Difference between revisions of "Part:BBa K3037008"

Revision as of 00:05, 22 October 2019

MBP+HRP

Overview

The TU Dresden 2019 team designed this fusion protein in order to increase the expression of HRP. In order to make it in the RFC 25 standard was used (more information).

HRP was inserted into the pSB1C3 vector for transformation and expressed with pOCC97 (BBa_K3037000) in Escherichia coli.

The HRP was made from BBa_K1800002 but adapted to the Freiburg RFC25 standard

Biology

The metalloenzyme horse radish peroxide is widely used in many biochemical and immunological applications. This Enzyme on its own does not give any visual read out but however upon addition of suitable substrate, HRP oxidizes this substrate and yields a colour change and this can be spectrophotometrically analysed. One such common example for chromogenic substrate is TMB (3,3',5,5'-Tetramethylbenzidine) that HRP oxidizes. Additionally, an advantage of using HRP includes its stability and small size, hence reducing the interference during conjugation reactions such as for secondary antibody detection and moreover it is economical in comparison with other alternative enzymes like the alkaline phosphatase.

MBP is a well-established, reliable protein-tag that is meant to increase the solubility of the proteins it is fused to. Therefore it limits the risk of accumulation of over expressed recombinant protein in inclusion bodies and increases the total protein yield. It can also improve expression of difficult enzymes like Cas9. It can further be used to purify proteins via affinity chromatography in amylose resin.[1]

You can find more information in the pages of the individual parts of this composite: BBa_K3037007 BBa_K3037001

Characterization

Outline

1) Expression in pOCC97, growth curve (BBa_3037000)

2) Expression in pOCC97, SDS-PAGE (BBa_3037000)

Experiments in Detail

1) Expression in pOCC97, growth curve (BBa_3037000)

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  Growth curve (Normal bacterial Growth curve is seen during the expression of MBP-HRP)

2) Expression in pOCC97, SDS-PAGE (BBa_3037000)

Expression of MBP-HRP seen in the graph (right)

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  SDS-PAGE (Comparison of expression of full construct (BBa_K3037003) and MBP-HRP (BBa_K3037008) in the pOCC97 (BBa_K3037000) before and after optimization)

Sequence

NOTE: Please be aware, that by combining all the basic parts used for this composite part, the registry automatically inserted a RFC23 scar. However, the design and our cloning strategy is based on RFC25.

Reference

[1] https://www.genscript.com/bacterial-soluble-protein-expression-MBP-tag.html