L9 - Vectors for Eukaryotes PDF

Eukaryotic Vectors What are vectors? Vectors are DNA molecules that act as destination for GOI. Vectors act as a vehicle to ultimately transfer the gene into host Host are like biofactory! Vectors can be: Plasmid, Bacteriophage, Cosmid/ phagemid, Transposon, Virus, Artificial chromosome...

Eukaryotic Vectors What are vectors? Vectors are DNA molecules that act as destination for GOI. Vectors act as a vehicle to ultimately transfer the gene into host Host are like biofactory! Vectors can be: Plasmid, Bacteriophage, Cosmid/ phagemid, Transposon, Virus, Artificial chromosome 2 2 Vectors for Yeast and Fungi 2 µm Plasmid: in EK! 6kb in size Copy number 70-200 Rep1 and Rep2 are involved in replication of the plasmid FLP codes for a protein that can convert the A form of the plasmid to B form in which the gene order changes via intramolecular recombination D? Selectable marker: methtrexate/Cu tolerance; Auxotrophic host: leu- host turns Leu+ 3  Vectors based on 2 µm Plasmid Yeast Episomal Plasmid (YEp) 10.7 kb Selectable Leu gene Shuttle vector: can replicate in both E. coli and Yeast Can exist as both plasmid or episome in yeast: So can be problematic during DNA isolation and sequencing To solve this problem the selection is done in bacteria; then introduced in Yeast Other types of yeast cloning vectors  Other types of yeast cloning vectors Yeast integrative plasmid (YIps) Ura3 selectable marker Yip can only replicate when it is integrated Yeast Replicative plasmid (YRps) They can multiply independently Selectable marker Trp1  Transformation frequency of vectors YEp has high transformation frequency; 10,000 to 100,000 per µg YRps have 1,000 to 10,000 per µg tansformation frequency YIp has 1000 per µg ; only 1-10 without special measures Copy number of vectors YEp has 20-50 copy number YRp has 5-100 copy number YIp usually only 1 copy number! Then why use YIp? Once Yip is integrated it is very rarely lost (daughter cells receive it inevitably)!  Artificial Chromosomes Yeast Artificial Chromosome Artificial Chromosomes  Yeast Artificial Chromosome The fragment between two Tel region (BamHI sites) is discarded SnaBI is Blunt end cutter; so the products to be integrated also has to be Blunt ended Protoplast transformation Applications Stable during segregation Long inserts can be inserted (even 1mb!) Gene library Vectors for Plants 11 Vectors for Plants 12 Vectors for expression There are two general use of vectors 1. Cloning for mapping and sequencing (Store) 2. Expressing the gene 13 Expression optimization 1. Transcription initiation and termination 2. Promoter strength 3. Plasmid copy number 4. Plasmid stability 5. Host-cell physiology 6. Translation initiation sequences 7. Codon choice 8. mRNA structure 9. Toxicity and metabolic drain or load 14 Metabolic load Overexpression of unnatural protein have detrimental effect on bacterial growth! Solution: Use of regulatory/inducible promoter, such as, λ PL, T7, trc (tac) or BAD. The trc and tac promoters are hybrid promoters derived from the lac and trp promoters and induced by lactose and IPTG. Eg. pET vector with T7 promoter 15 Schematic diagram of T7 promoter control in the pET vector T7 RNA pT7 Target gene TT polymerase Expression Vector IPTG mRNA Lac repressor plac olac T7 polymerase gene 1 TT mRNA placI lacI TT  The phage T7 promoter site located up steam of target gene is under the control of lac promoter in the chromosome of the E. coli. Under un-induced condition the lac gene produce repressor molecule which bind at the promoter region of the T7 RNA polymerase gene1. As a result the T7 RNA polymerase is not produce, thus the target gene remain silent. In presence of IPTG, the lacI protein fails to bind at the upstream position of the gene1. As a result the T7 RNA polymerase gene is expressed. The T7 RNA polymerase then get attached to the T7 promoter site upstream of the target gene and results in expression of the target gene. 17 Translation optimization The mRNA produced during transcription needs to be effectively translated into protein. Interaction of the ribosome with the bases upstream from the initiation codon of the gene is important. In bacteria, this sequence is called ‘ribosome binding site’ or ‘Shine-Dalgarno (S-D) sequence’. 18  Characteristics of the translation initiation sequence The degree of similarity to the consensus sequence The spacing between S-D sequence and the initiation codon needs to be 5-10 bases, with 8 being the optimal. Decreasing the distance below 4 bp or increasing it beyond 14 bp can reduce translation by several orders of magnitude. Translation is affected by the seq. of the bases that follow the S-D site. The presence of 4A residues or 4 T residues in this position gave the highest translational efficiency. The composition of the triplet immediately preceding the AUG start codon affect the efficiency of translation. e.g. for translation of β- galatosidase mRNA, the most favorable combinations of the bases are UAU and CUU. If UUC, UCA or AGG replaced by UAU or CUU, the level of expression increase 20 folds. 19 Characteristics of the translation initiation sequence Codon choice and biasness: The codon biasness is (i) correlated with tRNA availability in the cell, and (ii) Non-random choices between pyrimidine-ending codons. The translation stoppage is also dependent on codon biasness. UAA is a favored in genes expressed at high levels, whereas UAG and UGA are used more frequently in genes expressed at low level. 20 Protein Purification & Expression vector Use of Tag: 1. facilitate protein purification using affinity chromatography, 2. determine presence of the cloned protein and 3. determine size of the cloned protein. Example: GST tag (glutathion-S-transferase), MBP tag (Maltose- binding protein), His tag (multiple: mostly 6X histidine residues). After purification the Tag site has to be removed by protease if purification is stringent. Use of inteins (proteins that can digest itself) is useful!! 21 Use of His Tag For Protein Purification Target Gene Target Gene N-terminal tag C-terminal tag = 6X His protein tag So, tag can be at N- or C-terminal of the foreign protein Protein Purification & Expression vector Use of Antibody or immuno-assay. Antibody can be generated against the protein of interest and can be used via affinity chromatography or immuno assay to detect and purify them 23 Protein Purification & Expression vector Inclusion Body: Protein of interest can aggregate to form inclusion body in improperly folded conformation Factors responsible for inclusion body formation 1. Temperature 2. pH 3. Media composition 24 Protein Purification & Expression vector Solution to inclusion body Modification in the vector- 1.The host cell is engineered to over-express a chaperon. 2. Making minor change in the amino acid sequence of the cloned gene. Example, cys to ser in ‘fibroblast growth factor’ minimize inclusion body formation 3. Proteins that are insoluble in their native form may be produced as soluble protein when fused with certain soluble protein as fusion, like MBP, NusA, GrpE and thioredoxin 4. Export the recombinant protein to the periplasmic space. Add signal sequence for that!! Vector to Promote Protein Export Secreted proteins must be transported to outer membrane or even outside the bacterial cell Signal sequence/peptide: A signal sequence has three domains: a positive charged amino-terminal region, a hydrophobic core consisting of 5-15 hydrophobic amino acids, and a leader peptidase cleavage site. The signal sequence is present at the N-terminus. Many signal sequences derived from naturally occurring secretory proteins (e.g. OmpA, OmpT, PelB, β-lacramase etc.). 26