Directed Evolution of Proteins on the basis of Mutational Scanning

Contents

• Introduction
• Methods to obtain amino acid alphabet
• Materials
• Methods

Introduction

• In past few years, directed evolution emerged as the most efficient and effective protein engineering methods.
• It has many application in basic as well in biotechnology, synthetic organic chemistry.

This methods used for the improvement of some traits like:

• Protein activity
• Expression
• Folding affinity
• Solubility
• Fluorescence
• Inter alia
• Selectivity (enantio-, stereo-, and regioselectivity).
• Stability (pH, temperature, and organic solvents).

• It consists of repetitive cycles of mutagenesis, screening or selection or expression of random or focused gene of the fittest variant.
• Various methods are employed to create diversity in which the most common one being error prone repair (epPCR), Saturation Mutagenesis and homologous recombination based methods like DNA shuffling.
• Although, epPCR and DNA shuffling are the most common methods in directed evolution, from these methods semirational approaches have emerged because the same or better results are obtained with less effort.
• From these one of the methods are there which are depends on the Iterative Saturation Mutagenesis (ISM), in which careful designing of the library is crucial for the success.
• Activity can be increased by substrate scope and stereoselectivity and regioselectivity,  amino acids lining are grouped and randomized near the substrate binding pocket using the Combinatorial Active-site Saturation Test (CAST).

Strategies have been developed for selecting the optimal AAAs

1. Bioinformatics and computational method
2. Random methods
3. Rational method

Materials

Databases

Suitable database are used to obtain the nucleotide sequence of interest, e.g., GenBank.

Reagents

• Cloning or assembling of target gene into a suitable vector.
• In ultra-pure water or suitable buffer, good quality oligonucleotides are suspended at a concentration of 2-5 μM.
• PCR Master mixture prepared which includes buffer, dNTPs, and polymerases.
• Restriction enzyme is added such as DpnI.
• Expression host: E. coli BL21 (DE3) Gold.
• Ingredients of SOC medium: 20 g/L tryptone, 10 mM MgSO4, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, and 5 g/L yeast extract.
• Media was then sterilized properly by autoclaving, 20 mM glucose added using 2M of sterile stock solution.
• Luria Broth (LB) medium and LB agar plates prepared.
• On the basis of plasmid suitable antibiotic stocks solution(s) was added.
• Plasmid maxiprep, midiprep, and/or miniprep kit.

Equipment

• Gradient thermocycler suitable for 96-well microtiter plates (MTPs).
• High-throughput (HT) DNA electrophoresis.
• Incubator with shaker for MTPs.
• Incubator with shaker for Eppendorf tubes.
• Centrifuge for MTPs and Eppendorf tubes.
• Spectrophotometer for determining DNA concentration.
• Multichannel pipettes (8 or 12 channels).

Materials

• Standard plastic ware (PCR tubes, 1.5 and 2.0 mL plastic reaction tubes, 15 and 50 mL falcon tubes, petri dishes of 10 and 15 cm diameter).
• 96-Well MTPs for PCR.
• Agarose gels for HT DNA electrophoresis.
• Standard 14 mL round-bottom tubes for cell culturing.
• 2.2 mL deep-well 96-well MTPs used with paper or metal lids.
• 6-/12-Well multidish plates with flat-bottom well design.
• Glass beads of ca. 3 mm diameter.

Services

HT DNA plasmid extraction and sequencing in MTP format is provided by a company.

Software and Servers

• Programs used for designing of primers in HT format:
• • Deep scan is one of the program which is currently in use that allows introduction of degenerate or non-degenerate codons in the primers which are partially overlapped. In contrast, Quik Change mutagenesis which used for gene segments or complete genes.
  • AAscan is a technique which was primarily utilized for the finding of optimal primers for the alanine scanning.
  • MegaWHOP is a method which based on the method MegaPrimer.
• Software for alignment and processing of the files of multiple DNA sequencing.
• Software for the measurement of base peak heights from DNA chromatograms.
• To calculate library screening effort an online program applied.

Methods

Stages of this protocol are:

• Library Design in Silico
  • Choose optimal system
  • Define target region according to budget allocation
  • Design oligos
  • Order a few test oligos
• Optimisation conditions for library creation
  • PCR with test oligos
  • Template digestion, transformation, and cell plating optimization.
  • DNA sequencing
• Evaluation of preliminary libraries
  • Determine QQC and Q values
  • Q values should be less than 0.6
• Library creation
  • High-throughput mutagenesis PCR
  • Transformation and sequencing
  • Identity missing mutation
  • Order oligos for library completion
• Library completion
  • HT site directed mutagenesis for obtaining missing variants.
  • HT transforming in MTP
  • HT DNA sequencing in MTP
  • Library evaluation
  • Construction of master plate
• Investing sequence function relationships
  • Screening of master plate.
  • Construction and analysis of mutability landscape using excel and or Matlab.
  • Construction of combinatorial library based hotspot

Reference and Sources

• https://link.springer.com/protocol/10.1007/978-1-4939-7366-8_6
• https://www.thermofisher.com/order/catalog/product/15544034
• https://pubmed.ncbi.nlm.nih.gov/29086305/
• https://www.nature.com/articles/nrg3927
• https://www.biotechbotany.com/2020/09/03/luria-broth-lb-medium-and-lb-agar-for-e-coli-growth/

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