Biol328 - B3212 Molecular Biotechnology

Transcription

1 Prof. Fahd M. Nasr Faculty of Sciences Lebanese University Beirut, Lebanon Biol328 - B3212 Molecular Biotechnology Lectures 12 and 13 1

2 Manipulation of Gene Expression in Prokaryotes Objectives Expression of cloned gene by a host organism For production For modifying host properties Generally requires a specialized vector Promoter, TT, RBS, etc. The gene plasmid borne or integrated Expression systems Many are expressed in E. coli Other systems: B. subtilis, yeast, plant, etc. 2

3 Antibiotics commonly used as selective agents Antibiotic Ampicillin Ap, Amp Hygromycin B HygB Kanamycin Km, Kan Description Inhibits cell wall formation Inactivated by b-lactamase Blocks translocation from A site to P site Inactivated by a phosphotransferase Binds to 30S, prevents translocation Inactivated by a phosphotransferase Antibiotics commonly used as selective agents Antibiotic Neomycin Nm, Neo Streptomycin Sm, Str Tetracycline Tc, Tet Description Binds to 30S, inhibits protein synthesis Inactivated by a phosphotransferase Blocks protein initiation complex formation, misreading during translation Inactivated by a phosphotransferase Prevents binding of aa-trna to 30S Resistance gene inner cell mb protein antibiotic out and block entry 3

4 5 super-families of the microbial efflux systems 4

5 Chemical structures of tetracyclines Enzymes used in DNA technology Enzyme Activity DNase I dsdna degradation E. coli exo III 3' 5' exo Klenow fragment Pol + 3' 5' exo (of Pol I) Mung bean-nuclease ssdna + RNA endo RNase H Degrades RNA in RNA/DNA Taq DNA pol Heat-stable Pol (T. aquaticus) b-agarase Digests agarose 5

6 Enzymes used in DNA technology And many others Alkaline phosphatase Nuclease BAL31 Reverse transcriptase T7 DNA polymerase S1 nuclease T4 polynucleotide kinase Etc. Plasmids (vectors) Self-replicating dscccdna molecules Extrachromosomal entities Range in size from 1 500kb Each has an origin of replication Cloning vectors (pbr322, puc19, etc.) Expression vectors (pkk233-2, YEp13, etc.) 6

7 Plasmids (vectors) 10 to 100 copies/cell (high copy number) 1 to 4 copies/cell (low-copy number) Selfish plasmids of the same incompatibility group cannot coexist Some microorg. contain 8 to 10 diff plasmids Origin of replication Specific to one host (narrow-host-range plasmid) Less specific (broad-host-range plasmid) 7

8 Plasmids (vectors) Carry information Genes coding for transfer system F plasmids Genes coding for resistance to antibiotics R plasmids Genes for utilization of unusual metabolites Degradative plasmids Genes coding for bacteriocins Col plasmids Genes turning the bacterium into a pathogen Virulence plasmids Genes with no apparent functions Cryptic plasmids Plasmids as Vectors Used as potential vectors to carry DNA Naturally occurring plasmids lack some good features A small size efficiency of transformation MCS (unique RE sites) One or more selectable genetic markers Genetically engineered (many generations) 8

9 Strong and regulatable promoters Strong promoter high affinity to RNA pol. Regulatable promoter precision Promoter of lac operon and other promoters from a range of organisms Strong promoter concerns High and constitutive expression Detrimental to host cell energy drain Impair cellular functions Plasmid can be lost cells without plasmid grow faster Plasmid instability prevents large-scale production Strong and regulatable promoter specific stage and specified duration 9

10 Regulation of lac Operon Expression Off Off Regulatable promoter Negative regulation Positive regulation Modified lac Promoters tac promoter Has (-)10 box of lac, (-)35 box of trp separated by 16 bp trc promoter Has (-)10 box of lac, (-)35 box of trp separated by 17 bp trc and tac are 3x stronger than trp and 10X stronger than lac 10

11 Turn on or off the lac promoter Repression (turning off) by laci repressor Induction (turning on) by Addition of lactose Isopropyl-b-D-thiogalactopyranoside (IPTG) IPTG lactose IPTG is a gratuitous inducer IPTG is not degraded by b-galatosidase 11

12 Phage Promoters Phage l p L Strong promoter Regulated by ci (available as ts mutant) Very strong promoter when active, can be virtually completely shut down by ci Phage Promoters Phage l p L At nonpermissive temperature ci inactivated and promoter turned on Heat is relatively cheap!!! 12

13 Phage T7 Promoter Increasing Protein Production Increase target gene copy number Often by increasing temperature of culture Properly done can reach 20% of total protein (vs. 2% for most common natural protein) 13

14 Effect of temperature on the plasmid copy number of three Expression Vectors pcp3 Expression Vector 14

15 Increasing protein production T4 DNA ligase gene was inserted in MCS At 42 C, 20% of the cellular protein is T4 DNA ligase Large Scale Systems Heat useful/cheap for small scale production (1-5 liters) Heat expensive and slow for large scale productions (up to 200 or more liters) IPTG also costly But tryptophan is cheap 15

16 Two plasmid-system Control over the l pl promoter by regulating ci with tryptophan 1 st plasmid: p trp promoter next ci lowcopy nb plasmid 2 nd plasmid: pertinent gene under pl promoter No tryptophan p trp is turned on pl is turned off Dual Plasmid System Growth on inexpensive medium Allows for high level expression Good repression, cheap/simple induction on demand 16

17 Dual plasmid system Cells are grown on molasses and casein hydrolysate (little tryptophan) Induction by addition of tryptone (enough free tryptophan) Experiments b-galactosidase gene 21% of cellular protein Citrate synthase gene 24% of cellular protein Other Prokaryotic Systems E. coli not always best Must use promoter most appropriate for strain/species Broad host range promoters useful but may not give maximum expression in all species (best will vary by species) 17

18 ß-Galactosidase activity expressed by gramnegative bacteria carrying a plasmid vector with the E. coli lacz gene and a heterologous promoter Some conclusions All promoters are active in all bacteria The most active in E. coli is tac The least active in E. coli are S1 and Nm Nm is the most active in all other bacteria 18

19 Lactococcus Species Lactococcus spp. commonly used in food production (cheese, yogurt, etc.) Cannot alter product palatability, appearance, etc. so additives to modulate expression limited Lactococcus lactis promoters synthesized with random nucleotides between 10 and 35 and tested for activity Functional promoters with 400-fold range of strength identified Can now customize level of expression Fusion Proteins Foreign proteins in cells often degraded Combining polypeptide with a host polypeptide can increase stability Created by ligating two open reading frames together in frame Cleavage/release of host polypeptide after purification is generally necessary Specific cleavage preferable 19

20 Cleavage of fusion proteins Fusion protein as final product is undesirable Biological function Unsuitable for clinical use Strategy: remove all unwanted aa sequence Join target protein to stabilizing partner with aa sq recognizable by specific protease Factor X a Protease Blood coagulation factor X a protease cleaves only at a specific amino acid sequence Gly Gly Ser Ile Glu Gly Arg Engineer X a site between two polypeptides which make up fusion protein 20

21 Fusion Protein Cloning Vector ompf promoter, initiation of translation and secretion signal lacz present as out of frame reporter Cloned segment in frame with secretion peptide and which puts lacz in frame makes fusion protein and ß- galactosidase activity The end 21