Recombinant DNA Technology

1. Recombinant DNA Technology Dr. Hui LI Office : S408 Tel: 26538722 lihui80@szu.edu.cn

2. Isolation of target gene Selection and construction of vectors Ligation of target DNA and vector Transformation of target gene into receptor cell Screening for recombinant plasmids Expressing a cloned gene Process of cloning

3. Topic 5 Expression of recombinant gene (Eukaryotes)

4. Heterologous Protein Production In Eukaryotic Cells • Prokaryotic systems are generally cheaper, but… • Eukaryotic proteins produced in bacteria may be • Unstable or lack biological activity due to lack of posttranslational modifications or correct assembly • Possess unacceptable contaminants after purification

5. Posttranslational Modifications • Correct disulfide bond formation • Phosphorylation • Amino acid removal from initial polypeptide • O-linked or N-linked glycosylation • About 30% of eukaryotic proteins are glycosylated

6. Generalized Eukaryotic Cloning Vector Prokaryotic origin of replication, selectable marker Eukaryotic origin, selectable marker MCS with eukaryotic promoter and transcriptional terminator/polyadenylation signal

7. Yeast, animal & plant cells as receptor cells Foreign gene Promoter (eukaryotic or virus) MCS PolyA signal Terminator

8. I. Expression in yeasts 1. Expression vectors in yeast ① Cloning and amplification in E.coli(能在E.coli中克隆和扩增); Ori ② Selectable marker in E. coli(有大肠杆菌的选择标记); Ampr、Tetr。 ③Selectable marker in yeasts(有酵母的选择标记); Leu2+、His+、Ura3+、Trp1+; ④MCS(克隆位点)。

9. Dividing Saccharomyces cerevisiae (baker’s yeast) cells

10. （1） YIp(整合型载体) 由大肠杆菌质粒和酵母的DNA片断（选择标记）构成。 Ex: PYeleu10: ColE1(E.coli) Leu 2+ (Yeast)

11. Yip Vector for Chromosomal Insertion Use LEU2- host strain Vector provides functional gene Select on leucine-free media Double recombination with homologous host site inserts targeted portion of recombinant vector Linear DNA works Best

12. ①Low efficiency of transformation （1-10 transformant/ µgDNA）; ② Impossibility of self-replication in yeast; 载体上只有细菌的复制区，没有酵母的自主复制区。 ③ Integration in the chromosome of yeast 可与受体细胞的染色体DNA同源重组，随染色体一起复制。 ④Difficult to recover the plasmids from yeast

13. （2） YRp(复制型载体) 由大肠杆菌质粒和酵母的DNA片断(选择标记和酵母DNA自主复制顺序ARS）构成。 Marker(yeast) Plasmid (E.coli) ARS(yeast)

14. ARS ARS（automously replicating sequence）: 250bp ATTTTATATTTA Conservative region T G T

15. ① High efficiency of transformation （102-103transformant/ µg DNA); ②Easy to extract the plasmids from E.coli or yeast; ③ “Shuttle vector”（穿梭载体） 既能在大肠杆菌中复制，又能在酵母细胞中自主复制。 ④not very stable, risk of loss the foreign gene

16. （3） YCp (着丝粒质粒) YRp plasmid + Centromere region of yeast chromosome (酵母染色体的着丝粒区) YRp CEN ①Very stable; ② Exist as single copy (单拷贝存在); ③ Difficult to recover from yeast cell.

