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Study Guide 3 BIO 463

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Syracuse University
BIO 463

Chp 7: Eukaryotic expression: 1. Glycosylation difference: Glycosylation is the addition of specific sugars to certain amino acids to provide stability and distinctive binding properties to a protein. Proper protein glycoslation is important because it contributes to protein conformation by influencing protein folding, can target a protein to a particular location, for example through interaction with a specific receptor molecule; or can increase protein stability by protecting it from proteases. Oligosaccharides are attached to newly synthesized proteins in the endoplasmic reticulum and in the Golgi apparatus by specific enzymes known as glycosylases and glycosyltransferases. a. “o” glycosylation- attachment to specific sugars to hydroxyl group either serine or threonine. O-linked oligosaccharides have a number of arrangements with different combinations of sugars. b. “N” glycosylation- attachment of specific sugar to amide group of asparagine. All N-linked glycosylation in eukaryotes start with the same initial group, which is subsequently trimmed and then elaborated in diverse ways within and among species. c. Transfection? Denote inherited changes in animal cells that are due to addition of (forgein DNA) 2. General understanding of eukaryotic expression vector? a. Must have eukaryotic promoter that drives the transcription of the cloned gene of interest, eukaryotic transcriptional and translational stop signals. Since recombinate DNa is hard to carry out with eukaryotic cells, most eukaryotic vectors are shuttle vectors with two orgins of replication and selectable marker genes. 3. Why do you want to use Eukaryotic host organism instead of using E.coli? a. Fungi share features of other higher eukaryoties and a good choice for heterologous protein production. They have growth advatnages similar to those of prokyarotes. They generally do not require growth factors to be added to the growth medium, can correctly process eukaryotic proteins and secrete large amounts of heterologous proteins. 4. Yeast: a. Usefulness of yeast host? Properties of yeast suitable for production of heterologous protein? i. There are several strong promoters that are isolated from the yeast and characterized, and a naturally occurring plasmid, called the 2um plasmid, can be used as part of an endogenous yeast expression vector system ii. Capable of carrying out many posttranslational modifications iii. Normally secretes few proteins that when it is engineered for extracellular release of a recombinant protein, the product can be easily purified. 5. Types of yeast vectors: a. YEP? (Yeast episomal plasmids) i. based on a high copy number 2um plasmid b. YIP? (Yeast integrating plasmids) c. YAC? 6. 2 um vector – a small, circular plasmid found in the most natural strains of S. cerevisiase. The vector replicates independently of the host chromosome via a single origin of replication and is maintained in more than 30 copies per cell. 7. Can YIP vector replicate inside the host organism? Why or why not? a. Many S. cerevisiase selection schemes rely on mutant host strains that require a particular amino acid or nucleotide for growth. Such strains are said to be auxotrophic because minimal growth medium must be suplmented with a specific nutrient. In practice the vector is equipped with a functinaonl wild type versone of the gene that complements the mutaed gene in the host strain. 8. Cu/Zn SOD overproduction-How? And usefulness? Which vector is good? a. Overproduction of Cu/Zn SOD is used to minimize the cytotoxic effects of superoxide dismutase SOD. The Cu/Zn SOD scavenges the superoxide radical and combines it with a hydrogen ion to from hydrogen peroxide which I nturn is degraded to water and oxygen by catalase or peroxide. In addition, Cu/Zn SOD migh act as a therapeutic agent against inflammatory diseases. s 9. Pichia Pastoris: a. Why use this yeast over S. crevasse? i. Glycoylastion occurs to a lesser extent and the linages between sugar residues are of a 1,2 type which are not allergenic to humans. b. AOX 1 gene promoter is used for the expression of…? Transcription of AOX1 is tightly regulated; in the absence of methanol, the AOX1 gene is completely turned off but responds rapidly to the addition of methanol to the medium. Therefore, AOX1 promoter is an excellent candidate for producing large amounts of recombinant protein under controlled conditions. 10. How would you select a yeast strain transformed with a plasmid containing your gene of interest? Baculovirus: are a large, diverse group of viruses that specifically infect arthopods, including many insect species and are not infectious to other animals. Polyhedron- viral nucleocapsids (virons) are clustered in a matrix that is made up of protein polyhedron. The occluded virions packaged in this protein matrix are referred to as polyhedron and are protected from inactivation by envirionmental agents. polyhedron 1. AcMNPV is used for the production of genetically engineered virus? a. AcMNPV is commonly used cell line for genetically enginnered cells. In the cells, the polyhedron promoter is exceptionally active, and during infections with wild-type baculovirus, high levels of polyhedron are synthesized. 