L1 Expression vectors - Lecture notes 1 PDF

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MOLECULAR BIOLOGY II BY DR.Otieno...

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UNIVERSITY OF NAIROBI SCHOOL OF BIOLOGICAL SCIENCES SBT 415 MOLECULAR BIOLOGY II Lecture 1: Expression vectors: production of recombinant protein Specific objectives  By the end of this topic you should be able to: 1 Explain how an expression vector differs from a cloning vector. 2 List three elements that an expression vector should have for high-level inducible synthesis of proteins. 3 Explain the nature of a fusion protein 4 Describe how λgt   11 c an be used to produce a fusion protein. 5 Explain briefly how positive λgt   1 1clones in a cDNA library could be screened for expression of a protein 6 Explain what an open reading frame is. 7 State the advantage of inducibility of an expression vector Expression Vectors  ∙ Any piece of DNA capable of autonomous replication within a host cell into which other DNA sequences can be inserted and amplified is refereed to as a standard cloning vector. ∙ Unlike  a standard cloning vector, an expression vector efficiently expresses an inserted foreign gene by producing the protein product of the cloned gene, i.e. the recombinant protein. ∙ An  expression vector should be propagated in its host cell as a single copy genomic insert t o enhance its stability, i.e. should occur only once in the host cell genome. ∙ It should also respond to induction with rapid increase in transcription of the foreign DNA. Synthesis of a recombinant protein ∙ Depends on the presence of a collection of expression signals or short sequences of nucleotides surrounding the foreign gene. ∙ These expression signals are a promoter, terminator and ribosome binding site ∙ The signals must be recognizable by E.coli a nd provide instructions for the transcriptional and translational apparatus of the cell.  ∙ Foreign gene is placed under the control of the E.coli e xpression signals. ∙ Genes of higher organisms are also surrounded by expression signals, but their nucleotide sequences are not the same as the E. coli v ersions.  ∙ Foreign gene is inactive in E.coli b ecause the bacterium does not recognize its expression signals.

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 1. Promoter. ∙ The promoter marks the point at which transcription of the gene should start. ∙ It is recognized by the sigma subunit of the transcribing enzyme RNA polymerase. ∙ A  sigma factor is a protein component of prokaryotic RNA polymerases that binds loosely to the  core enzyme and restricts mRNA transcription to one of the two DNA strands and appropriate promoter region.  ∙ The promoter is the most important component of an expression vector because: o It  controls the first stage of gene expression i.e. the attachment of an RNA polymerase enzyme to the DNA o It determines the rate at which mRNA is synthesized.  o To some extent it determines the amount of recombinant protein ∙ A small variation has a major effect on the efficiency with which the promoter can direct transcription.  ∙ Most E.coli p romoters do not differ much from the consensus sequence (TTGACA ), e.g. TTTACA instead of TTGACA. ∙ Consensussequences areconservedbasesequences commoninregulatoryregionsofDNA e.g. promoter  regions,  where  each base occurs in a particular position in a large proportion of genomes of that species.  ∙ Examples   of consensus sequences are TATA   box in eukaryotes (about 25-30 base pairs upstream of  the sequence to be transcribed) and Pribnow box sequences in prokaryotes (TATAATG. also called a  –10  region,  becauseoftheinvariantTresidueatbase10upstream from the start of the transcribed region). 

 One can distinguish between strong promoters and weak promoters.

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(a) Strong promoters (e.g. from phage) are those which can sustain  a high rate of transcription. They usually control genes whose translation products are required in large amounts by the cell. (b) Weak promoters direct transcription of genes whose products are needed in only small amounts. They are relatively inefficient.  2. Terminator  (a) It marks the point at the end of the gene where transcription should stop.  (b) It is usually a nucleotide sequence that can base-pair with itself to form a stem-loop structure.  3. Ribosome binding site (RBS) (a) This is a short nucleotide s equence in mRNA molecule recognized by the ribosome as the point for its attachment.  (b) RBS includes a Shine-Dalgarno sequence (SDS),  which is a group of 6-8 purine-rich nucleotides (5’ AAGGAGGU 3’) located in mRNA 4-15 bases upstream (5’) from the initiator codon AUG. RBS is complementary to a region close to the 3’ end of 16S rRNA (the anti-SD sequence).

