Purification and immunity analysis of recombinant 6His-HPT protein expressed in E. coli.
OBJECTIVE
To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plant selective maker gene product expressed from E. coli and to prepare the polyclonal antibody (pAbs) against it.


METHODS
HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT was transferred into E. coli DH5alpha for HPT expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E. coli cells were lysed by sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Western blot respecitively.


RESULTS
Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E. coli accounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity. Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein).


CONCLUSIONS
HPT protein can be expressed and purified from E. coli by a relatively simple method, which has high immunity activity.
Yang
Li-Chen
LC
Key Laboratory of Trace Element Nutrition of the Ministry of Health, China, Institute of Nutrition and Food Safety, Chinese Center for Diseases Control and Prevention, Beijing 100050, China.
Zhu
Zhen
Z
Yang
Xiao-Guang
XG
D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005819:Genetic Markers; D030781:Organisms, Genetically Modified; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000276:immunology* / Q000302:isolation & purification*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D004269:DNA, Bacterial; D005819:Genetic Markers; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase