Manipulation of Gene Expression in Prokaryotes

1. Manipulation of Gene Expression in Prokaryotes

2. Gene Expression in Prokaryotes • The expression of the cloned gene in a selected host organism. • It does not necessarily ensure that it will be successfully expressed. • A high rate of production of the protein encoded by the cloned gene is required. • Specialized expression vectors have been created that provide genetic elements for controlling transcription, translation, protein stability, and secretion of the product from the host cell.

3. Manipulation of Gene Expression • The promoter and transcription terminator sequences. • The strength of the ribosome-binding site. • The number of copies of the cloned gene. • The gene is plasmid-borne or integrated into the genome of the host cell. • The final cellular location of the synthesized foreign protein. • The efficiency of translation in the host organism. • The intrinsic stability within the host cell of the protein encoded by the cloned gene.

4. Gene Expression from Strong and Regulatable Promoter • The strong promoter is one that has high affinity for RNA polymerase that the adjacent downstream region is frequently transcribed. • The ability to regulate a promoter enables the cell to control the extent of transcription in a precise manner. • The lac operon of E. coli has been used extensively for expressing cloned genes. • Many different promoters with distinctive properties have been isolated from a range of organisms.

5. Gene Expression from Strong and Regulatable Promoter • The most widely used are those from the E. coli lac and trp operons. • The tac promoter is constructed from -10 region of the lac promoter and -35 region of the trp promoter. • The pL promoter from bacteriophage λ. • The gene 10 promoter from bacteriophage T7. • Each of these promoters interacts with regulatory proteins which provide a controlable switch for either turning on or turning off specific transcription of adjacent cloned gene.

6. Gene Expression from Strong and Regulatable Promoter

7. Gene Expression from Strong and Regulatable Promoter

8. Gene Expression from Strong and Regulatable Promoter • The pL promoter is controlled by the cI repressor protein from bacteriophage λ. • A temperature-sensitive mutant of the cI repressor, cI857, is generally used to regulate pL-directed transcription. • Cells are first grown at 28 - 30ºC, at which the cI repressor prevent transcription. • When the cell culture reaches the desired stage of growth, the temperature is shifted to 42ºC. • The cI repressor is inactivated and transcription can proceed.

10. Gene Expression from Strong and Regulatable Promoter • The gene 10 promoter from bacteriophage T7 requires T7 RNA polymerase for transcription. • T7 RNA polymerase gene is inserted in the E. coli chromosome on a bacteriophage T7 lysogen under the control of the E. coli lac promoter. • In the absence of IPTG, the lac repressor represses the synthesis of T7 RNA polymerase. Therefore, the target gene is not transcribed. • When lactose or IPTG is added to the medium, it binds to the lac repressor. T7 RNA polymerase is transcribed and the target gene is transcribed.

12. When the portion of the spacer region from the E. coli lac promoter was mutated, the activity increased >40 fold in the absence of CRP. • The -20 to -13 region was altered from GC-rich to an AT-rich.

13. Increasing Protein Production • Plasmid pCP3 was created in an effort to obtain the highest possible level of foreign-protein production in a recombinant E. coli strain. • It contains the strong pL promoter, ampicillin resistance gene, a multiple cloning sequence immediately downstream from the promoter, and a temperature-sensitive origin of replication. • The plasmid’s copy number increases 5- to 10- fold when the temperature is increased to 42ºC.

14. At lower temperature, the cI repressor, integrated to E. coli chromosomal DNA, is functional. The pL promoter is turned off, and the plasmid copy number is normal. • At higher temperature, the cI repressor is inactivated, the pL promoter is active, and the plasmid copy number increases to around 600 copies per cell, increasing protein production.

15. Large-Scale Systems • In large-scale systems, shifting temperature requires time and energy, both of which can be costly. • The cost of chemical inducer, such as IPTG, make the overall process uneconomical. • To overcome some of the problems, a two-plasmid system has been developed. • The cI repressor was placed under the control of the trp promoter and inserted into a low-copy-number plasmid, ensuring that excess cI repressor molecules are not produced.

16. Large-Scale Systems • Cell can be grown on an inexpensive medium consisting of molasses and casein hydrolysate, which contain small amount of free trptophan. • The cloned gene can be induced by the addition of tryptone to the medium, which contains enough trptophan for efficient induction of transcription. • With no trptophan, the cI repressor is produced and thereby blocking the transcription of the cloned gene. • With tryptophan, the cI repressor is repressed, and the cloned gene is transcribed and translated.

