E.

coli as an expression system for

recombinant Trypsinogen
What are we going to talk about?
The analysis of the known properties of trypsin and trypsinogen of shrimp
Litopenaeus vannamei and together with the interest of applying them in the
aquaculture area, the expression system of Escherichia coli was selected to produce
shrimp trypsinogen

Introduction
Aquaculture
Aquatic organisms are propagated or cultivated under controlled conditions and
whose activity complements the demand for products obtained through the
traditional fishing industry.
Recombinant Protein
Thanks to the manipulation of genes, it has been possible to produce large amounts
of proteins, many of which are found in very low concentrations in their natural
environment
To choose a suitable expression system for the synthesis of a recombinant protein,
has to be considered the biological origin, the chemical and biological properties of
the protein and the application and the bioprocess that will be used in its production

Biological Function and Properties of Trypsin

Trypsin is present in the digestive tract of a wide variety of mammals and plays an
important role in the activation of digestive endopeptidases.
It catalyzes hydrolysis reactions of peptide bonds containing residues of basic amino
acids, like lysine and arginine, to make accessible for further degradation by other
proteases.
Trypsinogen (trypsin zymogen) is secreted into the intestine and activates trypsin by
a specific proteolytic cutting and can be catalyzed by the enteropeptidase or
enterokinase that is only produced in the intestine. Enterokinase is a serine protease
that specifically recognizes the amino acid sequence (Asp)4 Lys-↓-X and activates
trypsinogen releasing trypsin by breaking a small peptide
Many mammalian enzymes are also present in aquatic organisms. The differences
between this and mammals are molecular weight, amino acid composition, optimal
pH, optimal temperature, stability, inhibition characteristics and kinetic properties
Pacific White Shrimp’s Trypsinogen (Litopenaeus Vannamei)
Commercial trypsin is mainly obtained from bovine or porcine pancreatic tissue. This
obtained trypsin contains traces of other undesirable pancreatic proteases in some
industrial or laboratory processes and the strict guidelines and regulations of the
FDA
Due to the importance of this enzyme in the digestive processes of the Pacific White
shrimp L. vannamei, Worlds interest is to have a pure and homogeneous source of
this enzyme or its zymogen, for the studies and its potential application in the
aquaculture of this cultivable species.

Elements for our expression system

The E. coli strain
For prokaryotic cells, the chosen systems are those of easy use and low price,
although they could have their restrictions to produce eukaryotic proteins. Mostly
because lots of eukarya proteins suffer post translational modifications, like disulfide
bridge formation, glycosylations, phosphorylations, which are necessary for their
correct folding, stability and stability and biological activity.
The BL21 CodonPlus (DE3) strain is used for the production of recombinant proteins
because of the lead in recombinant expression genes containing rare codons for E.
coli, both prokaryotic and eukaryotic.
Plasmid
First, let's remember the basic parts of the plasmid.
• Replication origin (Ori): A
DNA sequence allows the bacteria to
make more copies of the plasmid as
it grows and divides.
• Antibiotic resistance gene: It
is a specific gene that allows the
bacteria with the plasmid to grow in
the presence of an antibiotic specific
to the gene.
• Gene: DNA sequence that
codifies for a particular interest
protein.
• Promoter: DNA sequence that
allows the cell to produce the protein encoded by the gene.
• Restriction sites: DNA sequences that allow a researcher to cut and paste
components of plasmids together.
To use BL21 strain the plasmid has to belong to it, so the chosen plasmid will allow
the expression of the trypsinogen
In this case was the vector "pET-28a" because it has a regulatory system by IPTG
or isopropyl β-D-1-thiogalactopyranoside. Which is a chemical reagent that mimics
allolactose, which eliminates a repressor of the lac operon to induce gene
expression, which allows us to manipulate this expression according to our interest.

• Antibiotic resistance gene:

Kanamycin
• Promoter: T7 promoter and lacI
promoter
• Restriction sites: Several restriction
sites

Our sequence
In the investigation, we found that the coding sequence for our protein is as follows
(along with its complementary):

