Screening Thermostability of Cytochrome P450 BM3
This specific example illustrates a thermostability screen based on the activity assay outlined in Chapter 15. 1. Ligate a cytochrome P450 BM-3 mutant library into the pCWOri 10 expression vector. 2. Pick colonies from the transformed library into 96-deep-well plates containing 300 yL of LB ampicillin per well. Include on each plate six wells containing the parent enzyme. Place lids over the plates and grow for 24 h in a humidified plate shaker 30 C, 250 rpm . 3. Use 20 yL of each LB culture to...
Biological and Chemical Materials Pvi
All chemicals were purchased from Sigma St. Louis, MO unless otherwise indicated. 1. Appropriate E. coli strain s . 2. Plasmid containing dehydrogenase gene under appropriate promoter. 3. LB Luria Broth and LB agar plates 14 . 4. TSS Transformation and Storage Solution 10 w v PEG6000, 5 dimethyl sulfoxide DMSO , 50 mM MgCl2 in LB, pH 6.5. 5. Liquid nitrogen or ethanol dry ice. 9. Proofreading polymerase such as Pfu Stratagene, La Jolla, CA . 10. dNTPs Boehringer-Mannheim, Indianapolis, IN . 11....
Introduction Tgs
Enzyme thermostability is a property of great importance in the era of designed biocatalysts. While enzymes are capable of catalyzing reactions with exquisite specificity and selectivity, they are often limited by insufficient stability. Improvements in enzyme activity through protein engineering often come at the cost of reduced stability. This is likely a result of both natural drift and a tradeoff that often exists between activity and stability for many single residue substitutions 1 ....
Preparation of TB Master Plates
1. Prepare a stock solution of 100 mM IPTG and sterilize by filtration see Note 4 . 2. Prepare a stock solution of 30 mg mL chloramphenicol in absolute ethanol see Note 4 . 3. Combine 47.6 g TB media, 15 g agar, 4 mL glycerol, and ddH2O to 1 L. Sterilize by autoclaving. 4. Cool the autoclaved solution to 50 C. Aseptically add 1 mL of IPTG stock solution, 1 mL of Cm stock solution and mix thoroughly. 5. In a sterile manner, pour the agar solution into sterile Petri dishes. Allow the agar to...
Image Acquisition Using a Flatbed Scanner
Use of a standard flatbed scanner is a simple and very inexpensive way to acquire images of colonies. This method is limited to colorimetric screens only, as wavelength filters cannot be used with a flatbed scanner. Flatbed scanners provide more than adequate image resolution for screening purposes. Often, the user can save time by imaging multiple plates at once on a flatbed scanner. 1. Place plates colony-face down onto the scanning surface. Any number of plates may be scanned at once up to...
Alexander V Tobias and John M Joern 1 Introduction
It is often possible to screen libraries of variant enzymes directly on agar plates. Colony-based solid-phase screening is an attractive option because of its relative ease and high throughput compared to liquid-phase screening in multi-well plates. As with any high throughput screening approach, a suitable colori-metric or fluorimetric assay must exist or be developed for the enzyme function in question. There are two major requirements that must be met for solid-phase screening to be...
Christopher R Otey and John M Joern 1 Introduction
The oxidation of aromatic compounds is important in producing chemical intermediates for the chemical and pharmaceutical industries 1,2 . Conventional aromatic oxidation reactions are prone to byproduct formation and often require heavy-metal catalysts, extremes of temperature and pressure, and explosive reagents 3 . In contrast, biocatalysts such as mono- and dioxygenases perform the same chemistry in water at ambient conditions, usually with higher regioselectivity than the analogous chemical...
Materials Nqw
9. Isopropy-P-D-thiogalactoside IPTG . 10. S-Aminolevulinic acid hydrochloride. 11. 90-mm nitrocellulose filter discs. 12. Nitroblue tetrazolium NBT . 13. Phenazine methosulfate PMS . 14. Trace elements 0.5 g MgCl2, 30.0 g FeCl3 6H2O, 1.0 g ZnCl2 4H20, 0.2 g CoCl2 6H2O, 1.0 g Na2MoO4 2H2O, 0.5 g CaCl2 2H2O, 1.0 g CuCl2 and 0.2 g Na3BO3 in 1 L HCl solution 90 v v distilled water concentrated HCl . 17. Color reagent 0.5 mg NBT and 0.03 mg PMS in 1 mL 0.1 M phosphate buffer, pH 8.0. 18. Deep-well...
