Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli Speaker: Yu-Chen Ku Professor: Ching-Tsan Huang, Ph. D. Source: Biochemical Journal (2007) 402,429-437 Date: December 4th, 2007 1 DCase (N-carbamoyl-D-amino acid amidohydrolase) β-lactam antibiotics: Inhibit the formation of peptidoglycan cross-links in the bacterial cell wall eg. penicillins, cephalosporins penicillin 2
Semi-synthesis of β-lactam antibiotics Semi-synthesis/partial chemical synthesis: uses compounds isolated from natural sources as starting materials. N-carbamoyl-D-p-hydroxyphenylglycine DCase D-p-hydroxyphenylglycine (D-HPG) 3 The poor solubility/misfolding of recombinant proteins Accumulate in inclusion bodies 4
Co-expression of foldase or chaperones Fusion of tags that contain a highly soluble polypeptide Not Good Enough! 5 Random mutation 34 18 18 30 Sequencing Site-directed mutagenesis 30 34 Characteristics analysis Structural analysis Solubility analysis 6
Random Mutation 7 Random Mutation 8
MgCl 2 (mm) Units of Taq polymerase dntps (mm) MnCl 2 (mm) Standard PCR 1.5 2.5 0.2 0 7 5 varying 0.075-0.15 Taq polymerase: low fidelity MgCl 2 (Magnesium chloride): stabilize noncomplementary pairs High concentration of Taq polymerase: promote chain extension beyond mismatch pairs MnCl 2 (Manganese chloride): mutagenic to polymerase 9 www.nyu.edu/classes/ytchang/book/c002.html 10
Random Mutation 11 β-gal http://www.langara.bc.ca/biology/mario/biol2315notes/biol2315chap11.html 12
Inducer: IPTG (isopropyl-β-d-thiogalactoside) IPTG allolactose X-gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside): substrate of β-gal The product of X-gal after β-gal digestion turned blue! 13 β-galactosidase (β-gal): divided into α- andϖ-fragments P: DCase Fusion protein: fuse the α-fragment to the C terminus of DCase Nature Biotechnology 19, 131-136 (2001) 14
Random Mutation 15 One unit of enzyme activity: the amount of enzyme that catalyses the formation of product at the rate of 1 μmol/min Native-PAGE Gel contains X-gal 16
Random Mutation 17 18: Alanine (A) to Threonine (T) 30: Tyrosine (Y) to Asparagine (N) 34: Lysine (K) to Glutamic acid (E) Ala 18, Tyr 30 and Lys 34 are involved in solubility. 18
Random Mutation Create double mutant (A18T/Y30N, A18T/K34E, Y30N/K34E) and triple mutant DCase-M3 (A18T/Y30N/K34E) 19 50% 35% 35% SDS-PAGE Coomassie Blue Single mutant Double mutant Triple mutant (DCase-M3) tot: total protein ppt: precipitant fraction sup: supernatant fraction 80% Cumulative effect! 20
Hydrophilicity affects solubility! Hydrophilicity of amino acids from weak to strong: Lue (L), Ala (A), Gly (G), Thr (T), Tyr (Y), His (H), Glu (E), Asp (D), Asn (N), Lys (K), Arg (R) D T : the density of total protein D S : the density of soluble protein S F : D S /D T S F : (S F of mutant S F of WT)/S F of WT The bigger S F, the higher solubility! 21 Random Mutation 22
V max = k cat *[E] K m : the Michaelis-Menten constant The substrate concentration needed to achieve a half-maximum enzyme velocity. Represents the affinity to its substrate k cat : the turnover number The maximum number of enzymatic reactions catalyzed per second. V max divided by the total enzyme concentration. 23 The fluorescence of tryptophan Urea: break H-bond G H2O : in the absence of urea C m : urea concentration at the midpoint of the transition m: G H2O /C m Mutations do not change the characteristics of DCase! 24
Random Mutation 25 The active site Mutations might not affect the function of DCase, consistent with results of the kinetic characteristics 26
Arginine (R): positive charge Lysine (K): positive charge Glutamic acid (E): negative charge Aspartic acid (D): negative charge Charge affects solubility! 27 Ala 18, Tyr 30 and Lys 34 of DCase are involved in its solubility. Mutations in the 18, 30 and 34 positions do not change the characteristics of DCase. Increasing the hydrophilicity or the negative charge of amino acids on the key positions improve the protein solubility. 28
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