Protein expression Flashcards
Compare cloning vectors and expression vectors by including their definitions, roles, types and features
A cloning vector is a small circular DNA that is used to introduce a foreign DNA into a host cell. An expression vector is a plasmid that introduces the gene of interest into a host and it facilitates the expression of the gene to produce protein. The role of a cloning vector is to obtain multiple copies of the gene of interest and an expression vector is used to analyze the gene product( RNA or protein). The types of cloning vectors include plasmids, cosmids, phages, YACs, BACs, MACs and expression vectors are just plasmids. The features of a cloning vector include an origin of replication, multiple cloning sites or a polylinker and an antibiotic resistance gene and an expression vector has the features of a cloning vector and regulatory elements, such as promoters, termination sequences, transcription initiation and translation initiation.
What is one of the most important uses of recombinant DNA technology
The production of proteins
Explain how bacterial proteins are produced
A structural gene is cloned and inserted into an expression vector. The expression vector contains the properly positioned control sequences for transcription and translation for the protein’s expression. With the use of a relaxed control plasmid and a promoter, the production of the protein of interest may reach 30% of the host bacterium’s total cellular protein. These genetically engineered organisms are called overproducers.
What are some of the problems that may be encountered when producing bacterial proteins in bacteria
> the bacterial cells often produce large amounts of proteins that are useless to the bacterium as insoluble and denatured inclusion bodies. A protein that is extracted from inclusion bodies must be renatured by dissolving it in a solution of urea or guanidinium ion and then removing the denaturant via a membrane via which the protein cannot pass through by dialysis or ultrafiltration. A strategy that can be used to avoid this step is to engineer the gene for the protein of interest so that it is preceded by a signal sequence. A signal sequence will ensure that the protein is secreted into the periplasmic space. The signal sequence is then removed by a bacterial protease.
The protein might be toxic to the host cell, which will kill the bacteria before sufficient amounts of protein can be produced. To prevent this from happening the gene that encodes the toxic protein can be put under the control of an inducible promoter, eg. the lac promoter in a plasmid that also induces the gene for the lac repressor. The binding of the lac repressor to the lac promoter will prevent the expression of the protein in the same way it prevents the expression of the lac operon genes. However after the cells have grown to a high concentration, an inducer is added which releases the repressor from the promoter and the protein will be produced. The cells are killed but not before they produce large amounts of the protein. The inducer is isopropylthiogalactoside (IPTG)- an analog of lac repressors natural inducer allolactose.
There is also a problem associated with inserting a DNA segment into a vector- DNA that is digested using 1 restriction enzyme can be ligated to a cloning vector cut with the same enzyme in any direction. Also, 50% of the structural genes will be inserted the wrong way in relation to the transcriptional and translational control sequences and it will result in the protein not being properly expressed. This can be corrected by using directional cloning in which 2 restriction enzymes are used.
What are the problems that occur when eukaryotic proteins are produced in bacterial cells
> The eukaryotic control elements for RNA and protein synthesis are not recognized by bacterial hosts. This can be eliminated by inserting the protein encoded region of the eukaryotic gene into a vector that contains correctly placed bacterial control elements.
Bacteria cannot carry out gene splicing. They do not have the cellular machinery to excise introns that occur in many eukaryotic transcripts. The need to excise introns can be eliminated by cloning the cDNA of the proteins mature mRNA. Alternatively, if the sequence is known small proteins can be chemically synthesized. Both these strategies are not universally applicable because mRNAs are not really abundant to be isolated and the genes that encode eukaryotic proteins are too large to be reliably synthesized.
Bacteria lack the enzyme systems to carry out post-translational processing that many eukaryotic proteins require in order to be active. Most bacteria do not glycosylate proteins. There is no general approach for post-translational modification of eukaryotic proteins.
Eukaryotic proteins may be degraded by bacterial proteases. This may be prevented by inserting the eukaryotic gene after a bacterial gene so that they both have the same reading frame. The resulting hybrid protein has an N-terminal polypeptide of bacterial origin and this can prevent the bacterial proteases from recognizing the eukaryotic segment as being foreign. This can also aid in the purification of the hybrid protein via a process called affinity chromatography. The normally insoluble C-terminal can become soluble because of the hybrid protein. The two polypeptide segments can later be separated by treatment with a specific protease that specifically cleaves the susceptible site that had been engineered into the boundary between the two segments.
