Cloning, Overexpression and Characterization of the FeSI Protein from Azotobacter vinelandii CA6

Year 2022, , 470 - 476, 30.06.2022

Burak Kabasakal

https://doi.org/10.35414/akufemubid.1124731

Abstract

The [2Fe-2S] protein from Azotobacter vinelandii CA6, also known as Shethna protein I or FeSI protein, was cloned and overexpressed in E. coli and purified. SDS-PAGE analysis showed a band at ~11 kDa, the monomeric size of the protein, at each stage of the purification. Gel filtration profile of FeSI indicates it forms a dimer in its native state. The UV-visible spectrum showed absorbances at signature wavelengths, 344, 418 and 464 nm, due to the iron-sulfur cluster. The sequence of A. vinelandii CA6 FeSI protein are similar to the sequences of [2Fe-2S] ferredoxins from nitrogen-fixing Clostridium pasteurianum and Aquifex aeolicus, which is not a nitrogen fixer, including conserved cysteine residues. These suggest that FeSI may or may not be involved in nitrogen fixation as there is no evidence although the FeSI gene is present in the major nif gene cluster in Azotobacter vinelandii CA6. This study will be beneficial for understanding the function of FeSI in nitrogen fixation and the relations with other [2Fe-2S] proteins.

Keywords

Iron-sulfur protein, Nitrogen fixation, Nitrogenase, Azotobacter vinelandii CA6

Supporting Institution

TUBITAK

Project Number

118C225

Azotobacter vinelandii CA6 FeSI Proteininin Klonlanması, İfadesi ve Karakterizasyonu

Abstract

Bu çalışmada, Azotobacter vinelandii CA6'dan Shethna protein I veya FeSI proteini olarak da bilinen [2Fe-2S] proteini izole edilmiş, E. coli'de aşırı ifade edilmiş ve saflaştırılmıştır. SDS-PAGE analizi, saflaştırmanın her aşamasında proteinin monomer boyutu olan ~11 kDa'da bir bant göstermiştir. FeSI jel filtrasyon profili, doğal halde dimer olduğunu işaret etmektedir. UV-görünür spektrumunda, demir-kükürt kümesine özgü 344, 418 ve 464 nm dalga boylarında absorbanslar ölçülmüştür. A. vinelandii CA6 FeSI proteininin dizisi, korunmuş sistein rezidüleri dahil olmak üzere, nitrojen sabitleyici Clostridium pasteurianum ve bir nitrojen sabitleyici olmayan Aquifex aeolicus'tan elde edilen [2Fe-2S] ferredoksinlerin dizilerine benzemektedir. Bu bulgular, FeSI geninin Azotobacter vinelandii CA6'daki majör nif gen kümesinde bulunmasına rağmen hiçbir kanıt olmadığı için FeSI'in nitrojen fiksasyonuna dahil olabileceğini veya olmayabileceğini düşündürmektedir. Bu çalışma, FeSI'in nitrojen fiksasyonundaki işlevini ve diğer [2Fe-2S] proteinleri ile olan ilişkilerini anlamak için faydalı olacaktır.

Keywords

Demir-kükürt proteini, Azot fiksasyonu, Nitrojenaz, Azotobacter vinelandii CA6

Project Number

118C225

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