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Fusion proteins the process to preparation and utilization in expression systems of recombinant proteins

A fusion protein and calcium-binding protein technology, applied in recombinant DNA technology, expression enhancement stability/folded protein fusion, peptides containing affinity tags, etc., can solve problems such as low yield, difficult to operate and precipitate

Inactive Publication Date: 2012-02-08
ESCOLA SUPERIOR AGRARIA DE COIMBRA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] One of the major difficulties in using these expression systems is the production of protein as inclusion bodies, conditions that often lead to very low yields, and the formation of unwieldy precipitates

Method used

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  • Fusion proteins the process to preparation and utilization in expression systems of recombinant proteins
  • Fusion proteins the process to preparation and utilization in expression systems of recombinant proteins
  • Fusion proteins the process to preparation and utilization in expression systems of recombinant proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] [Example 1: HCP12 and HIL5 constructs]

[0101] To obtain these constructs, we first obtained the H fragment. PCR was performed using primers H and Fh8Rev (see Table 1). Primer H allows insertion of a SacI restriction site after the codon for the 11th amino acid of Fh8. The PCR product was isolated and then digested with SacI restriction enzyme. The SacI-digested N-terminal fragment (H fragment) was then isolated.

[0102] Second, IL5 (primers IL5Sac and IL5Kpn) and CP12 (primers CP12Sac and CP12Kpn) fragments were amplified using isolated human genomic DNA (gDNA) and Cryptosporidium parvum gDNA as templates, respectively. After isolation of the PCR products, they were digested with SacI and the corresponding isolation of the Sac-digested fragments was performed.

[0103] Sac-digested H fragments were combined with SacI digested IL5 and CP12 fragments. The HIL5 fragment was obtained by PCR using this conjugate as a template, and primer H and primer IL5Kpn as primer...

Embodiment 2

[0104] [Example 2: HTgOWP construct]

[0105] To obtain the HTgOWP fragment, the pQEHCP12 vector was digested with KpnI and SacI, thereby removing the CP12 fragment from the vector, and thus obtaining pQE30H.

[0106] The TgOWP fragment was generated by PCR using gDNA of Toxoplasma gondii as a template, and primers TgOWPSac and TgOWPKpn.

[0107] The PCR reaction allows the addition of SacI and KpnI enzyme restriction sites to the DNA template sequence of TgOWP. This product was then purified on an agarose gel and bound to pGEM vector. Subsequently, digestion with KpnI and SacI restriction enzymes was performed, followed by isolation of the TgOWP fragment digested with SacI and KpnI. This fragment was incorporated into pQE30H vector digested with SacI and KpnI, thereby obtaining construct pQEHTgOWP.

[0108] This fragment was incorporated into pQE30H vector digested with SacI and KpnI, thereby obtaining construct pQEHTgOWP.

Embodiment 3

[0109] [Example 3: pQECP12, pQEIL5 and pQETgOWP constructs]

[0110] Vectors containing H fragments (pQEHCP12, pQEHIL5 and pQEHTgOWP) were used to prepare corresponding negative controls. HCP12, HIL5 and HTgOWP fragments were used as starting points for obtaining negative controls as described.

[0111] Since the procedures of the three vectors are similar, the following provides an example of preparing the pQECP12 recombinant vector. The pQEHCP12 vector was digested with Sad enzyme and then purified on an agarose gel.

[0112] After the purification step, the pQEHCP12 vector was also digested with EcoRI restriction enzyme, present in the pQE30 vector sequence. This digestion allowed removal of the H fragment as well as a small portion of the pQE30 vector. The pQEHCP12, pQEHIL5, and pQEHTgOWP vectors digested with SacI and EcoRI restriction enzymes were purified on agarose gels, followed by digested fragments of the pQE30 empty vector. For this purpose it was necessary to ...

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Abstract

The present application relates to improved recombinant protein production processes through the use of novel fusion tags. In particular, the present application discloses novel fused proteins comprising an aminoacidic sequence that corresponds to a fragment H or an aminoacidic sequence that corresponds to a calcium-binding protein excreted / secreted by the adult worm of Fasciola hepatisa, namely, Fh8 and Fh22 proteins, followed by an aminoacidic sequence that corresponds to a non-related protein fragment or protein, and methods for its preparation and its use thereof.

Description

【Technical field】 [0001] The present invention relates to fusion proteins, their preparation methods and their application in recombinant protein expression systems. 【Background technique】 [0002] The preparation of specific protein-enriched extracts readily occurs rarely from the organisms that produce them. Thus, the production of recombinant proteins is often the only possible way to produce those proteins in the required amounts and with the requisite level of activity. [0003] The expression of recombinant proteins is one of the most relevant and applicable methods at the research and development level as well as at the industrial level. The possibility to express foreign proteins in well-defined models using bacteria, fungi or eukaryotic cells, constitutes a tool of great potential and relevance in the field of biotechnological processing, allowing the production of large quantities of proteins of interest. [0004] Escherichia coli (E. coli) is already the most wi...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/67
CPCC07K14/43536C07K14/5409C07K2319/20C07K2319/35C12N15/62C12P21/02
Inventor M·A·佩雷拉达孔塞桑S·J·马克斯达科斯塔A·M·奥利韦拉卡斯特罗A·A·达席尔瓦阿尔梅达
Owner ESCOLA SUPERIOR AGRARIA DE COIMBRA
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