2. How would you modify this virus (AcMNPV) for the expression of your gene of interest? a. Create a transfer vector that includes ACMPNV DNA upstream and downstream, used for homologous recombinant w ACMPNV. Polyhedrin promoter region, MCS, Polyhedrin terminal. b. Next, insect cells in culture are cotransfected with ACMNPV DNA and transfer vector carring the clonged gene. c. Double-crossover recombination event occurs at homologous polyhedron gene sequences on the transfer vector and in the ACMNPV genomne and the cloned gene wit the polyhedron promoter and termination regions becomes integrated into the ACMNPV DNA. (7.17) 3. Why do you linearize the plasmid during cross over? a. Linearization increase the frequency of recombinant plaques. Two Bsu 361 sites that were placed on either side of the polyhedron gene. The segment of the essential gene is meissing so no viral replication occurs. A transfer vector constructed with GOI with intact versions of gene . the transfer vector is introduced into insect cells that were previously transfeted with linearized that is missing the segment between the two Bsu sites. Double crossover event both reestablishes a functional version of ORF and incorpaotes the coloned gene into the ACMNPV genone 4. What is Bacmid vector? Mammalian cell expression system: 1. Mammalian vector? a. Share same features i. Eukaryotic orgin of replication, usually from a nimal virus such as SV40 ii. Promoter sequences that drive expression of the cloned genes iii. Selectable marker genes iv. Transcription termination sequences (polyadenylation signals) v. 2. Why contain viral promoter? a. Inducible promoters are often used when continuous synthesis of the heterologous protein is toxic to the host cell. Expression of a gene of interest is often increased by placing the sequence for an intron between the promoter and the multiple cloning site within the transcribed region. 3. What is Kozak sequence? a. Initiation of translation in higher eukaryotic organism depend on a specific sequence of nucleotides surrounding the start (AUG) condon in the mRNA. The DNA sequence for Kozak sequence, which is often followed by a signal sequence to facilitate secretion, a protein sequence tag to enhance the purification of the heterologous protein nand a proteolytic leavage sequence that enables the tag to be removed from the heterologous protein. 4. (fig 7:23 and 7:24) 5. Explain 7:25, a. Two expression vectors each with the gene or cDNA for one of the subunits and a different selectable marker gene can be cotransfected into host cells. The transfected cells are treated with both selecting agents and the cells that surive carry both vectors. However, two vecotrs are not always maintinaed with the same copy number so one is overproduced relative to the other and yields of final product reduced. 6. 7:26, a. single vectors that carry two cloned genes have been developed. Two genes are placed under the control of independent promoters and polyadenylation signals. To ensure that equal amounts of recombinant proteins are synthesized, vectors (bicistronic vecotsrs) 7. 7:29 a. selection schemes are desgined not only to identify transfected cells, but to increase heterologous protein production by amplifying the copy number of the expression vector. 8. What is IRES and its function? internal ribosomal entry site they allow simultaneous translation of different proteins from a polycistronic mRNA molecule. 9. What is ‘bicistronic”? to ensure equal amounts of recombinant of recombinant proteins are synthesized, vectors have been constructed with two cloned genes separated from each other by a DNA sequence that contains an internal ribosomal entry site. a. SINGLE TWO GENE TRANSCRIPT SYNTHESIZED AND TRANSALTION PRODUCEDS from 5’ end of mRNA to produce one of the chains Molecular Diagnostics: chp 9 1. Generalized ELISA protocol? A way to determine whether an antibody has bound to its target antigen. Frequently used for diagnostic detection. a. Bind the sample being tested for the presence of a specific molecule or organism to a solid support. Wash support to remove unbound molecules b. Add a marker-specific antibody (primary antibody directed against the target antigen) to the bound material and den wash the support to remove unbound primary antibody. Wash the support to remove unbound primary antibody c. Add second antibody that binds to the primary antibody and not to the target molecule. d. Add the colorless substrate e. Observe or measure the amount of colored product. 2. Monoclonal vs polyclonal antibody? 3. Monoclonal a. Create a cell line that could be grown in culture and produce a single type of antibody molecule with a high affinity for a specific target antigen b. Such a cell line would provide a consistent and continuous source of identical antibody molecules. 4. Polyclonal antibody a. Drawbacks: i. The amounts of different antibodies within a polyclonal preparation may vary from one batch to the next. One batch of polyclonal antibody may have a low level of antibody molecules directed against a major epitope and not be as effective as a previous antibody preparation. ii. Polyclonal antibodies cannot be used to distinguish between two similar targets 5. Advantages and drawbacks? 6. Method of producing monoclonal antibody? a. ELISA protocol 7. How would you select hybrid cells? (hybridoma) 8. HAT medium? Why? Function of different chemicals? a. Growth medium containing hypoxanthine, aminopterin and thymidne b. Allows only the myeloma spleen fusion cells to grow because none of the other cell types can proliferate in this medium. Unfused spleen cells and spleen spleen fusion cells cannot grow in this medium. 9. HGRT- myelenoma cells? And their role? a. The splenic cell suspension is mixed with a suspension of myeloma cells that are genetically defective for the enzyme hypoxanthine guanine phosphoribosyltransferase
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