ACGCGGGTGACCAGTGACCAGCCATGAAGTCTCTCGTGCTCTGCCTGCTCCTCGCCGGGGCCTTCGCCGC
CCCCTCCAGGAAGCCCACCTTCCGTCGCGGTCTCAACAAGATCGTCGGAGGAAGTGAGGTCACTCCTGGG
GAGCTGCCTTACCAGCTCAGCTTCCAGGACAACTCCTGGGGGACCGCCTGGCATTTCTGCGGCGCCTCCA
TCTACAACGAGAATTGGGCCATCTGCGCTGGTCACTGCGTCCAGGGCGACGACTTTGATAATCCCAGCTA
CCTTCAGGTCGTGGCCGGAGAGCATAACTTCGACGTGAATGAGGGCAACGAGCAGACGGTCGTCCTCTCC
AAGATCATCCAACACGAGGACTACAACGGCTTCACCATCAGCAACGACATCTCCCTGCTCAAGTTTTCTC
AGCCTCTGAGCTTCAACGACTACGTTCGCGCCATCGATATTCCCGCTCAGGGTCACGCTGCCTCTGGCGA
CTGCATCGTGTCCGGCTGGGGCGCTCTCACTGAAGGAGGCAGCTCCCCCAGCGCCCTCCAGAAGGTGTCC
GTTCCCATCGTGTCTGACGACGAGTGTCGCGATGCTTATGGCCAGAGCGGCATTGAGGACTCCATGATCT
GTGCCGGAGTGCCCGAGGGCGGCAAGGACTCGTGCCAGGGTGACTCTGGCGGCCCCCTTGCCTGCTCTGA
CACCGGCTCCACCTACCTGGCCGGCATCGTGTCCTGGGGCTACGGCTGTGCCCGTCCCAACTACCCTGGC
GTGTACGCTGAGGTCTCCTACCATGTCGATTGGATCAAGGCCAATGCTGTTTAAATCTAACTGGATCAAA
TAATAAAAGAATCCATAATGGCAAAAAAAAAAAAAAAAAAAAAAAAA

TGCGCCCACTGGTCACTGGTCGGTACTTCAGAGAGCACGAGACGGACGAGGAGCGGCCCCGGAAGCGGCG
GGGGAGGTCCTTCGGGTGGAAGGCAGCGCCAGAGTTGTTCTAGCAGCCTCCTTCACTCCAGTGAGGACCC
CTCGACGGAATGGTCGAGTCGAAGGTCCTGTTGAGGACCCCCTGGCGGACCGTAAAGACGCCGCGGAGGT
AGATGTTGCTCTTAACCCGGTAGACGCGACCAGTGACGCAGGTCCCGCTGCTGAAACTATTAGGGTCGAT
GGAAGTCCAGCACCGGCCTCTCGTATTGAAGCTGCACTTACTCCCGTTGCTCGTCTGCCAGCAGGAGAGG
TTCTAGTAGGTTGTGCTCCTGATGTTGCCGAAGTGGTAGTCGTTGCTGTAGAGGGACGAGTTCAAAAGAG
TCGGAGACTCGAAGTTGCTGATGCAAGCGCGGTAGCTATAAGGGCGAGTCCCAGTGCGACGGAGACCGCT
GACGTAGCACAGGCCGACCCCGCGAGAGTGACTTCCTCCGTCGAGGGGGTCGCGGGAGGTCTTCCACAGG
CAAGGGTAGCACAGACTGCTGCTCACAGCGCTACGAATACCGGTCTCGCCGTAACTCCTGAGGTACTAGA
CACGGCCTCACGGGCTCCCGCCGTTCCTGAGCACGGTCCCACTGAGACCGCCGGGGGAACGGACGAGACT
GTGGCCGAGGTGGATGGACCGGCCGTAGCACAGGACCCCGATGCCGACACGGGCAGGGTTGATGGGACCG
CACATGCGACTCCAGAGGATGGTACAGCTAACCTAGTTCCGGTTACGACAAATTTAGATTGACCTAGTTT
ATTATTTTCTTAGGTATTACCGTTTTTTTTTTTTTTTTTTTTTTTTT
Why is it important to keep the sequence in mind?
Because we need to look for a restriction enzyme that does cut the plasmid, but not
our DNA strand. With the support of bioinformatics tools, the following were obtained:

For example, we cannot use the AlwNI, BglI, BlpI, BspDI, BstEII, ClaI, NruI enzyme,
because it not only cuts the plasmid, but also our DNA strand.
 Therefore, the chosen restriction enzyme is Eco53kI because it is immediately by our
promoter T7.
Also, from what you can tell, it is a blunt cut restriction
enzyme, because the characteristic of our DNA is that
it has many adenines, there is no way that a cohesive
cut can bind to our strand, since restriction enzymes are palindromic and with
different nitrogenous bases.

