Latest revision as of 08:56, 17 October 2021

pPfuT7, a plasmid for the overexpression of Pfu.

Description

pFU

This plasmid was designed as part of the AdAPTED project from iGEM Athens 2021. The goal of this part is to result in the expression of Pfu polymerase, when E. coli BL21 bacteria are transformed, using heat shock. The backbone is plasmid pGGA and inserted is the sequence encoding Pfu polymerase, in addition to a histidine tag. The pFU plasmid also contains a T7 constitutive promoter, an RBS, specific for E. coli bacteria, as well as a terminator.

pGGA

This backbone contains a Chloramphenicol resistance gene under the control of a cat promoter. Additionally it contains a high-copy number ori and a SP6 promoter and a T7 promoter flanking two bsaI cut sites. Lastly, it contains two MCS between outside the two bsaI cut sites.

Pfu Structure and Function

The DNA polymerase Pfu (Pfu polymerase), can be found in Pyrococcus furiosus which is an extremophilic species of Archaea and it is a vital protein that is responsible for adding new nucleotides during DNA replication [1, 2]. DNA polymerase consists of 775 amino acids and a molecular weight of 90 kDa. It has been found that it has a homologous structure to the α-like DNA polymerases of humans and to the DNA polymerase II which is found in E. coli [3]. Due to its thermotolerans it has been widely used for amplification of genes through polymerase chain reaction (PCR) [4].

Proofreading

The main advantage of this enzyme is that it possesses 3´→5´ exonuclease activity (“proof-reading”) which allows it to reverse its direction and correct mismatched bases [1]. Thus its fidelity is much better (1.3 x 10^-6 mutation frequency/bp/duplication) than other thermotolerant DNA polymerases like Taq (8 x 10^-6 mutation frequency/bp/duplication) [5].

Pfu variants

Since error rate is important to be minimum in a number of techniques, there are several attempts to improve Pfu polymerase in that aspect with most importantly the Phusion variant [6]. Pfu can be attached to Sso7d which is a 7 kDa protein originating from Sulfolobus solfataricus, a hyperthermophilic archaebacterium [7]. Fusion to Sso7d has been proven to increase processivity, enabling longer amplifications and greater amplification speed [8].

Isolation and Purification

Since Pfu is an important enzyme worldwide there have been reported many attempts to produce it and purify it. Originally Pfu polymerase was isolated directly from Pyrococcus furiosus, but growing this species is a challenge especially in large quantities [9]. Thus, it has been successfully attempted to express that enzyme in E. coli BL21 [10]. The purification of the protein has been done with many different ways like His-tag purification and with the use of weak cation exchange resins [11, 12]. Recently a new simple method utilizing the tolerance to heat of the enzyme has been used successfully introducing a new level of simplicity for isolation and purification of thermotolerant proteins [13].

Histidine Tag

Lastly, some extra elements are added to each part. In the Pfu encoding gene, a histidine tag is added to isolate the protein for future applications. Purification of Pfu using polyhistidine affinity tags was selected as it is a rapid and efficient method, resulting in 100-fold enrichment and up to 95% purities in a single purification step (Bornhorst et. al. 2000).
The TU of Pfu was successfully ligated and transformed, as well as identified. SDS-PAGE for Pfu purification solutions was performed three times from different colonies to confirm the results. The average protein concentration after chromatography was 0.312 (g of Pfu polymerase)/L that corresponds to 4.4 (mg of Pfu polymerase)/(L of liquid culture).
The Pfu polymerase purification was performed using a gravity flow column chromatography with Nickel resin to collect the protein in imidazole from three different colonies to confirm the results. The elution was performed with a gradually increasing concentration of imidazole. In Figure C1 the gel from SDS-PAGE can be seen for the 10 mM, 100 mM, 100 mM imidazole elution for colony labeled as 2, 10 mM, 100 mM imidazole elution for colony labeled as 2’ and 10 mM, 100 mM imidazole elution for colony labeled as 3. The bands at 92 kDa for the 10 mM imidazole solutions show that a protein with His-tag was isolated. For the three 10mM solutions the absorbance was measured: A2=0.258, A2’=0.268, A3=0.364 and using Lambert–Beer law the concentrations were calculated: C2=0.271 g/L, C2’=0.282 g/L, C3=0.383 g/L, or an average of 0.313 g/L. Pfu polymerase was concentrated in 7 ml from 500 mL liquid culture, resulting in a concentration of 4.4 (mg of Pfu polymerase)/(L of liquid culture).

T7 promoter (BBa_J64997)

The part T7 consensus -10 and rest serves as a T7 promoter that allows the binding of T7 RNA Polymerase to initiate transcription (Arnaud-Barbe, 1998).

RBS (BBa_B0030) and

The RBS was chosen from the existing parts of the iGEM Registry, due to its high popularity and efficiency for E. coli.

Source of the part

NCBI code: WP_011011325

References

[1] Lundberg, K. S., Shoemaker, D. D., Adams, M. W., Short, J. M., Sorge, J. A., & Mathur, E. J. (1991). High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene, 108(1), 1–6. https://doi.org/10.1016/0378-1119(91)90480-y
[2] Zheng, W., Wang, Q. & Bi, Q. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli . Protein J 35, 145–153 (2016). https://doi.org/10.1007/s10930-016-9651-4
[3] Uemori, T., Ishino, Y., Toh, H., Asada, K., & Kato, I. (1993). Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucleic acids research, 21(2), 259–265. https://doi.org/10.1093/nar/21.2.259
[4] Pavlov, A. R., Pavlova, N. V., Kozyavkin, S. A., & Slesarev, A. I. (2004). Recent developments in the optimization of thermostable DNA polymerases for efficient applications. Trends in biotechnology, 22(5), 253–260. https://doi.org/10.1016/j.tibtech.2004.02.011
[5] Cline, J., Braman, J. C., & Hogrefe, H. H. (1996). PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic acids research, 24(18), 3546–3551. https://doi.org/10.1093/nar/24.18.3546
[6] McInerney, P., Adams, P., & Hadi, M. Z. (2014). Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. In Molecular Biology International (Vol. 2014, pp. 1–8). Hindawi Limited. https://doi.org/10.1155/2014/287430
[7] Agback,P., Baumann,H., Knapp,S., Ladenstein,R. and Hard,T. (1998) Architecture of nonspecific protein‐DNA interactions in the Sso7d‐DNA complex. Nature Struct. Biol., 5, 579–584.
[8] Wang Y (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 32, 1197–1207.
[9] Fiala G, Stetter KO (1986) Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 °C. Arch Microbiol 145:56–61
[10] Lu CL, Erickson HP (1997) Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus. Protein Expres Purif 11:179–184
[11] Dabrowski S, Kur J. Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei. Protein Expr Purif 1998;14:131–138.
[12] Sun Z, Cai J. Purification of recombinant Pfu DNA polymerase using a new JK110 resin. Korean J Chem Eng 2006;23:607–609.
[13] Sankar, P. S., Citartan, M., Siti, A. A., Skryabin, B. V., Rozhdestvensky, T. S., Khor, G. H., & Tang, T. H. (2019). A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification. Iranian journal of microbiology, 11(2), 181–186.