 Factors to be consider when constructing an expression vector. 1. The expression vectors should carry a strong promoter, so that the cloned gene is transcribed at the highest possible rate  2. It should have a strong translation initiation site.  3. It should be possible for the expression vector to regulate the promoter.

4 There are two major types of gene regulation in E.coli, namely, induction and repression. (a) Induction is the switching on of expression of a gene or group of genes in response to a chemical or other stimulus. An inducible gene is one whose transcription is switched on by addition of a chemical to the growth medium. Often this chemical is one of the substrates for the enzyme coded by the inducible gene. 

 (b) Repression is the switching off of expression of a gene or group of genes in response to a chemical or other stimulus. A repressible gene is switched off by the addition of the regulatory chemical.

 Many of the sequences important in induction and repression lie in the region surrounding the promoter and are  therefore also present in an expression vector. It is therefore possible to extend the regulation to the expression vector, so that the chemical that induces or represses the gene normally controlled by the promoter is also able to regulate expression of the cloned gene.  The regulation of the cloned gene by the chemical that also induces or represses the gene normally controlled by the promoter is an advantage in the production of recombinantprotein for two reasons. 1. If the recombinant protein has a harmful effect on the bacterium, then its synthesis must be carefully monitored to  prevent accumulation of toxic levels. This  can be achieved by judicious use of the regulatory chemical to control expression of the cloned gene. 2. Even if the recombinant protein has no harmful effects on the host cell, regulation of the cloned gene is still desirable, as a continuously high level of transcription may affect the ability of the recombinant plasmid to replicate, leading to its eventual loss from the culture.  Expression vectors permit the expression of the cloned sequences by fusing them to transcription and translation start signals.  Expression vector that produces a fusion proteins e.g. λgt  11 ∙ λgt 11 is a phage designed specifically as an expression vector.   gene (the E.coli gene that encodes β  - ∙ It contains the lac c ontrol region followed by the lac Z galactosidase )  ∙ The cloning sites are located within the lac Z  gene, so products of a gene inserted into this vector will be fusion proteins with a leader of β-galactosidase.    ∙ The gene to be expressed is inserted into the EcoR1 s ite near the end of the lacZ coding region just

before the transcription terminator.

5 ∙ Upon induction of the lac Z  gene by IPTG, a fused mRNA results, containing the inserted coding region just downstream from that of the β-galactosidase.    ∙ This mRNA is translated by the host cell to yield a fusion protein.  ∙ (A    fusion protein is a protein resulting from the expression of a recombinant DNA containing two open reading frames (ORFs) fused together. An open reading frame is a reading frame that is uninterrupted by translation stop codons).

 Detection of positive λgt  11 clones  ∙ λgt 11 is suitable for making and screening cDNA libraries.  ∙ It allows for direct screening of a group of clones for the expression of the right protein. ∙ The  main components required for this procedure are a cDNA library in λgt  11 and an antiserum  directed against the protein of interest. ∙ The λ phages with various cDNAs are plated and the proteins released by each clone are blotted onto a support such as nitrocellulose.  ∙ The host cells lyse and in doing so release their products, making it easy to transfer proteins from thousands of clones simultaneously, simply by touching a nitrocellulose filter to the surface of a petri dish containing the plaques.  ∙ Once the proteins from each plaque are transferred to nitrocellulose, they are probed with the antiserum.  ∙ Next, the antibody bound to protein from a particular plaque is probed by use of radioactive protein A from Staphylococcus aureus.  ∙ This protein binds tightly to antibody and makes the corresponding spot on the nitrocellulose radioactive.  ∙ The radioactivity is detected by autoradiography. The corresponding plaque is then picked from the

master plate. Note  ∙ A fusion protein is being detected and not the cloned protein by itself.

