INTEGRATED BIOINFORMATICS BNFO 601
Virginia Commonwealth University
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This 4 page Class Notes was uploaded by Vivienne Dickens on Wednesday October 28, 2015. The Class Notes belongs to BNFO 601 at Virginia Commonwealth University taught by Staff in Fall. Since its upload, it has received 15 views. For similar materials see /class/230696/bnfo-601-virginia-commonwealth-university in BioInformatics at Virginia Commonwealth University.
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Date Created: 10/28/15
BIOL591 Introduction to Bioinformatics 2003 Protein Overexpression Structure vs Function 1 Overview of protein overexpression Take a look at that Pepsi nondiet high fructose corn syrup Well corn syrup doesn t have an appreciable amount of fructose in it just lots of glucose So where does the fructose come from It comes from the enzyme glucose isomerase originally from ground up Bacillus coagulans which equilibrates glucose to glucose fructose Considering the gazillion liters of soft drink in the world that s a lot of enzyme that s required Or what about human insulin which since 1982 has been available for use by diabetics Where does that come from Not from ground up babies These and many other proteins are produced within bacteria for industrial use Industrial use implies industrial quantities implying a need to get bacteria to produce more of the desired protein than nature might otherwise dictate The overproduction of protein raises several issues some of which are How can the gene of a eukaryote be expressed within a bacterium How can the gene encoding the desired protein be transcribed at a very high rate How can the transcript be translated at a very high rate 0 How can an unnaturally large amount of protein in small cells be convinced to maintain normal solubility After considering these questions we ll turn to the question that will be of the greatest concern to us 0 How can bioinformatics contribute to the enhancement of protein production by microbes IA How can the gene of a eukaryote be expressed within a bacterium Antibodies certain peptide hormones and many other human protein are not naturally found in bacteria If we wish to produce large quantities of them there are a number of choices One choice is not human tissue culture 7 it isn t possible to grow up enough economically One can turn instead to recombinant cows express the genes such that the protein appears in the cows milk plants yeast or bacteria Since humans have had the greatest experience since antiquity growing microbes for industrial purposes eg various fermentative processes microbes are the most familiar choice Bacteria pose a number of challenges in expressing eukaryotic genes 0 Eukaryotes and prokaryotes have different mechanisms for initiating transcription A eukaryotic gene to be expressed in a prokaryote must be supplied with a prokaryotic promoter They also have different mechanisms for initiating translation A eukaryotic gene to be expressed in a prokaryote must be supplied with a prokaryotic ribosome binding site Protein overexpression 1 0 While the genetic code in prokaryotes is the same as in eukaryotes some protein are modi ed after translation and these modi cations will not be found in eukaryotic proteins expressed in prokaryotes In those cases where modi cation is important bacteria are excluded as sites of production IIB How to express large amounts of protein in bacteria On one hand you d like to make as much protein as possible so as to harvest the greatest amount of product in the least time On the other hand stuf ng a bacterium with foreign protein is not compatible with good growth To get rapid growth you d like to have little expression of the foreign protein How to reconcile these two opposite needs Transcriptional fusions have proven useful in helping us regulate the amount of foreign protein expressed in a bacterial cell One of the oldest yet still amongst the most effective ways of doing this is to fuse a souped up version of the regulatory region of the lacZ gene to the foreign gene to be expressed Figure 1 Once this is accomplished the foreign gene will not be expressed when the lac repressor binds to the regulatory region but will be highly expressed when the repressor is removed Since the binding of the repressor is sensitive to sugars in the environment the process is under our control ribosome binding site TCACACmAACAGCT I GTGTGTCCTTTGTCG Bi ding site for lac repressor Fig 1 Transcriptional Fusion between the lac regulatory region and a foreign protein SQl How can transcriptional fusion with the lac regulatory region help express foreign protein IIC Problems with solubility In general if the protein is transcribed and translated it will fold on its own to the three dimensional structure that gives it activity However expressing an abnormally high level of protein in a cell can lead to unwanted structures for two reasons First in some cases proteins need help in folding from auxiliary proteins called chaperones Very high expression of a protein can sometimes saturate the A level 0 r 39 39 J expression of chaperones can help solve this problem Second weak interactions between protein that are insigni cant at normal concentrations may become overwhelming when the cell is stuffed with