How can all these problems be overcome
The development of cloning vectors that propagate in eukaryotic hosts, such as yeast or cultured animal cells, have eliminated these problems. Baculovirus-based vectors, which replicate in cultured insect cells, have been pretty successful. Shuttle vectors can propagate in both yeast and E-coli and can transfer genes between the two types of cells.
What is an application of recombinant DNA technology used to produce proteins
The ability to synthesize a given protein in large quantities has had an enormous medical, agricultural and industrial impact. Human insulin is used in the treatment of diabetes, the human growth hormone is used in the treatment of children that are abnormally short, the synthesis of the hepatitis B vaccine and in the synthesis of blood clotting factors for the treatment of hemophilia.
Explain site-directed mutagenesis
This is used to tailor proteins to specific applications by altering their amino acid sequence at specific sites. In this technique an oligonucleotide that contains the desired gene segment with the altered base sequence corresponding to the amino acid sequence is used as a primer. The primer is extended with DNA polymerase I in the replication of the mutated gene. The primer can be made to hybridize to the corresponding wild-type sequence if there are only a few mismatched base pairs and DNA polymerase I extends it to yield the altered gene. The altered gene can then be inserted into a suitable organism and cloned in quantity, PCR may also be used. Using site-directed mutagenesis, the development of a variant form of the bacterial protease subtilisin(Met222 is replaced with Ala) has permitted its use in laundry detergent that contains bleach. The wild-type subtilisin would be inactivated by bleach.
What is a reporter gene
A reporter gene is a known gene whose RNA and protein levels can be measured easily and accurately. It is often used to replace other genes whose protein levels are difficult to determine quantitatively. An example of a reporter gene is the lacZ gene in the presence of X-gal because its level of expression can be seen in the intensity of the blue color that is generated. The most used reporter gene encode green fluorescent protein(GFP). GFP is a product of a bioluminescent jellyfish. It fluoresces green when irradiated by UV or blue light. The fluorescing is the result of the spontaneous cyclization and oxidation by oxygen of three consecutive residues, Ser-Tyr-Gly, to yield a conjugated system of double bonds that gives the protein its properties. GFP does not require a substrate or cofactor to fluoresce. Its presence can be monitored through the use of UV light or a fluorometer and its cellular location can be determined by fluorescence microscopy. When the GFP gene is placed under the control of the gene expressing a particular protein- GFPs fluorescence is unaffected by the formation of a fusion protein- it can be used to determine the expressional activity. A number of GFP variants with distinct sets of excitation and emission wavelengths were developed- this allows for the expressional activities of several different genes to be monitored simultaneously.
Explain reporter genes and fusion proteins
Reporter genes can be attached to other sequences so that the reporter protein is synthesized fused to another protein. Often a short peptide sequence that serves as an affinity or epitope tag(antigenic determinant) is used.
What is a transgenic organism
It is preferable to tailor an intact organism not just a protein(true genetic engineering). A transgenic organism is a multicellular organism expressing a foreign gene(from another organism), Their transplanted foreign genes are known as transgenes. For the change to be permanent(heritable), a transgene must be stably integrated into the organisms germ cells.
Explain the use of cloning transgenic animals
An application of transgenic farm animals is for them to secrete pharmaceutically useful proteins, such as human growth hormone and blood clotting factors in their milk.
Explain the use of transgenic plants
Crop plants have been generated with increased herbicide resistance, resistance to viruses, bacteria and fungi, control of ripening. Altered plant architecture such as height(improves crop productivity).Increased tolerance to environmental stresses such as cold, heat, salinity. Improved nutritional properties- eg. a strain of rice has been developed that contains the foreign genes encoding the enzymes that synthesize beta-carotene. This is an orange pigment that is the precursor of vitamin A. This genetically modified rice is named golden rice and helps with vitamin A deficiency. Vitamin A deficiency affects millions of people and can lead to blindness and death.
Define gene therapy and the types
Gene therapy is the transfer of new genetic material to the cells of an individual resulting in therapeutic benefit to that individual. There are 3 types of gene therapy:
> Ex vivo(out of the body approach)- the cells are removed from the body, incubated with a vector and then returned to the body, eg. bone marrow cells- precursors of blood cells.
> In situ approach- the vector is applied directly to affected tissues. eg. to treat cystic fibrosis by inhaling an aerosol containing a vector encoding the normal protein
> In vivo(in the body) approach- the vector would be injected directly into the bloodstream.