6 ∙ It does not matter if one has cloned a whole cDNA or not. The antiserum is a mixture of antibodies that will react with several differentpartsof ourprotein,soeven a partialgene willdo, aslongas its coding region is cloned in the same orientation and reading frame as the leading β  galactosidase coding region.  ∙ For instance, some vectors have restriction sites located just next to the control region for the lac genes, which has been spliced into the vector. These restriction sites permit foreign DNA to be spliced into the vector for cloning next to the lac c ontrol regions.  ∙ This allows expression of the cloned gene by using the lac t ranscription and translation start signals and control of expression by the lac r epressor.  ∙ Some vectors are bifunctional , allowing expression in two different hosts. For example, a certain plasmid contain the origin of replication of the plasmid pBR322 and of the animal virus SV40. ∙ These origins allow replication in E.coli a nd some cultured mammalian cell lines, respectively.  ∙ These plasmids are called shuttle vectors, because they can transfer genes back and forth from one type of cell to another.  ∙ The pUC vectors place inserted DNA under the control of the lac promoter, which lies upstream from the multiple cloning site.  ∙ If an inserted DNA happens to be in the same reading frame as the lac g ene it interrupts, a fusion protein will result.  ∙ It will have a partial β  -galactosidase protein sequence at its amino end and another protein sequence, encoded in the inserted DNA, at its carboxyl end. The  foreign polypeptide  can be recovered from the fusionproteinbycleavingatthejunctionbetween the two components with cyanogen bromide, which   cuts polypeptides specifically at methionine residues. The   methionine residue at the fusion junction must be the only one present in the entire polypeptide. If others are present then cyanogen bromide will cleave the fusion protein into more than two fragments. 

 Promoters often used in expression vectors

(a) The lac promoter  gene coding for β-galactosidase (and also This is the sequence that controls transcription of the lac Z  the lac Z’ gene fragment carried by the pUC and M13mp vectors. The lac p romoter is induced by

7 IPTG, so addition of this chemical into the growth medium switches on transcription of a gene inserted downstream of the lac p romoter carried by an expression vector. 

 (b) The trp promoter  ∙ The trp promoter is normally upstream of the cluster of genes coding for several of the enzymes involved in biosynthesis of the amino acid tryptophan.  ∙ The trp promoter is repressed by tryptophan, but is easily induced by 3- -indoleacrylic acid.

 (c) The tac promoter This is hybrid between the trp and lac promoters that is stronger than either, but still induced by IPTG.

 (d) The PL promoter This is one of the promoters responsible for transcription of the λ DNA molecule. λPL is  a very strong promoter that is recognized by the E.coli R  NA polymerase, which is subverted by λ into transcribing the bacteriophage DNA. The promoter is repressed by the product of the λcl gene. Expression vectors that carry the λPL promoter are used with a mutant E.coli h ost that synthesizes a  temperature-sensitive form of the c l protein. At low temperature (less than 30o C) this mutant cl  protein is able to repress the λPL promoter. At higher temperatures the protein is inactivated resulting in transcription of the cloned gene.

 ∙ It is usually advantageous to keep a cloned gene turned off until one is ready to express it. There are three reasons for this: (a) Eukaryotic proteins produced in large quantities in bacteria can be toxic.  (b) Even if the eukaryotic proteins are not toxic, they can build up to such great levels that they interfere with bacterial growth.  (c)  If  the  cloned were allowed to remain turned on constantly, the  bacteria bearing the gene would never growtoagreat enough concentrationtoproducemeaningful quantities of protein product. 

∙ The   solution is keep the cloned gene turned off by placing it behind an inducible   promoter that can  be turned off. One strategy is to use a very tightly controlled promoter such as the λ  phage promoter PL.  ∙ An example of a vector with such promoter is the pKC30. (a) The gene to be expressed in inserted into the unique Hpa I site of pKC30 vector, downstream region. from the λ OLPL operator/promoter 

8 (b) The host cell used is a λ lysogen  bearing a temperature-sensitive λ repressor gene (cI 8 57). (c) When the temperature of these cells is kept relatively low (32o C), the repressor functions, and no expression takes place. (d) When the temperature is raised to the nonpermissive level (42oC), the temperature-sensitive repressor is inactivated and removed from OL, allowing  transcription of the cloned gene to  occur. 

