In-house production of Cas9 protein using prokaryotic and eukaryotic expression systems

Abstract

Recombinant protein expression of Cas9 was performed at BioInvent International AB using prokaryotic and eukaryotic expression systems. For the eukaryotic expression, two types of mammalian cells were used namely HEK293 cells and CHO cells. The first expressions were performed using the pIRO-KL vector encoding the kappa leader secretory sequence. The kappa secretory sequence is responsible for translocating the expressed protein into the ER space and further on secretion out of the cell. Interestingly, the expressed Cas9 was not secreted, so two other overexpression vectors were tested lacking a leader sequence. Although they additionally were lacking a kozak sequence, which is important to initiate translation efficiently, they showed expression of Cas9 protein. Upon addition of a kozak sequence to both vectors, we observed significantly better expression compared to the non-kozak vectors. Interestingly, the CHO cells did not show any signs of Cas9 expression while in HEK293 cells a clear expression was detected. In parallel, prokaryotic expression of Cas9 was performed using a pET303 vector in the BL21 DE3 expression strain. pET303 has a pelB secretory sequence responsible for directing the protein into the periplasm. Unfortunately, only slight expression was observed in the periplasm at induction conditions OD600=3, 28°C and 0.5 mM IPTG. Removing the pelB sequence led to a drastic improvement of expression, and improved induction conditions showed promising expression
results. In conclusion, I succeeded in expressing the Cas9 protein in both expression systems, but there was not sufficient time to purify Cas9 completely from neither of the systems.

Popular Abstract

Recombinant DNA technology is a powerful tool that was discovered in the 1970s. The concept of
this technology is to manipulate the DNA sequence e.g. by making changes through deleting some
endogenous genes or inserting new ones. The new DNA construct will elucidate targeted
modifications to have the desired function in the system. It has been leading to vital improvements in
biology and life science research. For instance, drug discovery and medicines were revolutionized by
this technology to cure many diseases such as AIDS, cancer, and many other diseases. By
manipulating the DNA, the scientists could also produce recombinant proteins. This is based on
insertion of the gene encoding the protein of interest (insert) into appropriate vector and this
construct called a plasmid. Thereafter, the plasmid is transformed into the cells of an expression
strain where the protein of interest is produced. During my project I had to express Cas9 protein
which is a part of CRISPR-Cas9 system. The CRISPR-Cas9 system is a recent gene editing technology
that was awarded the noble prize in chemistry 2020. This system is described to be the key
component of the adaptive immune system of the Streptococcus pyogenes bacterium. The system is
activated by foreign DNA sequences of invading pathogens, so called bacteriophages. The CRISPR-
Cas9 system consists of combinations of short RNAs and a DNase, which can induce double strand
breaks in the targeted sequence of DNA. This system inspired scientists to target virtually any DNA,
including the human genome to repair mutated and disease causing sequences. For instance, cancer
immunotherapy is one of the major therapies that have been promoted since the arising of CRIPSR-
Cas9 system. During my work I tried to produce the Cas9 protein at BioInvent using two expression
systems, namely an eukaryotic and a prokaryotic system. I first started the expression of Cas9 using
the eukaryotic system and specifically mammalian cells because the proteins that are expressed
using this system normally undergo post translational modifications. These modifications are
required for some proteins to be functional. More importantly, the proteins expressed by eukaryotic
cells are generally endotoxin free and can directly be introduced into other mammalian cells. I used
two types of mammalian cells, which were the Human Kidney Embryonic cells (HEK293 cells) and the
Chinese Hamster Ovary cells (CHO cells). I observed better expression of Cas9 using the HEK293 cells
compared to the CHO cells. I designed many vectors for the eukaryotic expression, the first one was
pIRO-KL-Cas9, which had a kappa leader secretory sequence. The kappa leader is responsible for
placing the expressed protein into the endoplasmic reticulum space from where the protein will be
secreted further to the outside of the cell. This would facilitate the purification of the expressed
protein from the supernatant of the cell culture. However, the Cas9 protein was expressed but not
secreted and remained inside the cells. So, I decided to design new vectors without any secretory
sequences because the protein folding might have been affected due to the environmental
conditions in the endoplasmic reticulum space. The new vectors were pIRO-Linker-2-Cas9 and Ori-
amp-C-0, whereof I achieved higher production of Cas9 using pIRO-Linker-2. I had many troubles to
express the protein at sufficiently high levels using the eukaryotic system, so I tried to produce Cas9
using a prokaryotic system. The prokaryotic expression systems are much faster, easier, and often
gives higher yield of the protein expressed. I had already designed the plasmid pET303-Cas9 with
pelB secretory sequence, which will secrete the expressed protein into the periplasm. The expression
using this vector was controlled through a T7 lac operon and I used BL21 DE3 strain for expression.
The protein was not detected in the periplasm, possibly for the same reason as in the eukaryotic
expression with a secretory leader. Hence, I reconstructed the plasmid without pelB. Moreover, I
investigated different expression conditions such as varying the growth stage and the strength of
induction, as well as the growth temperature of the induced culture. The best induction conditions I
found was using a more gentle induction (0.5 mM IPTG) at a not too dense stage of the culture
(Optical Density at 600 nm: 3) and at a lower temperature (28°C). I performed a partial purification of
the expressed Cas9 using affinity nickel chromatography taking advantage of a His-tag extension that
was included in the recombinant protein. Because of time limitation I was not able to get completely
pure Cas9. In summary, I succeeded in expressing the protein using both systems and a partial
purification was achieved, which are the initial steps towards obtaining larger amounts of pure
isolated Cas9.

• author: Dowah, Hesham
• supervisor: David Ermert
• organization: Department of Chemistry; Computational Chemistry
• course: KEMR20 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Cas9, protein, expression, biochemistry
• language: English
• id: 9091513
• date added to LUP: 2022-06-23 11:47:48
• date last changed: 2022-06-23 11:47:48