18. Promoters that are induced when cells enter stationary phase may be useful in the design of expression vectors that are useful for large-scale application. • The house keeping RNA polymerase sigma factor, σD, and stationary-phase sigma factor, σS, recognize a similar sequence. • The cells would be grown to a high density, as the cells entered the stationary phase, gene expression would be induced.

19. Expression in Other Microorganisms • Cell can be grown on an inexpensive medium consisting of molasses and casein hydrolysate, which contain small amount of free trptophan. • The cloned gene can be induced by the addition of tryptone to the medium, which contains enough trptophan for efficient induction of transcription. • With no trptophan, the cI repressor is produced and thereby blocking the transcription of the cloned gene.

20. Expression in Other Microorganisms

21. Fusion Proteins • Foreign proteins, especially small ones, often are degraded in the host cell. • A DNA construct that encodes a target protein is in frame with stable host protein. • Fusion protein protects cloned gene product from attack by host cell proteases. • Fusion proteins are stable because the target proteins are fused with proteins that are not especially susceptible to proteolysis.

23. Fusion Proteins for Purification

24. Fusion Proteins for Purification • The target protein is fused to the marker peptide. • The secrete protein can be purified in a single step by immunoaffinity chromatography. • The marker peptide is relatively small, it will not interfere with the production of the target protein.

25. Immunoaffinity chromatography purification of a fusion protein

26. Fusion Proteins for Purification • As an alternative, it has become popular to generate a fusion protein containing six or eight histidine residue attached to either N- or C- terminal end of the target protein. • The histidine-tagged protein and other cellular proteins are then passed over an affinity column of nickel-nitrilotriacetic acid that bound to histidine-tagged protein. • The elution of the bound protein is done by adding of imidazole (the side chain of histidine.)

27. Cleavage of Fusion Proteins • The marker protein may be undesirable. Thus, strategies have been developed to remove unwanted amino acid sequence from the target protein. • Specific sequence that encode short stretches of amino acids that are recognized by a specific nonbacterial protease.

28. Cleavage of Fusion Proteins • The most commonly used proteases are enterokinase, tobacco etch virus protease, thrombin, and factor Xa. • However, it is necessary to perform additional purification steps in order to separate both the protease and the fusion protein from the protein of interest. • The protease may cleave the protein of interest at unintended sites. • The cleavage reaction may not go to completion. • To avoid the problems, the self-splicing inteins is used.

29. Purification of a protein of interest from an intein-containing fusion protein bound to a chitin chromatography column through a chitin-binding domain. • Cleavages occurs upon the addition of dithiothreitol.

30. Surface Display • The expressed target protein is fused to bacteriophage M13 protein pIII near its N terminus. • After M13 replication in E. coli cells, the plaques are assayed immunologically using antibodies that detect the present of target protein. • This is an extremely powerful selection system to find cDNAs for very rarely expressed but important proteins.

31. Surface Display • Fusions between the genes for the target protein and for an outer surface protein are created to export proteins to the surface of a gram-negative bacteria.

32. Translation Expression Vectors • Putting cloned gene under the control of a regulatable and strong promoter may not be sufficient to maximize the yield of the cloned gene product. • The efficiency of translation and the stability of the newly synthesized cloned-gene protein, may also affect the amount of product. • The stronger binding of the mRNA to the ribosomal RNA, the greater the efficiency of translational initiation. • The expression vectors have been designed to ensure that the mRNA of a cloned gene contains a strong ribosome-binding site.

33. Translation Expression Vectors • For each cloned gene, it is important to establish that the ribosome-binding site is properly placed and that the secondary structure of the mRNA does not prevent its access to the ribosome. • AUG – start codon • GGGGG – ribosome-binding site • G can also pair with U

34. The Expression Vectors pKK233-2 • Ampicillin resistance gene as a selectable marker. • ptac regulatable promoter • Ribosome binding site • Cloning site (NcoI, PstI, and HindIII) • Start codon and transcription terminators.

