1. 蛋白質工程於生物技術 之應用與發展 Protein Engineering Applications and Progress in Biotechnology

2. Structure Structural support: collagen ( 膠原蛋白 ) Transport Hemoglobin ( 血紅素 ): transports oxygen from the lungs to cells Storage Myoglobin ( 肌紅蛋白 ): stores oxygen in muscles Hormones Insulin ( 胰島素 ): protein hormone controls blood glucose level Enzymes ( 酵素 ) Alcohol dehydrogenase ( 醇脫氫酶 ) that breaks down alcohols Protein Functions

3. Proteins Protein is synthesized via the following processes: Each protein is made from the 20 standard amino acids and fold into a specific 3-D structure. DNA RNAProtein GeneFunction

4. Proteins

5. Bioinformatics Target identification and cloning Protein expression test Protein purification and production Applications Principle in Protein Biotechnology

6. Bioinformatics: exploitation of the genome Bioinformatics is central to the interpretation and exploitation of the wealth of biological data generated in genome projects Exploitation of the wealth of information from the genomes of human and model organism is critical to biotechnology research Applications:  sequence analysis  Search for conserved domains  protein structure analysis

7. E. coli genome

8. Data sources NCBI: www.ncbi.nlm.nih.gov National Center for Biotechnology Information  GenBank Files and a relational database with web access  Extensively integrated sequence information  Structure and alignments

15. Bioinformatics Target identification and cloning Protein expression test Protein purification and production Applications Principle in Protein Biotechnology

16. Cloning and expression of target gene: + Recombinant Vector Gene of Interest Expression Vector Expression of Fusion Protein

17. SDS-PAGE electrophoresis Protein separation: check purity and MW

18. I : cell extract of induction N : cell extract of no-induction S : solubility 116 66 45 I N S I N S I N S I N S I N S I N S 1 (32.2KDa) 2 (51KDa) 3 (92.7KDa) 4 (33.4KDa) 6 (47KDa) 35 25 5* (73.3KDa) Protein Expression Test

19. Bioinformatics Target identification and cloning Protein expression test Protein purification and production Applications Principle in Protein Biotechnology

20. Column Chromatography Protein separation and purification

21. Ion Exchange

22. Gel Filtration

23. Affinity Chromatography

24. SDS PAGE of Purification 1.Total proteins 2.High Salt 3.Ion exchange 4.Gel-filtration 5.Affinity

25. Bioinformatics Target identification and cloning Protein expression test Protein purification and production Applications Principle in Protein Biotechnology

26. Applications  Functional Studies  Enzymatic Assays  Protein-protein interactions  Protein Ligand Interactions  Structural Studies Protein Crystallography & NMR Structure Determination  Target Proteins for Rational Drug Design  Therapeutic Proteins – Preclinical Studies

27. Protein Engineering Ulmer, K. M. (1983) “ Protein Engineering ”, Science, 219: 666-671. Deliberate design and production of proteins with novel or altered structure and properties, that are not found in natural proteins.

28. Why Engineering Proteins ? To study protein structure and function Applications in industry (enzymes) and medicine (drugs) -- New and improved proteins are always wanted. Example: Extremophilic proteins have been found in nature (temperatures, salt concentrations, pH values) could be useful.

29. Factors that make the proteins from thermophilic microorganisms more stable Thermophilic enzymes usually exhibit optimal activity at a higher temperature than the mesophilic enzymes. No general rules revealed (the best way is to measure experimentally).

30. General features of the thermostable enzymes:  Increase of compactness and better packing  Increase in electrostatic interactions (with the formation of additional ion pairs)  Additional H-bonds  Additional disulphide bridges  Increasing internal hydrophobic interactions.

