scholarly journals Random-priming in vitro recombination: An effective tool for directed evolution

1998 ◽  
Vol 26 (2) ◽  
pp. 681-683 ◽  
Author(s):  
Z. Shao ◽  
H. Zhao ◽  
L. Giver ◽  
F. H. Arnold
Keyword(s):  
Directed Evolution ◽  
In Vitro Recombination ◽  
Random Priming

scholarly journals OP0039 ALPN-303, AN ENHANCED, POTENT DUAL BAFF/APRIL ANTAGONIST ENGINEERED BY DIRECTED EVOLUTION FOR THE TREATMENT OF SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND OTHER B CELL-RELATED AUTOIMMUNE DISEASES

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 21.2-21
Author(s):  
S. R. Dillon ◽  
L. S. Evans ◽  
K. E. Lewis ◽  
J. Yang ◽  
M. W. Rixon ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Directed Evolution ◽  
Human Lymphocyte ◽  
Lupus Erythematosus ◽  
Plasma Cells ◽  
Systemic Lupus ◽  
Cynomolgus Monkeys

Background:BAFF and APRIL are TNF superfamily members that form homo- and heteromultimers that bind TACI and BCMA on B cells; BAFF also binds BAFF-R. BAFF and APRIL support B cell development, differentiation, and survival, particularly for plasmablasts and plasma cells, and play critical roles in the pathogenesis of B cell-related autoimmune diseases. In nonclinical models, inhibition of either BAFF or APRIL alone mediates relatively modest effects, whereas their co-neutralization dramatically reduces B cell function, including antibody production. Fc fusions of wild-type (WT) TACI (e.g. atacicept and telitacicept) target both BAFF and APRIL and have demonstrated promising clinical potential in e.g. systemic lupus erythematosus (SLE) and IgA nephropathy but have not yet clearly exhibited long-term and/or complete disease remissions.Objectives:To generate a dual BAFF/APRIL antagonist with inhibitory activity superior to WT TACI and BCMA and with the potential to improve clinical outcomes in B cell-mediated diseases.Methods:Our directed evolution platform was used to identify a potent variant TNFR domain (vTD) of TACI that exhibits significantly enhanced affinity for BAFF and APRIL as compared to WT TACI; this TACI vTD domain was fused to a human IgG Fc to generate the therapeutic candidate ALPN-303. ALPN-303 was evaluated for functional activity in: 1) human lymphocyte assays, 2) the NOD.Aec1Aec2 spontaneous model of Sjogren’s syndrome (SjS), 3) the bm12-induced mouse model of lupus, 4) the (NZB/NZW)F1 spontaneous model of lupus, and 5) preclinical rodent and cynomolgus monkey pharmacokinetic/pharmacodynamic studies.Results:ALPN-303 inhibited BAFF- and APRIL-mediated signaling in vitro in human lymphocyte assays, with significantly lower IC50 values than WT TACI-Fc and belimumab comparators. In all mouse models evaluated, administration of ALPN-303 rapidly and significantly reduced key lymphocyte subsets including plasma cells, germinal center B cells, and follicular T helper cells. ALPN-303 significantly reduced autoantibodies and sialadenitis in the spontaneous SjS model, inhibited glomerular IgG deposition in the bm12-induced model of lupus, and potently suppressed anti-dsDNA autoAbs, blood urea nitrogen levels, proteinuria, sialadenitis, kidney lesions, and renal immune complex deposition in the NZB/W lupus model. As compared to WT TACI-Fc, ALPN-303 exhibited higher serum exposure and significantly and persistently decreased titers of serum IgM, IgG, and IgA antibodies in mice and cynomolgus monkeys (Figure 1).Figure 1.ALPN-303 induces more potent suppression, as compared to WT TACI-Fc, of serum immunoglobulins following a single 9 mg/kg IV infusion (on Day 0; arrows) in female cynomolgus monkeys.Conclusion:ALPN-303 is a potent BAFF/APRIL antagonist derived from our directed evolution platform that consistently demonstrates encouraging immunomodulatory activity and efficacy in vitro and in vivo, superior in preclinical studies to anti-BAFF antibody and WT TACI-Fc. This novel Fc fusion molecule demonstrates favorable preliminary developability characteristics, including higher serum exposures and more potent immunosuppressive activities, which may enable lower clinical doses and/or longer dosing intervals than WT TACI-Fc therapeutics. ALPN-303 may thus be an attractive development candidate for the treatment of multiple autoimmune and inflammatory diseases, particularly B cell-related diseases such as SLE, SjS, and other connective tissue diseases. Preclinical development is underway to enable the initiation of clinical trials later this year.Disclosure of Interests:Stacey R. Dillon Shareholder of: Alpine Immune Sciences, Bristol Myers Squibb, Employee of: Alpine Immune Sciences, Bristol Myers Squibb, Lawrence S. Evans Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Katherine E. Lewis Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Jing Yang Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Mark W. Rixon Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Joe Kuijper Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Dan Demonte Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Janhavi Bhandari Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Steve Levin Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Kayla Kleist Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Sherri Mudri Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Susan Bort Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Daniel Ardourel Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Michelle A. Seaberg Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Rachel Wang Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Chelsea Gudgeon Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Russell Sanderson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Martin F. Wolfson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Jan Hillson Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences, Stanford L. Peng Shareholder of: Alpine Immune Sciences, Employee of: Alpine Immune Sciences

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Characterization of an ATP-dependent DNA strand transferase from human cells.

1987 ◽  
Vol 7 (9) ◽  
pp. 3124-3130 ◽  
Author(s):  
D Ganea ◽  
P Moore ◽  
L Chekuri ◽  
R Kucherlapati
Keyword(s):  
Dna Sequences ◽  
Atp Hydrolysis ◽  
Reca Protein ◽  
Strand Exchange ◽  
Cell Nuclei ◽  
In Vitro Recombination ◽  
Dna Strand ◽  
Exchange Activity

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.

Directed evolution approaches for optogenetic tool development

2021 ◽  
Author(s):  
Jaewan Jang ◽  
G. Andrew Woolley
Keyword(s):  
Directed Evolution ◽  
Recent Progress ◽  
Tool Development ◽  
Future Directions ◽  
Molecular Events

Photoswitchable proteins enable specific molecular events occurring in complex biological settings to be probed in a rapid and reversible fashion. Recent progress in the development of photoswitchable proteins as components of optogenetic tools has been greatly facilitated by directed evolution approaches in vitro, in bacteria, or in yeast. We review these developments and suggest future directions for this rapidly advancing field.

scholarly journals Creating Hybrid Genes by Homologous Recombination

2000 ◽  
Vol 16 (1-2) ◽  
pp. 3-13 ◽  
Author(s):  
Peter L. Wang
Keyword(s):  
Directed Evolution ◽  
High Efficiency ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Strand Breaks ◽  
E Coli ◽  
Hybrid Genes ◽  
Distinct Features

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution.In vitrorecombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties.In vivorecombination in bothE. coliand yeast is greatly enhanced by double-strand breaks; forE. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombinationIn vivohave distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recentlyin vivorecombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

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