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Protein Biotechnology Protein Biotech Spring 2008 Ziad W Jaradat 1 Construct design for high-level transgene expression in animal cells When ever a recombinant protein is needed to be produced the ultimate goal will be the production of the maximum yield. Therefore, the following considerations should apply when a high level of expression is required: The use of strong and constitutive promoter The inclusion of an intron The inclusion of a polyadenylation signal The removal of unnecessary untranslated sequence Optimization of the transgene for translational efficiency The incorporation of a targeting signal Protein Biotech Spring 2008 Ziad W Jaradat 2 The use of strong and constitutive promoter Very active promoters produce the highest level of transgene expression. In viral systems, the transgenes are expressed under the strongest endogenous promoter such as; Baculovirus polyhedrin promoter Adenoviral E1 promoter Vaccinia virus p 7.5 promoter Protein Biotech Spring 2008 Ziad W Jaradat 3 The inclusion of an Intron The presence of an intron in a eukaryotic expression system usually enhances expression. Currently most mammalian expression systems incorporate a heterologous intron such as the SV40 small t- antigen intron or the human growth hormone intron. However, introns may not by used in some expression systems such as vaccinia virus. Protein Biotech Spring 2008 Ziad W Jaradat 4 The inclusion of a polyadenylation signal Polyadenylation signals (terminators) are required in eukaryotic genes to generate a defined 3’ end of the mRNA. This is normally done by the addition of several hundred adenosine residues to generate a poly A tail. This tail is required for the export of mRNA into the cytoplasm and to increase its stability. It is found that in the absence of the 3’ end with the poly A sites of the mRNA, the level of expressed protein fall down by as much as 90%. Protein Biotech Spring 2008 Ziad W Jaradat 5 The inclusion of a polyadenylation signal… cont. Example; the poly A site from the SV40 transcription unit or the mouse beta globin gene are often incorporated into mammalian expression vectors to enhance expression. Protein Biotech Spring 2008 Ziad W Jaradat 6 The removal of unnecessary untranslated sequence Eukaryotic mRNAs comprise a coding sequence (gene) bracketed by untranslated regions (UTRs) of variable lengths. Both the 5’ and 3’ UTRs can influence gene expression in a number of ways; 5’ UTR may contain one or more AUG codons upstream of the authentic translational start site. Such codons are detrimental to the translation initiation. The 3’ UTR may contain regulatory elements (AU-rich) that control mRNA stability. Both the 5’ and 3’ UTRs may be rich in secondary structure that prevents efficient translation. Therefore, UTR sequences are generically removed from transgene construct to maximize expression Protein Biotech Spring 2008 Ziad W Jaradat 7 Optimization of the transgene for translational efficiency The sequence around the translational initiation site should conform to Kozak’s consensus, which is defined as 5’-CCRCCAUGG-3’. Of great importance is the purine at the -3 position ® and the guanidine at the +4 position. The adinosine in the AUG initiation codon is given the number +1 and the immediately preceding base is defined as 1. The expression of foreign genes in animals can be inefficient in some cases due to suboptimal codon choice. i.e different organisms prefer to use different codons to specify the same amino acid. Therefore, if a transgene contains codon that is commonly used in the source organism but rarely used in the host, the translation may pause at that codon due to the scarcity of the corresponding tRNA. This will reduce level of protein synthesis or may even lead to the truncation of the protein or frame shifting. Protein Biotech Spring 2008 Ziad W Jaradat 8 The incorporation of a targeting signal If the goal of the expression is to produce large amount of functional protein, then it is necessary to consider whether it should be post-translationally modified or not. Example glycosylation might be needed for therapeutic proteins. This is important for the correct function of the protein and to minimize the immune response against it. It is important to target the protein to the correct compartment to be appropriately modified. This can be done by incorporating a signal peptide if the protein that has to be glycosylated. Protein Biotech Spring 2008 Ziad W Jaradat 9 Many mammalian expression vectors are available for this purpose. Example, the Invitrogen vector pSecTag2 which incorporates a sequence encoding the murine immunoglobulin light chain signal peptide for targeting the produced protein to the secretory pathway. In addition, the C terminus of the recombinant protein is expressed as a fusion protein to two different tags to facilitate its purification. Protein Biotech Spring 2008 Ziad W Jaradat 10 Purification of a cloned gene product can be facilitated by use of purification tags Many cloning vectors have been engineered so that the protein being expressed is fused to another protein called tag to facilitate its purification. Examples of such tags are; glutathione S-transferase MalE (maltose binding) protein Multiple histidine residues (his tag) which can be easily purified by metal affinity chromatography. The vectors are usually constructed so that the coding sequence for an amino acid which is cleaved by specific protease is incorporated between the coding for the tag and the gene being expressed. It is also possible to include with the tag a protein that can be assayed easily to facilitate detection of the protein. Protein Biotech Spring 2008 Ziad W Jaradat 11 Examples of the tags The use of polyhistidine fusion for purification; the gene of interest is first engineered into a vector in which there is a polylinker downstream of six histidine residues and a proteolytic cleavage site which could be a site for enterokinase. 