35. Translation Expression Vectors • Codon usage might interfere with efficient translation when a clone gene has codons that are rarely used by the host cells. • The host cell may not produce enough of the tRNAs that recognize these rarely used codons, and the yield of cloned-gene protein is much lower than expected. • An insufficient supply of certain tRNAs may lead to either a reduction in the amount of the protein synthesized or the incorporation of incorrect amino acids into the protein.

36. Translation Expression Vectors • There are several approached to alleviate this problem. • If the target gene is eukaryote, it may be cloned and expressed in an eukaryotic host cell. • A new version of the target gene containing codons that are commonly used by the host cell may be chemically synthesized (codon optimization.) • A host cell that has been engineered to over express several rare tRNAs may be employed.

39. Increasing Protein Stability Intrinsic Protein Stability • Under normal growing conditions, the half-lives of different proteins range from a few minutes to hours. • The presence of a single extra amino acid at the N-terminal end is sufficient to stabilize a target protein. • Long-lived proteins can accumulate in cells and thereby increase the yield of the product. • This phenomenon occurs in both prokaryotes and eukaryotes.

40. Increasing Protein Stability Intrinsic Protein Stability

41. Increasing Protein Stability Intrinsic Protein Stability • In contrast, there are internal amino acid sequences that make a protein more susceptible to proteolytic degradation. • This region is called PEST sequence (proline, glutamine, serine, and threonine.) • They are often flanked by clusters of positively charged amino acid, such as lysine and arginine. • This may act to mark proteins for degradation within the cell.

42. Increasing Protein Stability Facilitating Protein Folding • Proteins produced in E. coli accumulate in the form of insoluble, intracellular, biologically inactive inclusion bodies because of incorrect folding. • The extraction procedure requires expensive and time consuming protein solubilization and refolding procedures. • Fusion proteins that contain thioredoxin as the fusion partner remain soluble. • The target gene is cloned into a multiple cloning site just downstream from the thioredoxin gene.

43. Increasing Protein Stability Facilitating Protein Folding • Without tryptophan, cI repressor is synthesized to prevent transcription from the pL promoter and therefore prevent the production of fusion protein. • When tryptophan is added, the trp promoter is turned off, the cI repressor is not synthesized, allowing transcription and translation of the fusion protein. • The soluble fusion protein is selectively released by osmotic shock from E. coli cells into growth medium. • The native form of protein can be released by treatment with the enzyme enterokinase.

44. Increasing Protein Stability Facilitating Protein Folding

45. Increasing Protein Stability Facilitating Protein Folding • Foreign proteins that contain three or more disulfides generally do not fold correctly in bacteria and often form inclusion bodies. • The gene coded for human tissue plasminogen activator was coexpressed with gene for either rat or yeast protein disulfide isomerase to assist protein folding. However, it did not affect the amount of the protein that could be obtained. • Overproduction of DscB results in correctly folded and active human tPA.

46. Increasing Protein Stability Facilitating Protein Folding

47. Increasing Protein Stability Coexpression Strategies • The expression of foreign proteins in E. coli results in the formation of inclusion bodies of inactive proteins. • Cultivation of recombinant strains at low temperatures, resulting improper protein folding, often significantly increases the amount of active protein. • However, E. coli grow very slow at low temperatures. • Recombinant strain of E. coli containing the chaperonin 60 gene and cochaperonin 10 gene can grow at 4 -10ºC. • However, this is the first step of expressing system for temperature sensitive proteins.

48. Overcoming Oxygen Limitation Protease-Deficient Host Strains • One possible way to stabilize foreign proteins produced in E. coli is to use host strains that are deficient in the production of proteolytic enzymes. • However, this is not as simple because E. coli has at least 25 different proteases, and only a few have been studied. • These enzymes are necessary for the degradation of abnormal or defective proteins. • Thus, decreasing protease activity caused cells to be debilitated.

49. Overcoming Oxygen Limitation Bacterial Hemoglobin • Some strains of Vitreoscilla bacterium normally live in oxygen-poor environments. • These bacteria synthesized a hemoglobin-like molecule that binds oxygen from environment and increases the level of available oxygen inside the cells. • When the gene was cloned and expressed in E. coli, the transformants displayed higher levels of synthesis of both cellular and recombinant proteins, higher level of cellular respiration, and higher level of ATP contents.

50. Overcoming Oxygen Limitation Bacterial Hemoglobin