Our steps so far

• Amplify our fraction of interest by PCR: The design of our oligos is pretty easy,
because is the area of the chain where many adenines are found.
5’-ACGCGGGTGACCAGTGACCA-3’
5’-AAAAAAAAAAAAAAAAAAAA-3’
• Digestion with restriction enzymes: We have already discussed this one, and
it is our Eco53kI gene that will only cut our plasmid.
• Ligation of the DNA segment to be expressed with the vector, using ligases.
• Bacterial transformation

Denaturation Alignment Extension

Products
Transcription and translation
The DNA of a gene serves as a
template for complementary base-
pairing, and an enzyme called RNA
polymerase II catalyzes the
formation of a pre-mRNA molecule,
which is then processed to form
mature mRNA. The resulting mRNA
is a single-stranded copy of the
gene, which will be translated into a
protein molecule.
Transcription
• Beginning: the RNA
polymerase joins to the
promoter at the beginning of
the gene. Each gene has its
own. The polymerase job is to
separate the DNA chains to
give the space needed for
transcription to happen
• Elongation: The mold chain
acts as a guide for the RNA polymerase, which produces an RNA molecule
from the complementary nucleotides, and it grows from 5’ to 3’
• Ending: Specific sequences called terminators indicate that RNA transcription
has been completed. Once transcribed, these sequences cause the transcript
to be released from RNA polymerase.
AUGAAGUCUCUCGUGCUCUGCCUGCUCCUCGCCGGGGCCUUCGCCGCCCCCUCCAGGAAGCCCACCUUCCGUCGCGGUCUCAACAAGA
UCGUCGGAGGAAGUGAGGUCACUCCUGGGGAGCUGCCUUACCAGCUCAGCUUCCAGGACAACUCCUGGGGGACCGCCUGGCAUUUCUG
CGGCGCCUCCAUCUACAACGAGAAUUGGGCCAUCUGCGCUGGUCACUGCGUCCAGGGCGACGACUUUGAUAAUCCCAGCUACCUUCAG
GUCGUGGCCGGAGAGCAUAACUUCGACGUGAAUGAGGGCAACGAGCAGACGGUCGUCCUCUCCAAGAUCAUCCAACACGAGGACUACA
ACGGCUUCACCAUCAGCAACGACAUCUCCCUGCUCAAGUUUUCUCAGCCUCUGAGCUUCAACGACUACGUUCGCGCCAUCGAUAUUCC
CGCUCAGGGUCACGCUGCCUCUGGCGACUGCAUCGUGUCCGGCUGGGGCGCUCUCACUGAAGGAGGCAGCUCCCCCAGCGCCCUCCAG
AAGGUGUCCGUUCCCAUCGUGUCUGACGACGAGUGUCGCGAUGCUUAUGGCCAGAGCGGCAUUGAGGACUCCAUGAUCUGUGCCGGAG
UGCCCGAGGGCGGCAAGGACUCGUGCCAGGGUGACUCUGGCGGCCCCCUUGCCUGCUCUGACACCGGCUCCACCUACCUGGCCGGCAU
CGUGUCCUGGGGCUACGGCUGUGCCCGUCCCAACUACCCUGGCGUGUACGCUGAGGUCUCCUACCAUGUCGAUUGGAUCAAGGCCAAU
GCUGUUUAA

Translation
• Beginning: here the ribosome is reunited with the mRNA and the first tRNA
so that translation can begin.
In bacteria, the small ribosomal subunit does not start at the 5' end of the
mRNA and travels towards the 3' end. Instead, it binds directly to certain
sequences on the mRNA. These Shine-Delgarno sequences are found just
before the initiation codons and "signal" them to the ribosome. Bacterial
genes are often transcribed in groups called operons. A Shine-Delgarno
 sequence marks the start of each coding sequence, allowing the ribosome to
find the correct start codon for each gene.
• Elongation: the tRNAs bring the amino acids to the ribosome and these are
joined to form a chain.
• Ending: the finished polypeptide is released to go and perform its function in
the cell.
The translation ends in a process known as termination. Termination occurs
when a stop codon in the mRNA (UAA, UAG, or AGA) enters the A site.

MKSLVLCLLLAGAFAAPSRKPTFRRGLNKIVGGSEVTPGELPYQLSFQDNSWGTAWHFCGASIYNEN
WAICAGHCVQGDDFDNPSYLQVVAGEHNFDVNEGNEQTVVLSKIIQHEDYNGFTISNDISLLKFSQPL
SFNDYVRAIDIPAQGHAASGDCIVSGWGALTEGGSSPSALQKVSVPIVSDDECRDAYGQSGIEDSMIC
AGVPEGGKDSCQGDSGGPLACSDTGSTYLAGIVSWGYGCARPNYPGVYAEVSYHVDWIKANAV