1. After induction of synthesis of the fusion protein, the cells are lysed and the viscosity of the lysate is reduced by neuclease treatment. The lysate is then applied to the column containing immobilized divalent nickel that selectively binds to the polyhistidine tag. After washing away any none specific material, the fusion protein is eluted from the column and will be treated with enterokinase to release the cloned gene product. Protein Biotech Spring 2008 Ziad W Jaradat 12 To facilitate the assay of the fusion protein, short antibody recognition sequences can be incorporated in the tag between the affinity label and the protease cleavage site. Example of these sequences: 1. Glu-Gln-…………Asp-LeuAnti-myc antibody 2. His-His-His-His-His-His-COOH Anti-His (Cterminal) antibody 3. Gly-Lys-………………Asp-Ser-ThrAnti-V5 antibody Protein Biotech Spring 2008 Ziad W Jaradat 13 An example of a purification tag is the incorporation of biotin carboxylase carrier protein. A protein that has the biotin tag can be purified by a streptavidin affinity column. E. coli expresses a single endogenous biotinylated protein but it does not bind to streptavidin in its native conformation making the affinity purification highly specific for the recombinant fusion proteins. In addition, the biotin on the fusion protein can be detected by enzymes coupled to streptavidin. Protein Biotech Spring 2008 Ziad W Jaradat 14 Vectors that promote solubilization of expressed proteins One of the problems associated with production of over expressed proteins is the formation of the inclusion bodies. How to get rid of the problem? 1. Culture methods such as reducing the temperature and changing media composition and pH values to reduce growth rate can minimize the problem. Or growing bacteria under osmotic stress (4% NaCl + 0.5 M sorbitol ) in addition, adding glycine betaine which protects proteins at high salt concentrations lead to generation of correctly folded proteins. Protein Biotech Spring 2008 Ziad W Jaradat 15 2. Genetic methods; 1. Host cell is engineered to overproduce chaperons + the protein (even though there is no guarantee of proper folding of the protein). 2. Making minor changes to the amino acid sequence of the target protein e.g cysteine-to-serine changes in fibroblast growth factor minimized inclusion-body formation. 3. Synthesis of target proteins (who aggregates in their native form) as thioredoxin fusion proteins or as maltose-binding protein or as fusion with some other proteins such as NusA, GrpE proteins or bacterioferritin. Enterokinase Cleavage site is incorporated to facilitate the purification of these fusion proteins later. Protein Biotech Spring 2008 Ziad W Jaradat 16 1. An alternative way to keep proteins soluble is to export them to the periplasmic space Ps. After synthesis, the fusion protein is released by osmotic shock and purified, then cleaved by the enterokinase to release the target protein. Protein Biotech Spring 2008 Ziad W Jaradat 17 Exporting proteins to periplasmic space In E. coli protein export to the periplasmic space or to the outer membrane is achieved by a universal mechanism known as the General Export Pathway (GEP). Proteins that enter the GEP are synthesized in the cytoplasm with a signal sequence at the N-terminus. This sequence is cleaved by a signal or leader peptidase during transport. The signal sequence has three domains; +ve charged amino terminal region, hydrophobic core and a leader peptidase cleavage site. Protein Biotech Spring 2008 Ziad W Jaradat 18 Exporting proteins to periplasmic space.. Cont. A signal sequence attached to a normally cytoplasmic protein will direct it to the export pathway. Many signal sequences that are derived from naturally occurring secretory proteins (ompA, ompT, AP… etc) support the efficient translocation of heterologous peptides across the inner membrane when fused to their amino terminus. Protein Biotech Spring 2008 Ziad W Jaradat 19 Optimizing Translation High level expression of genes requires more than a strong promoter. There are many factors that can influence the rate of translation: 1. The interaction of the ribosome with bases immediately upstream from the initiation codon of the gene (most important). In bacteria a key sequence is the ribosome binding site or Shine-Dalgarno (S-D) sequence. 2. The degree of complementarity of this sequence with the 16S rRNA can affect the rate of translation. Protein Biotech Spring 2008 Ziad W Jaradat 20 Optimizing Translation …..con. The spacing between the S-D sequence and the initiation codon is also important. Usually there are 5 to 10 basis with 8 being optimal. Decreasing the distance below 4 or increasing it above 14 can reduce translation several fold. 3. Translation can also be affected by sequence following the S-D site. The presence of 4 A or 4 T residues in this position gave the highest translation efficiency while the translational efficiency decreased to 50 or 25% when 4 C or 4 G residues where present. 4. 5. The composition of the triplet immediately proceeding the AUG start codon or the composition following the AUG also affects the efficiency of translation. Highly expressed genes have AAA (Lys) or GCU (Ala) as the second codons. Protein Biotech Spring 2008 Ziad W Jaradat 21 Optimizing Translation…. Cont. 6. Sequence upstream from the S-D can also affect the translation of certain genes. e.g in E. coli md gene there is a run of 8 uracil residues. Changing two to 5 of these residues had no effect on mRNA level, but reduces translation by 95%. Protein Biotech Spring 2008 Ziad W Jaradat 22 End of chapter Protein Biotech Spring 2008 Ziad W Jaradat 23 Protein Biotech Spring 2008 Ziad W Jaradat 24 Protein Biotech Spring 2008 Ziad W Jaradat 25 Protein Biotech Spring 2008 Ziad W Jaradat 26 Protein Biotech Spring 2008 Ziad W Jaradat 27 Protein Biotech Spring 2008 Ziad W Jaradat 28 Protein Biotech Spring 2008 Ziad W Jaradat 29