Team:UESTC Life/Results and discussion

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Results and discussion


Contents

TCP Biodegradation achieved

γ-HCH Biodegradation and F2A cleaving achieved

P2A being an excellent linker in chimeric protein

Polycistronic co-expression system constructed

Our vectors

Future Work









FIG.1 Proposed pathway for the metabolism of lindane (-yhexachlorocyclohexane) in P. paucimobilis UT26 (352). 1,-y-Hexachlorocyclohexane; 2, y-pentachlorocyclohexene; 3, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (chemically unstable); 4, 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (chemically unstable); 5, 2,5dichloro-2,5-cyclohexadiene-1,4-diol;







DhaA and HheC

DhaA and HheC also are cofactor-free dehalohygenase. DhaA from Rhodococcus sp hydrolyzes carbon-halogen bonds in a wide range of haloalkanes, including TCP, to the corresponding haloalcohol, releasing halide ions. Haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 is a potentially useful enzyme involved in the degradation of several important environmental pollutants, such as 1,3-dichloro-2-propanol, 2,3-dichloropropa- nol, 1-chloropropanol, epichlorohydrin and so on. Additionally, HheC has highly enantioselective dehalogenation of vicinal haloalcohols to epoxides, as well as the reverse reaction, the enantioselective and â-regioselective nucleophilic ring opening of epoxides by pseudo-halides such as azide and cyanide. In the synthesis of enantioselective medicine HheC being a important instrument puts it into a high gear. For the efficient degradation, we select two mutants DhaA31 and HheC/W239P, which have higher activity.18,22

FIG.2

Strategy

2A peptide Sequence

In foot-and-mouth disease virus (FMDV) and some other picornaviruses the oligopeptide (~20 amino acid) 2A region of the polyprotein mediates "cleavage" at its own C-terminus to release it from the 2B region. 2A is also active when placed between reporter proteins and in all eukaryotic systems tested - it acts as an autonomous element, making it an important tool for co-ordinated synthesis of multiple proteins from one open reading frame and making a ribosome jumping. (FIG.3)In recent year, there is little report about 2A peptide sequence that is active in prokaryotic systems, even some paper stated 2A sequence can’t work in prokaryotic. But Indian Scientist Dechamma et.1 have found the T2A peptide can work in E.coli, which give us creative advice.

Polycistroinc expression

Modular bacterial polycistronic expression system allows coexpression and copurification of multiple polypeptides in E. coli from a single expression plasmid. The system is comprised of the polycistronic expression vector and a transfer vector which facilitates subcloning of component genes into the polycistronic expression vector. Restriction sites present in the polycistronic expression vector allow both affinity tagged and untagged complexes to be overexpressed. In this project, 2A peptide sequence working in E.coli is just a exploration without guaranty from sufficient evident. Polycistroinc expression is the next way to achieve multi-enzyme co-expression.

Reference


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