31. Methods for protein engineering Chemical or Genetic?

32. Chemical modifications -- in vitro engineering One of the first way and a re-emerged method for altering protein properties. Polyethylene glycol (PEG) modification of protein surface amino groups reduces immunoreactivity, prolongs clearance times, improve biostability, increase the solubility and activity of enzymes in organic solvents. DeSantis, G. and Jones, J.B. (1999) “Chemical modification of enzymes for enhanced functionality”, Current Opinion in Biotechnology, 10(4):324-330

33. Genetic modification -- in vivo engineering Genes (DNA) encoding proteins are mutagenized Irrational engineering (random mutagenesis) and rational engineering (site-directed mutagenesis) DNA RNAProtein

34. PCR: Polymerase Chain Reaction

37. Proteins with new properties can be obtained by random mutagenesis DNA in cells are randomly mutated: chemical mutagens (e.g., hydroxyamine, sodium bisulfite), enzymatic synthesis, mutagenic strains of bacteria (with deficient repairing systems). Can be applied when the current theories are inadequate to predict which structural changes will give improvement on certain property. Appropriate procedures for screening or selecting for desired properties are needed

38. Protein could be made to evolve in vitro DNA shuffling: in vitro homologous recombination and in vitro protein evolution. Random mutagenesis by error-prone PCR(with excess of one dNTP) to generate diversity of templates (naturally occurring homologous genes can also be used). Selection under increasing selective pressures (antibiotics, pH, organic solvent). Combination with High-throughput screening

39. DNA shuffling: a method for in vitro homologous recombination between mutant genes. x x x x x x x x DNAse I In shuffling, the products are degraded to random small fragments with DNAse I.

40. x x x x Then full-length sequences are re-assembled by enzymatic DNA synthesis Denature, reanneal, enzymatic DNA synthesis Some products consist of full-length sequences containing several mutations. Recombinants with improved functions are selected

41. Example: Development of novel  -lactamases with increased activity towards certain substrates.  The  -lactam antibiotic cefotaxime is poorly hydrolysed by TEM  -lactamase.  Mutant  -lactamase genes were shuffled to produce new recombinant genes.  The 1st round of shuffling yielded enzymes conferring resistance to 0.32 - 0.64  g/ml cefotaxime.  Shuffling of these genes yielded enzymes resistant to 5 to 10  g/ml.

42.  A 3rd round of shuffling yielded enzymes resistant to 640  g/ml cefotaxime.  Sequencing of cefotaxime R genes revealed several point mutations.  Six AA replacements were found to confer the high resistance phenotype. ALA42 GLY92 GLY104 MET182 GLY238 ARG241 GLY SER LYS THR SER HIS Nature. 1994;370(6488):389-91

43. One more example: Improved GFP was generated by DNA shuffling Started with a synthetic GFP gene Performed recursive cycles of DNA shuffling. Screened for the brightest E.coli colonies (using UV light). After 3 cycles of DNA shuffling, a mutant was obtained with 45-fold greater fluorescence. Nature Biotechnology, 1996, 14:315-319

44. Comparison of the fluorescence of different GFP constructs in whole E. coli cells No GFPClontechwt cycle 2 mutant cycle 3 mutant

45. High-throughput screening

46. Proteins can be engineered using site- directed mutagenesis Nucleotide residues to be mutated need to be first identified: by using information from 3-D structure, homology comparison, and etc. Nucleotide and Amino acid residues can be replaced, deleted or added.

47. PCR technology can be used to carry out site-specific mutagenesis * * a b c d a *b *c d * * A) B) C)

48. Applications in Engineering Proteins Engineering of industrial enzymes Re-design of substrate specificity Folding and stability Custom-designed proteins Chimeric protein constructions

49. Novel proteins may be generated by de novo design Computer Modeling Gene construction Protein production characterization De novo design of proteins: The attempt to choose an amino acid sequence that is unrelated to any natural sequence, but will fold into a desired 3-D structure with desired properties.

50. Interesting Examples?

51. A fluorescent protein that changes color with time was generated from the red fluorescent protein (RFP) "Fluorescent Timer": Protein That Changes Color with Time Science, 24 November, 2000, Vol.290:1585-1599.

52. The RFP gene was mutated by error-prone PCR. Mutants exhibiting a green intermediate fluorescence were screened visually by using a fluorescent microscope. Mutants with various properties, such as faster maturation, double emission (green and red), or exclusive green fluorescence were isolated. One mutant protein (E5) changes color over time: initially bright green, then to yellow, orange, and finally red. E5 has two replacements: V105A and S197T.

53. Time course of green and red fluorescence in E5 RFP (in vitro analysis).

54. E5 used as a fluorescent clock: heat shock- regulated expression of the E5 mutant RFP in C. elegans.