Tutorial 8 Protein Engineering Flashcards
Discuss the importance of the usage of protein engineering in the industrial purposes?
- Due to the high value of natural products in industries, engineering their biosynthesis has attracted extensive attention.
- There are limitations in the development of natural product biosynthesis as the integration of natural enzymes is limited by enzymatic activities, narrow substrate ranges, poor stabilities and even loss of function in heterologous hosts.
- The use of natural enzymes make it challenging to further improve productivity or expand product spectra. Even the mining of novel enzymes from natural resources to solve these issues to too time-consuming and labor intensive.
- Hence, improving existing enzymes through protein engineering is effective in the development of biosynthetic approaches to produce industrial products.
What are the steps of general process of protein engineering that could be used to modify the industrial enzymes?
The library is established by either rational design aided by computational stimulation and/or crystal structures or random mutagenesis or directed evolution (mutations). With the assist of efficient screening methods, variants with different characteristics such as enhanced catalytic activity and increased stability towards heat, acid or alkaline can be selected and analyzed.
If no candidate shows the desired feature, it may be necessary to regenerate the library and repeat the process until the desired variant is obtained.
Mutagenesis could be further classified as the in vivo mutagenesis and in vitro mutagenesis.
Describe both terms and what is the main difference between the above mentioned methods?
In in vivo mutagenesis, gene targeting offers well-crafted, site mutagenesis within living cells. In vitro mutagenesis, can involve essentially random approaches to mutagenesis, which may be valuable in producing libraries of new mutants. It will result in the alteration of a specific amino acid or small component of the gene product in a predetermined way.
Site directed mutagenesis (SDM) is widely used to engineer the protein of interest, that are used for the industrial purposes. There are various methods that we could utilize to achieve site directed mutagenesis. Briefly mention the main 4 PCR based methods used for the SDM
Traditional PCR:
Primers designed to include desired change, which could be base substitution, addition or deletion.
Inverse PCR:
Inverse PCR enables amplification of a region of unknown sequence using primers oriented in the reverse direction. An adaptation of this method can be used to introduce mutations in the previously cloned sequences.
Prime extension:
Incorporates mutagenic primers in independent, nested PCRs before combining them in the final product.
Depending on your opinion, what method of protein engineering will mostly be beneficial on
industrial purposes? Rational design or Directed evolution?
Directed evolution
Rational design takes advantage of prior knowledge of the 3-dimensional structure of the enzyme, as well as structure/function and sequence information to predict in a rational/logical way, sites on the enzyme to obtain desired property of the enzyme. Once the crucial amino acids are identified, site-directed mutagenesis is applied and expressed mutants are screened for desired properties.
Directed evolution does not require such prior sequence or three dimensional structure knowledge, it employs random-mutagenesis protocols to engineer enzymes that are screened for desired properties. Further round of mutation and selection are applied and produces superior results than rational design.
For the error prone PCR technology, we utilize the taq DNA polymerase enzyme. What is the
main difference of that enzyme compared to the general taq polymerase uses in DNA
replication?
Error prone PCR utilizes the Taq DNA polymerase which lacks 3’ to 5’ exonuclease activity.
Error prone PCR method utilizes special chemical environments during the PCR amplification.
What are the parameters that we adjust during the process?
Change nucleotide concentration which would make the concentrations of dCTP and cTTP against dGTP and dATP.
Another method is to increase concentration of magnesium and manganese to carry out error prone PCR.
What is the main mutagenesis method that will result the tandem repeats? Describe that technique with further information?
Rolling circle error-prone PCR:
1. Mutation are introduced by first cloning the target sequence into an appropriate plasmid.
- The amplification process begins using random hexamer primers and DNA polymerase under error prone rolling circle amplification conditions.
- MnCl2 added into reaction mixture to promote random point mutations in the DNA strands.
This method results in linear DNA duplexes and these fragments contain tandem repeats of circular DNA called concatamers which can be transformed into bacterial strains.
EMS is a well-known chemical mutagen used for the protein modification. What is the main function of EMS during the enzyme engineering?
Ethyl Methane Sulfonate:
This chemical agent alkylates guanidine residues. This alteration causes errors during DNA replication.
What are the transposons? What is the function of transposons during the genetic modifications related to protein engineering?
Transposons are a group of mobile genetic elements that are defined as a DNA sequence. They are important in maintaining the integrity of new genomes. However, they have limited function. During the movement of transposon, they alter gene expression and rearrangement of genes. Some mutations result in the inactivation of transposons and other mutations could lead to desired properties.
Explain how you would simply conduct an DNA shuffling to enhance the properties of an enzyme? (Consider the property as catalytic activity)
DNA shuffling begins with the digestion of homologous parental genes into small fragments by DNase1.
These small fragments are purified from undigested parental genes.
Purified fragments are reassembled using primer-less PCR.
This PCR involves homologous fragments from different parental genes priming for each other resulting chimeric DNA.
The chimeric DNA of parental size is then amplified using end terminal primers in regular PCR.
Ligation of DNA into fragment into a vector
Insertion of of DNA into Host cell
Checking Catalytic activity.
What are the advantage of the random priming in vitro recombination method over the DNA shuffling?
There is no use of DNase 1, thus there is no bias for recombination next to a pyrimidine nucleotide.
It also use synthetic random primers which are uniform in length and lack biases.
Screening and selection of the desired protein after the bioengineering process is a crucial step.
Mention in steps , how you design a method to perform the screening and selection techniques?
(Use your protein of interest as Protein A)
Method 1: Phage display method
- Involve the fusion of genes encoding the variant polypeptides with phage coat protein genes.
- Protein variants expressed on phage surfaces are selected by binding with immobilized targets in vitro.
- Phages with selected protein variants are then amplified in bacteria, followed by the identification of positive clones by enzyme linked immunosorbent assay.
- These selected phages are then subjected to DNA sequencing.