the protein This can lead to precipitation and formation of what are called inclusion bodies In general protein that have precipitated are not active Techniques have been developed to recover activity from misfolded or precipitated protein but these are tedious and not suitable for some automated processes Protein overexpression 2 III The qrensirin39 111A The goal Overproduced enzyme As you read in the Scenario you re on track to make an industrial amount of artificial cartilage WNW m chondroitin SuLfate To do this you 11 need to make a major component of cartilage chondroitin sulfate It is glycosaminoglycan that is an important pressive resistance in protein Cum connective tissues such as cartilage chondroitin tissue engineering to construct threedimensional I I Figure 2 of damaged connective tissues such as cartilage 0mm r A 44 L of UDpglucuronate To make lots of chondroitin sulfate you ll need lots of UDPglucuronate preferably from a relatively cheap source Organic synthesis of the compound is prohibitively expensive e biological quot 39 39 the reaction illustrated in Figure 3 catalyzed by the enzyme UDpglucose dehydrogenase fully the substrate for the reaction is a derivative of glucose dirt cheap with the help of an enzyme that putsUDP on glucose quot 39 n 39 r 4 aspartofthe You isolate the gene encoding UDPglucose dehydrogenase from the bacterium Mzsmhizobmm 1021 chosen because ofthe superior qualities of the enzyme in industrial applications The gene is transcriptionall fused to a modifiedzoc regulatory region as shown in Fi e 1 and clonedin E 5011 DNA cloning involves the insertion of a gene into a selfreplicating genetic element typically a plasmid A 39 39439 h 39 circular r39 DNA f und in bacteria During cloning a DNA fragment containing the gene of interest is joined to plasmid DNA by digestion with restriction enzymes The vector DNA and e DNA fragment with the gene are cut by the same restriction enzyme leaving complementary cohesive ends on each strand A L DNA 39 39 L 39 I I Next uii eerur 0 quotDP GM 3 m 2NADHo gt Wm 2NADH2H Dzhydmgeme DH OUDF DUDP C et a1 Figures 39 Chemlsvy Vol 4No lpp 13171421996 Protein overEXpresslon r 3 into bacterial cells through transformation The bacterial cells replicate the newly acquired DNA along with their own genome during repeated cellular divisions Markers on the vector such as antibiotic resistance are used to ensure that the bacterium takes up and retains the plasmid containing the gene of interest Now you have as much E coli as you want expressing large amounts of UDPglucose dehydrogenase SQZ What is the function of UDP glucose dehydrogenase SQ3 Can you visualize each step in the cloning process SQ4 What was the purpose of cloning the gene IIIB Overcoming precipitation through random mutagenesis Unfortunately while there is lots of the protein in the E coli you ve constructed there s very little UDP glucose dehydrogenase activity The protein is being made in an inactive form A look through the microscope reveals crystalline bodies 7 inclusion bodies 7 that resolves the mystery The overexpressed protein is precipitating removing it from any possibility of catalyzing chemical reactions You reason thusly if precipitation is caused by weak interactions between copies of the protein perhaps you can prevent this by altering the amino acids on the enzyme s surface Which amino acids to change If you knew the answer to this question you could perform sitespecific mutagenesis of specific codons to alter the key amino acid residues But you don t so you try pot luck random mutagenesis If you tried to make mutant enzyme by exposing the E coli to a mutagen like UV light or a chemical mutagen you d expose every gene in the cell to the same process Either you d use so little mutagen that mutations in all genes would be rare and you d have a difficult time finding the mutant enzyme you re after or you d increase the amount of mutagen and end up killing the cell You need a method to mutagenize specifically the gene in question There are many ways of randomly mutagenizing a specific gene One simple means is to use PCR to amplify the gene and then reinsert it into the plasmid PCR employs a heatstable DNA polymerase often Taq polymerase derived from the hot springs bacterium Thermus aequalis Unlike the DNA polymerase used to routinely replicate DNA Taq polymerase does not have proofreading functions hence it makes frequent mistakes By frequent I mean about one error for every 105 nucleotides replicated This may not seem like much but the DNA polymerase used in replication has an error rate of one mutation in 109 nucleotides SQS What is the expected number of mutations suffered by the gene encoding UDP glucose dehydrogenase roughly 1200 nucleotides after ampli cation for 25 rounds of PCR Each round of PCR doubles the amount of DNA What fraction of the genes do you expect to have at least one mutation You can answer these questions approximately or as precisely as you like Maybe one or more of the mutations will prevent precipitation but how will you find out You re not going to put 1000 s of mutants under the microscope looking for inclusion bodies and the thought of doing 1000 s of enzyme assays is no better You need a surrogate method Protein overexpression 4
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