17. （4） YEp(附加体型载体) Plasmid (E.coli) + 2m plamid + Yeast DNA Plasmid (E.coli) 2m plasmid Selectable Marker (Yeast) Ex: pYF92: pBR322 2m Yeast his 3+ 2m plamid: 酿酒酵母的内源质粒，长度是2m 。含有自主复制起始区ori和STB序列（使质粒在供体中维持稳定）。

18. ① High efficiency of transformation （103-105transformant/ µg DNA); ② High copy number （25-100 copies/cell）。 ③ More stable than YRp

19. YEp24

20. 2. General process of yeast transformation and expression CaCl2、 PEG 酶去壁 Leu-酵母 原生质体 感受态 提取 转化 大肠杆菌 酵母载体 插入外源基因 Leu营养缺陷型 培养基筛选 整合到染色体上， 或独立在酵母细胞内 转化子克隆生长 外源基因产物 鉴定克隆 发酵表达 分离、纯化

21. Recombinant Proteins Successfully Produced in S. cerevisiae For a range of reasons as expressed previously each of these represented a better product than was obtainable using a prokaryotic expression system

22. Heterologous Protein Secretion by S. cerevisiae Gene must encode leader to pass through secretory system Also aids in correct disulfide bond formation, proteolytic cleavage of leader, etc. occur Over expression of PDI (protein disulfide isomerase )secretory enzyme also helps (up to 10X)

23. Why Other Yeast Species? S. cerevisiae sometimes hyperglycosylates proteins Proteins also sometimes retained in periplasmic space S. cerevisiae also produces ethanol at high cell densities Toxic to cells

24. P. Pastoris (嗜甲烷酵母) Highly efficient promoters available Tight control (e.g. AOX1 promoter) Produce up to 30% of total cell protein by wt. AOX1 (alcohol oxidase for methanol metabolism) promoter easily turned on by methanol Does not produce ethanol at high cell density Secretes few proteins, simplifying purification

25. Baculovirus (杆状病毒)Systems Infect invertebrates, including many insects Polyhedron (多角体蛋白) gene is not essential for life cycle (protects virus in environment) Commonly used with cultured insect eggs

26. Baculovirus Vector Vector has sequences for expression using polyhedron gene expression system Sequences also present for integration into baculovirus (AcMNPV) genome via recombination Prokaryotic sequences not shown 苜蓿银纹夜蛾细胞核型多角体病毒（Autographa california multiple nuclear polyhedrosis virus，AcMNPV）

27. Baculovirus Transfer Vector Done in cell culture Screening for recombinants tedious by PCR

28. Examples of Proteins Successfully Produced by Baculovirus Systems

29. E. coli Baculovirus Shuttle Vector - Bacmids Shuttle vectors allow ease of transfer between systems Genetic manipulations in one system, expression in another

30. Bacmid Construction (2) attR and attL are lambda sequences to give high efficiency and specific transposition

31. Bacmid Construction (3) Bacmid now operates efficiently in both E. coli and insect cells

32. Modifying the Insect Cell Host Some enzymes simply not present Genetic engineering of the host for proper expression Add missing glycosylation enzymes Add proteolytic processing enzymes

33. Mammalian Systems Sometimes insect cells simply don’t carry out proper/necessary glycosylations Other processing may also not occur Mammalian cell systems are more expensive by may be required for active product

34. Mammalian Expression Vector “I” is an intron that enhances expression Other signals similar to insect and prokaryotic vectors

35. Translation Control Elements K - Kozak Sequence (equiv. To rbs) S - for secretion signal peptide T – tag peptide for purification P – proteolytic cleavage sequence SC – stop codon for translation 3’UTR – proper sequences for efficient translation and mRNA stability (e.g. polyadenylation sequence)

36. Two Vector Expression System • Useful for proteins of two different polypeptides

37. Two Gene Expression Vector

38. Selectable Markers for Mammalian Systems Most commonly used to select for transformed cells (killing nonresistant ones) Can be used for increasing expression of heterologous proteins

39. Selective marker gene systems for mammalian cells

40. Use of Selectable Markers for Increasing Heterologous Protein Production in Mammalian Systems Methotrexate (MTX,甲氨蝶呤 ) inhibits dihydrofolate reductase (DHFR,二氢叶酸还原酶 ) DHFR- host cell with DHFR gene on cloning vector (i.e. linked to target gene) Gradually increase MTX concentration in culture Gene copy number of DHFR and linked target gene increase to compensate for inhibition of DHFR (more protein that is less active gives cell enough metabolic through put to survive)

41. Thanks, and see you next time!