References Hyw
1. Kinast, G. and Schedel, M. 1981 A four-stage synthesis of 1-deoxynojirimycin with a biotransformation as the central reaction step. Angew. Chem. 93, 799-800. 2. Fessner, W. D. 1998 Enzyme mediated C-C bond formation. Curr. Opin. Chem. Biol. 2, 85-97. 3. Neuberg, C. and Hirsch, J. 1921 ber ein Kohlenstoffketten kn pfendes Ferment. Biochem. Z. 115, 282-310. 4. Neuberg, C. and Ohle, H. 1922 Zur Kenntnis der Carboligase IV Mitteilung. Weitere Feststellungen ber die biosynthetischen...
Volker Sieber 1 Introduction
The low solubility of a protein is one of the most frequent impediments for its structural and functional analysis and, on a more practical aspect, for its application as an industrial enzyme. The reason for low solubility can lie in low conformational stability 1 , in a high number of surface-exposed hydrophobic amino acids 2 or in certain structural features, such as membrane binding regions 3 . By changing the amino acid sequence of these proteins, their solubility can be significantly...
Thermostability Assay General Protocol
1. Prepare 96-well assay plates for the addition of enzyme lysate. Two assay plates must be prepared for each plate of lysates to be screened one for initial activity and one for residual activity . It is useful to use multi-channel pipets or a pipetting robot for preparing the plates. For example, as described in Subheading 3.4., 96-well plates are prepared by adding buffer and dimethyl sulfoxide DMSO containing substrate to each well. 2. Using a pipetting robot, transfer an appropriate volume...
with Gibbs Reagent to Dioxygenases
Initial oxidation of aromatic compounds by dioxygenase results in arene cis-dihydrodiols, as shown in Fig. 1D. These compounds are difficult to detect in the background of a cell extract or supernatant, but are easily converted to detectable phenolic compounds using one of two methods. One is to convert the cis-dihydrodiol to a catechol by coexpressing the cis-dihydrodiol dehydro-genase that resides on the dioxygenase cistron. The dehydrogenase from the toluene dioxygenase cistron of...
Notes Rrl
1. When selecting or designing primers for DNA amplification it is not necessary that the primers anneal within the gene of interest. During successive rounds of biopanning, DNA fragments that encode peptide sequences that do not bind the target protein will be selected against and removed from the library 5 . 2. If QIAquick spin columns are not available, DNA purification can be performed using a low-melt agarose gel 10 . 3. The nebulizer technique used here was modified from a protocol that...
Edgardo T Farinas 1 Introduction
The United States consumes approximately 16-17 million barrels of crude oil per day, and the majority is used for electricity, heating, and transportation fuel 1 . The main constituents of crude oil are linear aliphatics. Clearly, the selective hydroxylation of alkanes to more valuable products would have a worldwide economic impact. Unfortunately, the chemical methods for the oxidation of alkanes are energy intensive, and the reagents and byproducts are hazardous to the environment 2 . In...
Expression and Complementation
When verifying expression of a protein where an antibody is available, Western blots are preferable 11 . Since no commercial antibody is available for E. coli NrdB, verification of expression can be confirmed via complementation of an E. coli strain that is deficient in NrdB expression and displays hypersensitivity to hydroxyurea see Note 7 . A similar functional complementation may be required for verification of other genes. Complemenation of sensitivity of E. coli strain KK446 to hydroxyurea...
ABTS Assay for Screening Laccase Activity
Standard laccase activity is determined by oxidation of ABTS at room temperature see Note 1, Fig. 1A . 1. Dilute the laccase sample with B amp R-buffer, pH 6.0, or enzyme diluent, if necessary see Note 2 . 2. Add 20 pL of the laccase sample into microtiter plate wells. 3. Add 180 pL of ABTS solution to every well. Mix sample and reagent thoroughly using the plate reader or a pipettor see Note 3 . 4. Follow the oxidation of ABTS by measuring the absorbance increase at 418 nm see Note 4 . One...
