Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

Hughes, Marcus D.; Zhang, Zhan-Ren; Sutherland, Andrew; Santos, Albert F.; Hine, Anna V.

doi:10.1093/nar/gni031

Cited by 15 publications

(7 citation statements)

References 37 publications

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“…Much of the existing probabilistic literature on saturation mutagenesis highlights the probability of full coverage as a criterion (1,5,11). Indeed, this criterion is used in practice (7). We have shown for the first time, to our knowledge, that using this criterion leads to needlessly large libraries.…”

Section: Discussionmentioning

confidence: 94%

“…To decrease the chances of introducing a premature stop codon, reduced codon sets are often used: NNB, NNS, and NNK codons (where N ϭ A/C/G/T, B ϭ C/G/T, S ϭ C/G, and K ϭ G/T) are popular choices that still encode all 20 amino acids, but the use of codon sets encoding fewer amino acids has been also advocated (9,15). More sophisticated randomization schemes, such as MAX (6,7), result in equal probabilities for all 20 amino acids (or for some predetermined subset thereof) without encoding stop codons. Either way, a large number of random variants, which together constitute a library, are produced and then screened in an attempt to discover a highly active variant among them.…”

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confidence: 99%

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When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

Nov

2012

Appl Environ Microbiol

103

107

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We developed new criteria for determining the library size in a saturation mutagenesis experiment. When the number of all possible distinct variants is large, any of the top-performing variants (e.g., any of the top three) is likely to meet the design requirements, so the probability that the library contains at least one of them is a sensible criterion for determining the library size. By using a criterion of this type, one may significantly reduce the library size and thus save costs and labor while minimally compromising the quality of the best variant discovered. We present the probabilistic tools underlying these criteria and use them to compare the efficiencies of four randomization schemes: NNN, which uses all 64 codons; NNB, which uses 48 codons; NNK, which uses 32 codons; and MAX, which assigns equal probabilities to each of the 20 amino acids. MAX was found to be the most efficient randomization scheme and NNN the least efficient. TopLib, a computer program for carrying out the related calculations, is available through a user-friendly Web server. Saturation mutagenesis (also called oligonucleotide-directed randomization) is a protein-engineering technique that has been used widely and successfully to improve protein properties such as catalytic activity, enantioselectivity, thermostability, and binding affinity (3,12,14,16). We use the term "activity" for the protein's property under optimization, but the methodology developed below is aimed at any desirable protein feature that may be influenced by mutation.In saturation mutagenesis, one or more positions along the protein sequence are identified as likely to accommodate beneficial mutations and are then randomized, i.e., the amino acids at these positions are replaced by random ones. The randomization originates at the DNA level, typically via degenerate primers containing a mixture of sequences at the chosen codons. To decrease the chances of introducing a premature stop codon, reduced codon sets are often used: NNB, NNS, and NNK codons (where N ϭ A/C/G/T, B ϭ C/G/T, S ϭ C/G, and K ϭ G/T) are popular choices that still encode all 20 amino acids, but the use of codon sets encoding fewer amino acids has been also advocated (9, 15). More sophisticated randomization schemes, such as MAX (6, 7), result in equal probabilities for all 20 amino acids (or for some predetermined subset thereof) without encoding stop codons. Either way, a large number of random variants, which together constitute a library, are produced and then screened in an attempt to discover a highly active variant among them. Clearly, the larger the library, the higher the probability of exploring more distinct variants. We shall use the term "variant space" to denote the set of all possible distinct variants in a given experiment; this space is determined by the number of positions randomized and the randomization scheme.The probabilistic literature on saturation mutagenesis (1, 5, 8, 11) focuses mainly on two mathematical quantities: the first is the expected percentage of variant spa...

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Section: Discussionmentioning

confidence: 94%

mentioning

confidence: 99%

When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

Nov

2012

Appl Environ Microbiol

103

107

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“…Recently Gendaq/Sangamo used this strategy in automated format to create large archives of two-finger modules of known binding specificities (Jamieson et al, 2003). Also various other bacterial and yeast selection and library screening systems were developed to select zinc fingers of novel DNA binding specificities (Cheng et al, 1997;Bartsevich and Juliano, 2000;Joung et al, 2000;Hurt et al, 2003;Hughes et al, 2005).…”

Section: Methods Of Generating Designer Zinc Fingersmentioning

confidence: 99%

Designer zinc-finger proteins and their applications

Papworth

Kolasinska

Minczuk

2006

Gene

130

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“…It may be possible to further enhance the biosensing sensitivity by employing lightly etched LPFG structures as we demonstrated before [10]. It will also be interesting to look at the advantage of this biosensor in further biomolecular interaction, for example, in developing a probe to discriminate between single nucleotide polymorphisms [14]. Probe none GCA CAG TCA GTC GCC NH 2 Target none GGC GAC TGA CTG TGC none …”

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confidence: 99%

Real-time detection of DNA interactions with long-period fiber-grating-based biosensor

et al. 2007

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Using an optical biosensor based on a dual-peak long-period fiber grating, we have demonstrated the detection of interactions between biomolecules in real time. Silanization of the grating surface was successfully realized for the covalent immobilization of probe DNA, which was subsequently hybridized with the complementary target DNA sequence. It is interesting to note that the DNA biosensor was reusable after being stripped off the hybridized target DNA from the grating surface, demonstrating a function of multiple usability. © However, some of these demonstrated optical biosensors have limitations for real-time hybridization studies and monitoring hybridization kinetics. Here we implement an optical biosensor based on a dualpeak long-period fiber grating (LPFG) for detecting biomolecular interactions at a silica-liquid interface with the advantages of high sensitivity, real-time monitoring, and reusability. To achieve high-sensitivity detection of the designed biomolecular interaction, the most sensitive LPFG structures should be used. It has been reported that the coupling condition of LPFGs with relatively short periods are close to the dispersion turning points, resulting in conjugate dual-peak cladding modes that are extremely sensitive to external perturbations [9,10]. Several dual-peak LPFGs with a relatively small period ͑ϳ160 m͒ were UV inscribed in H 2 -loaded SMF-28 fibers employing the point-by-point method and a frequency-doubled Ar laser. All the gratings were annealed at 80°C for 48 h to stabilize their optical properties. Figure 1 shows the spectral evolution of a 30 mm long LPFG with a period of 161 m during the UV inscription process. It is clear that with increasing UV exposure the conjugate dual peaks were increasing in strength and moving close to each other as the coupling condition approached the dispersion turning point.The ability of LPFGs to couple light from the fiber core mode to cladding modes allows optically detecting the change in refractive index at the grating surface. This thus provides an optical detection method to monitor biochemical and biomolecular interactions. Figure 2 shows the basic scheme of the functionalization of a LPFG as a DNA-array biosensor. Silanization is a process for modification of the glass surface to adsorb biomolecules. The LPFG surface is first silanized, followed by the activation of cross linkers to facilitate the immobilization of probe DNA and then to be used to monitor in situ the hybridization of targeted DNA. The DNA hybridization process modifies the refractive index of the LPFG surface, thus resulting in its spectral shift. By demodulating the spectral shift, the designed DNA hybridization can be monitored with high sensitivity.All the biochemical experiments were performed in a fume cupboard. To minimize the bend cross sensitivity, the dual-peak LPFG with a 161 m period was placed straight in a V-groove container on a Teflon plate, and all the chemicals and solvents were added and withdrawn from the container by carefully pipetting.Prior ...

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Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

Cited by 15 publications

References 37 publications

When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis

Designer zinc-finger proteins and their applications

Real-time detection of DNA interactions with long-period fiber-grating-based biosensor

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