Dente, E. Santonico, L. Castagnoli, G. Cesareni, J Mol Biol 307 , 1329 (2001)], then displayed at high density for panning experiments.