scholarly journals EASINESS: E. coli Assisted Speedy affINity-maturation Evolution SyStem

2021 ◽  
Vol 12 ◽  
Author(s):  
Hai-nan Zhang ◽  
Jun-biao Xue ◽  
Aru Ze-ling Wang ◽  
He-wei Jiang ◽  
Siva Bhararth Merugu ◽  
...  
Keyword(s):  
Basic Research ◽  
Affinity Maturation ◽  
Evolution System ◽  
Antibody Affinity ◽  
Dna Polymerase I ◽  
E Coli ◽  
Protein Protein Interaction ◽  
Pol I ◽  
Mutation System ◽  
Polymerase I

Antibodies are one of the most important groups of biomolecules for both clinical and basic research and have been developed as potential therapeutics. Affinity is the key feature for biological activity and clinical efficacy of an antibody, especially of therapeutic antibodies, and thus antibody affinity improvement is indispensable and still remains challenging. To address this issue, we developed the E. coliAssisted Speed affINity-maturation Evolution SyStem (EASINESS) for continuous directed evolution of Ag–Ab interactions. Two key components of EASINESS include a mutation system modified from error-prone DNA polymerase I (Pol I) that selectively mutates ColE1 plasmids in E. coli and a protein–protein interaction selection system from mDHFR split fragments. We designed a GCN4 variant which barely forms a homodimer, and during a single round of evolution, we reversed the homodimer formation activity from the GCN4 variant to verify the feasibility of EASINESS. We then selected a potential therapeutic antibody 18A4Hu and improved the affinity of the antibody (18A4Hu) to its target (ARG2) 12-fold in 7 days while requiring very limited hands-on time. Remarkably, these variants of 18A4Hu revealed a significant improved ability to inhibit melanoma pulmonary metastasis in a mouse model. These results indicate EASINESS could be as an attractive choice for antibody affinity maturation.

Polymerase Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Herpes Simplex Virus Type 1 DNA Polymerase: Their Predicted Three-Dimensional Structures and some Putative Functions in Comparison with E. Coli DNA Polymerase I. A Critical Survey

1992 ◽  
Vol 3 (4) ◽  
pp. 223-241 ◽  
Author(s):  
B. Lindborg
Keyword(s):  
Dna Polymerase ◽  
Three Dimensional ◽  
Dna Polymerase I ◽  
E Coli ◽  
Polymerase Domain ◽  
Pol I ◽  
Hiv Rt ◽  
Polymerase I ◽  
Β Sheet

Hypothetical three-dimensional models for the entire polymerase domain of HIV-1 reverse transcriptase (HIV RT) and conserved regions of HSV-1 DNA polymerase (HSV pol) were created, primarily from literature data on mutations and principles of protein structure, and compared with those of E. coli DNA polymerase I (E. coli pol I). The corresponding parts, performing similar functions, were found to be analogous, not homologous, in structure with different β topologies and sequential arrangement. The polymerase domain of HSV pol is shown to form an anti-parallel β-sheet with α-helices, but with a topology different from that of the Klenow fragment of E. coli pol I. The main part of the polymerase domain of HIV RT is made up of a basically parallel β-sheet and α-helices with a topology similar to the nucleotide-binding p21 ras proteins. The putative functions of some conserved or invariant amino acids in the three polymerase families are discussed.

Site-specific modification of E. coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Biochemistry ◽  
1984 ◽  
Vol 23 (9) ◽  
pp. 2073-2078 ◽  
Author(s):  
Anup K. Hazra ◽  
Sevilla Detera-Wadleigh ◽  
Samuel H. Wilson
Keyword(s):  
Dna Polymerase ◽  
Large Fragment ◽  
Dna Polymerase I ◽  
Site Specific ◽  
E Coli ◽  
Specific Modification ◽  
Polymerase I

scholarly journals Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 213-220 ◽  
Author(s):  
L J Reha-Krantz
Keyword(s):  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Rnase H ◽  
Ribonuclease H ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Dna Polymerase I ◽  
E Coli ◽  
Intragenic Complementation ◽  
Polymerase I

Abstract Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.

How E. coli DNA polymerase I (klenow fragment) distinguishes between deoxy- and dideoxynucleotides

1998 ◽  
Vol 278 (1) ◽  
pp. 147-165 ◽  
Author(s):  
Mekbib Astatke ◽  
Nigel D.F Grindley ◽  
Catherine M Joyce
Keyword(s):  
Dna Polymerase ◽  
Klenow Fragment ◽  
Dna Polymerase I ◽  
E Coli ◽  
Polymerase I

scholarly journals DNA Polymerase I Is Not Required for Replication of Linear Chromosomes in Streptomyces

2007 ◽  
Vol 190 (2) ◽  
pp. 755-758 ◽  
Author(s):  
Tzu-Wen Huang ◽  
Carton W. Chen
Keyword(s):  
Dna Polymerase ◽  
Double Mutant ◽  
Slow Growth ◽  
Dna Polymerase I ◽  
Uv Sensitivity ◽  
Pol I ◽  
Linear Chromosomes ◽  
Polymerase I

ABSTRACT Both polA (encoding DNA polymerase I; Pol I) and a paralog were deleted from Streptomyces strains. Despite the UV sensitivity and slow growth caused by the ΔpolA mutation, the double mutant was viable. Thus, in contrast to a previous postulate, Pol I and its paralog are not essential for replication of Streptomyces chromosomes.

Inhibition of E. coli DNA polymerase I by 1,10-phenanthroline

1977 ◽  
Vol 78 (1) ◽  
pp. 170-176 ◽  
Author(s):  
Vito D'Aurora ◽  
Andrew M. Stern ◽  
David S. Sigman
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase I ◽  
E Coli ◽  
Polymerase I

scholarly journals Interaction between the plasmid R6K andEscherichia coliwith defective DNA polymerase I

1978 ◽  
Vol 32 (1) ◽  
pp. 25-35 ◽  
Author(s):  
D. J. Tweats ◽  
J. T. Smith
Keyword(s):  
Cell Division ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase I ◽  
E Coli ◽  
R Factor ◽  
Polymerase I

SUMMARYInitial experiments demonstrated that the plasmid R6K cannot be transferred to or maintained readily in theE. coliDNA polymerase I deficient strain JG138polA1. Results withE. coliMM386polA12(R6K), which has a temperature sensitive polymerase I enzyme, showed cell division becomes abnormal when the polymerase I enzyme of the host bacteria is inactivated at the restrictive temperature. Under conditions of polymerase I deficiency, R6K replication, as measured by monitoring R-factor-mediated β-lactamase activity, also becomes abnormal with the loss of multiple R6K copies per cell and the apparent maintenance of a single R-factor copy per cell.

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