Summary: Mapping protein-protein interactions occurring in a living cell can provide answers to plethora of questions in biological research. Split Beta-lactamase based protein fragment complementation assay provides an opportunity to analyze such interactions in both in vitro and in vivo environments. This flexible and robust assay holds potential to create a difference in our understanding of cellular processes.
Introduction
Protein-protein interactions form the basis of cellular processes that occur inside a living cell. Recent advances in biological research have strongly instigated the need to map biochemical networks for improved understanding of living cells. Apart from prediction and determination of protein structures to ascribe
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Protein fragment Complementation: What’s in the name?
Protein fragment Complementation assays (PCAs) have emerged as an exciting option for such applications and tend to offer complete solution to study protein-protein interactions in a high-throughput format. These assays comprise of a protein molecule, which is rationally split into two fragments that do not interact with each other independently but can refold and reconstitute the activity of the native protein when brought together by interacting partners fused to them covalently. The reconstituted activity thus obtained can be used to select for such interacting pairs (Figure 1). To name a few, PCAs employing proteins like murine Dihydrofolate reductase (mDHFR), TEM-1 Beta lactamase (TEM-1 Bla), luciferase, Beta galactosidase, GFP, g3p of M13 filamentous phage have been used successfully for studying protein- protein interactions in bacterial hosts.
TEM -1 Beta lactamase: The molecule of choice
The focus of this review is the use of one of the most celebrated molecule the history of biology “TEM-1 Beta lactamase” (TEM-1 Bla; EC: 3.5.2.6) in PCAs. TEM-1 Bla is an E.coli derived plasmid borne resistance determinant to Beta lactam based drugs which was discovered in 1965 and named after the patient Temorina suffering from urinary tract infection untreatable with ampicillin. Although, clinically it has remained a barrier in the
4. Complement (p.458)(complement system): Set of serum proteins designated numerically according to their order of discovery
A protein has multiple existing structures, these are the primary, secondary, tertiary and quaternary structures which occur progressively. A protein is essentially a sequence of amino acids which are bonded adjacently, and interact with one another in various ways depending on the R group that the amino acid contains. There are 20 different amino acids which are able to be arranged in any given order, thus giving rise to a potential 2.433x1018 (4.s.f) different combinations, and therefore interactions between the various amino acids.
Second, in order to further confirm the information about characteristics and function of the targeting protein that we have
For this experiment, E. coli was best for genetic engineering because of their size, and their fast reproduction (Spilios, 2017). E. coli will be genetically transformed using an engineered plasmid. A plasmid is a circular piece of DNA which independently replicates and multiplies because it has its own origin of replication (Spilios, 2017). The pGLO is the plasmid used in this experiment. Plasmids are used as vectors and they contain manipulated genes such as genes coding for antibiotic resistance for drugs like ampicillin. This antibiotic resistance of such serves as the selectable marker in genetic transformation and for genetic transformation to proceed, the cell must reach competency which is the physiological state that is required for the vector plasmid to get into the cell for transformation (Spilios, 2017). While competency can be reached naturally in some organism, it must be reached artificially in E. coli through treatment with CaCl2 and exposing them to heat shock using incubation (Spilios, 2017).
There are thousands of chemical reactions that occur in a cell at every moment. These chemical reactions do not occur randomly, they are highly under the control of biological catalysts called enzymes. Most of these enzymes are proteins. These proteins have certain primary structures directed by
The TEM-1 type of Beta-lactamase is the most common -lactamase enzyme found in E. coli. More importantly, this enzyme is highly interactive with antibiotics by inhibiting antibiotics from accomplishing their purpose of: halting the synthesis of bacteria cell walls to cease the spread and existence of the bacteria. This makes TEM type
Plasmid map of pRSETB. Primers were designed to amplify via PCR to cDNA. The PCR product was digested with XhoI and EcoRI enzymes and ligated into the pRSETB plasmid. The pRSETB plasmid contains a T7 promoter region, is ampicillin resistant, inducible with Isopropyl β-D-1-thiogalactopyranoside IPTG, a molecular mimic of a lactose metabolite that triggers transcription of the lac operon, and has XhoI and EcoRI cut sites. The pRSETB plasmid is transformed into dH5α E. coli and plated on carbenicillin plates. Colonies are selected and grown on a carbenicillin plate while PCR is used to check that the plasmid that was up-taken was not
Therefore, to validate this link in the A. thaliana-TuMV pathosystem, we conducted bimolecular fluorescence complementation (BiFC) assays to test the direct interaction between TuHC and AtCA1. The
Methods: Firstly in this experiment, one had to get familiar with how to use many of the basic commands in FirstGlance in Jmol. To get familiar with the program, one did a tutorial analysis of protein PDB ID: 1LGD at http://bioinformatics.org/firstglance/fgij//, following the specific step listed in the handout.1
Heparanase has three binding domains located in its 50 kDa fragment. The first domain contains Lys158–Asp171 residues that bind to heparin. The second domain binds to heparin sulfate. It is located in the Gln270– Lys280 residues. The third domain, located in Lys411–Arg432, does not interact with heparin/ heparin sulfate39. It has been suggested that the third domain facilitates the second step of consecutive and gapped cleavage through allostric binding
The cell is the smallest unit able to sustain life, and they are often referred to as the building blocks of life. There are two primary types of cell, which are categorized according to the way their genetic material is packaged, rather than size or shape. These are:
The primary protein structure can be likened to a human chain in which each person is assumed to be an amino acid and their hands viewed as the carboxyl and amino groups. The person on one end of the chain, who has a free left hand, is assumed to be the free carboxyl group. The person on the other end, who has a free right hand, is assumed to be the free amino group. Everyone in this chain has a left hand linked to somebody’s right hand and a right hand linked to somebody else’s left hand forming peptide bonds. The heads and legs just like the side chains and hydrogens, do not take part in the linking.
Mannose binding lectin is a member of a family of proteins called collectins (CLs), which
Serine proteases are a group of important proteases which can fracture the peptide bond in the macromolecules and proteins. The serine proteases take a great part in mammal lives especially in digestion, coagulation and the complement system appears. The activation of serine proteases are all because of the change of a set of amino acid residues which includes at least one serine and that’s why the name became. Although there are about one third of the known proteolytic enzymes are serine proteases, the principle of interactions are really same: a complicate combined interaction in the catalytic triad--Ser-His-Asp. This essay will firstly talk about the mechanisms of the serine proteases and secondly mention the relationship of the protein structure, function, and evolution during the serine proteases interact. In the first part, the chymotrypsin interaction will be analyzed as an example to explain the mechanism of serine proteases interact.
In this study, we wanted to use all forms of protein interaction data available, which requires combining of different types of experiments, such as yeast two-hybrid and co-immunoprecipitation. Two-hybrid results are naturally pairwise, whereas copurification results are sets of one or more identified proteins. For a copurification result, only a set of size 2 can be directly considered a pairwise communication, otherwise it must be modeled as a set of hypothetical interactions. Biochemical copurifications can be thought of as populations of complexes with some hidden pairwise protein interaction topology that is unknown from the experiment. In the general case of the purification used by Gavin et al., one weakness tagged protein was used as bait to pull associated proteins out of a yeast cell lysate. The two intense cases for the topology underlying the population of complexes from a single purification experiment are a minimally connected 'spoke ' model, where the data are modeled as explicit bait-associated protein pairwise synergy, and a maximally connected 'matrix ' model, where the data are modeled as all proteins connected to all others in the set. The real topology of the set of proteins must lie around between these two extremes.