 Eukaryotic Expression Systems Incompatibility of eukaryotic genes in prokaryotic cells Eukaryotic genes are not compatible (not at home) in prokaryotic cells, even when they are expressed under the control of the prokaryotic vectors. There are several reasons for this incompatibility. 1. E.coli c ells frequently recognize the protein products of cloned eukaryotic genes as outsiders and destroy them.  2. Prokaryotes don not carry out the same kinds of postranslational modification as do eukaryotes. For example, a protein that would ordinarily be coupled to sugars in a eukaryotic cell will be expressed  as  a  bare protein when  cloned  in  bacteria. This  can affect a protein’s activity or stability, or at least its response to antibodies. 3. The interior of a bacterial cell is not as conducive to proper protein folding as the interior of a eukaryotic cell. Frequently, the result is improperly folded, inactive products of cloned genes. 4. A cloned gene can expressed at high level in bacteria, but the product forms highly insoluble,  inactive granules that are of no use unless one can somehow get the protein to dissolve and regain it activity. How to avoid incompatibility between a cloned gene and its host. ∙ This is done by expressing a gene in a eukaryotic cell.  ∙ The initial cloning is done in E. coli , using a shuttle vector that can replicate in both bacterial and eukaryotic cells.

∙ The recombinant DNA is then transferred to the eukaryote of choice by transformation. The eukaryote commonly used for this purpose is yeast.  ∙ There are several advantages of using yeast. (a) Like bacteria, yeast grows rapidly and is easy to culture.  (b) By splicing (linking) the cloned gene to the coding region for yeast export signal peptide , one ensures that the gene product will be secreted to the growth medium. (A signal peptide is a stretch of about twenty amino acids, usually at the amino terminus of a polypeptide, that helps to anchor the nascent polypeptide and its ribosome on the endoplasmic reticulum. Polypeptides with a signal peptide are destined for packaging in the Golgi apparatus

9 and are usually exported from the cell. The nascent polypeptide is extruded across the lipid bilayer, where a s ignal peptidase cleaves the signal from the protein ) .  (c) Purification of the protein is easy. The yeast cells can be removed by centrifugation, leaving relatively pure secreted gene product behind in the medium. The yeast-based shuttle vectors ∙ They are based on the 2-micron plasmid that normally inhabits yeast cells. ∙ The plasmid provides the origin of replication needed by any vector that must replicate in yeast. ∙ The  yeast-based shuttle vectors also contain the pBR322 origin of replication, so that they can replicate in E.coli .   ∙ They also contain a strong yeast promoter. Another eukaryotic vector is derived from the nuclear polyhedrosisvirus(NPV)thatinfects the  caterpillar known  as the  alfalfa looper. Viruses  in  this  class  have  a  rather large circular DNA genome, approximately 130 kb in length. The major viral structural protein, polyhedrin, is made in large quantities in infected cells. IthasbeenestimatedthatwhenacaterpillardiesofaNPV infection, up to 10% of the dry mass of the dead insect is this one protein. This indicates that the polyhedrin gene must be very active apparently due to its powerful promoter. General problems with the production of recombinant protein in E.coli Although expression vectors have been developed, there are still many difficultiesassociatedwiththe production of protein from foreign genes cloned in E.coli.  Theseproblemscan be grouped intothose that are due to:  (a) The sequence of the foreign gene, and  (b) Problems that stem from inherent properties of E. coli Problems resulting from the sequence of the foreign gene There are three ways in which the nucleotide sequence might prevent efficient expression of a foreign gene cloned in E. coli.  (a) The foreign gene might contain introns. This would be a major problem as E. coli genes do not contain introns and  the bacterium therefore does not possess the necessarymachineryfor removing introns from transcripts.

 (b)  The  foreign  gene  might  contain  sequences  that  act  as  termination  signals  in  E.  coli .  These sequences are perfectly innocuous in the normal host cell but in the bacterium result in premature termination and loss of gene expression.

10

 (c) The codon usage of the gene may not be ideal for translationinE.coli .  Although virtuallyall organisms  use  the  same  genetic  code,  each organism has a bias towards preferred codons. This bias reflects the efficiency with which the tRNA molecules in the organisms are ableto recognize  the  different codons. If a cloned gene contains a high proportion of unfavoured codons...