Stein M, Jacquot J P, Jeannette E, Decottignies P, Hodges M, Lancelin J M, Mittard V, Schmitter J M, Miginiac-Maslow M
Laboratoire de Physiologie Végétale Moléculaire, URA 1128 CNRS, Université de Paris-Sud, Orsay, France.
Plant Mol Biol. 1995 Jun;28(3):487-503. doi: 10.1007/BF00020396.
Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15,101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11,817 Da and no signal sequence. The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.
基于已知的氨基酸序列，通过聚合酶链式反应（PCR）生成了探针，并用于随后分离莱茵衣藻两种硫氧还蛋白（m和h）的编码cDNA和基因。硫氧还蛋白m是一种叶绿体蛋白，编码为140个氨基酸（15,101道尔顿）的前体蛋白，含有34个氨基酸的转运肽，其中丙氨酸（Ala）和精氨酸（Arg）残基含量非常高。硫氧还蛋白h的序列编码一个113个氨基酸的蛋白质，分子量为11,817道尔顿，且没有信号序列。硫氧还蛋白m基因包含一个内含子，其结构似乎比硫氧还蛋白h基因更古老，硫氧还蛋白h基因被分成4个外显子。编码莱茵衣藻硫氧还蛋白m和h的cDNA序列已整合到pET-3d表达载体中，该载体可在大肠杆菌细胞中高效生产蛋白质。获得了重组硫氧还蛋白的高表达水平（高达50毫克/升培养物）。这使我们能够研究这两种重组蛋白的生化/生物物理特性。有趣的是，虽然发现m型硫氧还蛋白的特性与原核硫氧还蛋白非常接近，但h型硫氧还蛋白在动力学行为方面，尤其是在热变性特性方面却有很大不同。