Expression Vector pCWOri HRP
The expression vector used for the expression of HRP-C is based on the pCWOri vector 7,8 . This system is inducible by IPTG, but leaky protein expression is also observed in the absence of IPTG. Taking advantage of the unique restriction sites present in pCWOri , the pCWOri HRP vector was constructed by cloning a cDNA copy of the HRP-C gene into the pCWOri vector using standard molecular biology techniques 6 . Two copies of a pelB sequence were placed directly upstream of the HRP-C coding...
Gary W Rudgers and Timothy Palzkill 1 Introduction
Protein-protein interactions are involved in most biological processes and are an important target for drug design. Over the past decade, there has been an increased interest in the design of small molecules that mimic functional epitopes in protein-protein interactions. However, the design of small molecules that disrupt protein-protein interactions remains a considerable challenge 1,2 . Progress has been achieved towards minimizing proteins into significantly smaller polypeptides that retain...
Materials Bnx
1. Escherichia coli BL21-Gold DE3 competent cells Stratagene, La Jolla, CA . 2. pET21b Novagen, Madison, WI derived plasmid library carrying mutant, wild-type or truncated pyrococcal a-amylase genes. 5. Ampicillin used for both liquid and agar cultivation at a concentration of 200 g mL . 6. 96-well flat-bottom microtiter plates Rainin, Emeryville, CA with a 3-mm glass ball in each well. 7. 20 x 20 cm 'QTray' Genetix, New Milton, UK . 8. Colony picking equipment sterile toothpicks or colony...
Thomas Bulter Volker Sieber and Miguel Alcalde 1 Introduction
Functional gene expression is a prerequisite for directed evolution with Escherichia coli E. coli , the preferred host organism. However, bacterial expression of eukaryotic genes can be impossible, or produce proteins with substantially altered properties, because of differences between bacterial and native expression systems 1 . Different codon usage, missing chaperones, and posttranslational modifications like disulfide bridges or glycosylation can all cause low expression levels, misfolding,...
Oriana Salazar and Lianhong Sun 1 Introduction
Directed evolution by sequential cycles of random mutagenesis and screening has proven to be useful for producing new or improved enzyme properties 1,2 . The first step is construction of a mutant library, usually accomplished by random point mutagenesis with error-prone PCR 3 or by DNA shuffling recombination 4,5 . The second, and most critical step is finding the desired mutants by screening or selection of the libraries. Screens for enzyme activity usually operate via detection of optical...
The T7 RNA Polymerase Autogene
A T7 RNA polymerase autogene is a construct where the gene for T7 RNA polymerase is cloned downstream of its own cognate promoter. Expression systems based on T7 RNA polymerase are very useful because the enzyme is both highly active T7 RNA polymerase is five times more efficient than E. coli RNA polymerase in elongating transcripts and highly specific for its own promoter. Hence, T7 RNA polymerase can be used to overexpress particular proteins without expressing host cell genes or interfering...
Manel Camps and Lawrence A Loeb 1 Introduction
The E. coli JS200 strain carries a temperature-sensitive allele of DNA polymerase I that renders this strain conditional lethal. Growth under restrictive conditions is restored by small amounts of DNA polymerase activity. Even mutants with greatly reduced 1-10 of wild-type catalytic activity or distantly-related polymerases of bacterial, eukaryotic, or viral origin effectively complement JS200 cells. The versatility of this complementation system makes it advantageous for selection of active...
Jessica L Sneeden and Lawrence A Loeb 1 Introduction
Genetic selection provides a powerful tool for the study of cellular processes. It is particularly useful in analyzing protein sequence constraints when used in conjunction with directed molecular evolution. Our lab has used this approach to analyze the function of enzymes involved in DNA metabolism, to study the mutability of protein domains, and to generate mutant proteins possessing properties different from those selected by natural evolution 1-4 . To illustrate the concept, this chapter...