| Thymosinα1 (Tα1), with its ability to act as an immunomodulator, is a safe and effective treatment for chronic hepatitis B when used alone or in combination with interferon. The synthetic version of Tα1 is now launched in about forty countries by SciClone company for the treatment of HBV under the trade name Zadaxin. Primary research indicates that Zadaxin may be useful in treating a number of other diseases as well, including hepatitis C, non-small cell lung cancer, melanoma, and HIV/AIDS. In addition, Zadaxin is also indicated as a vaccine adjuvant, to enhance the effectiveness of influenza and hepatitis B vaccines. In order to reduce the production cost, this study will establish a biosythetic technology for preparation of Tα1. Tα1 is a 28-amino acid peptide with an N-terminal acetylation. Its primary structure is: Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN. There are two main obstacles for preparation of Tα1, one is the N-terminal acetylation, the other is the expression of small peptides.N-terminal acetylation of proteins is catalyzed by N-terminal acetyltransferases (NATs), which transfer acetyl groups (CH3CO-, MW43.018 Da) from acetyl-coenzyme A (AcCoA) to theα-amino group of the N-terminal residues. There are at least two requirements for the N-terminal acetylation of proteins. First, a permissible N-terminal sequence appears necessary. This is why some proteins are acetylated and others are not. Second, an appropriate NAT activity must be present in the host cell expression system.In order to express Tα1 by recombinant E. coli, we constructed fusion proteins of Tα1 with three known endogenous N-terminally-acetylated ribosomal proteins L12, S5 and S18 of E. coli, respectively, named as A2 (Tα1-Cys-L12), A5 (Tα1-Cys-S5), and A8 (Tα1-Cys-S18). Tα1 is located at the N terminus of the fusion proteins, and a Cys residue is following it. As there is only one Cys residue in the fusion proteins, so Tα1 will be released from them after chemical cleavage with CDAP ( 1-Cyano-4- dimethyl- aminopyridinium tetrafluoroborate ). The fusion genes were cloned into plasmid pET22b and three expressing vectors, namely pET-A2, pET-A5 and pET-A8 were obtained. A His6-tag was fused to the C terminus of the fusion proteins for easy purifiction. After expression and purification, Q-TOF mass spectrometry showed that the fusion proteins A2 and A8 were partly acetylated ( A5 was not analyzed for its poor purity). After digestion with trypsin, the N-terminal 1-14 peptide fragment was subjected to sequence by tandem mass spectrometry. The acetylation was demonstrated to be located at the N-terminal residue serine. That is to say that Tα1-fused protein can be N-terminally acetylated when expressed in Escherichia coli. However, Tα1-fused protein was only partly acetylated when expressed in Escherichia coli. There were some of recombinant fusion proteins are in the unacetylated form. The fullest extent of acetylation of recombinant Tα1-fused protein was necessary condition for the preparation of Tα1 by genetic engineering. As N-terminal acetylation is a process catalyzed by NATs, so if we know which NAT is responsible for N-terminal acetylation of Tα1-fused proteins, then the fully acetylated recombinant fusion proteins could be obtained by co-expressing with this NAT.In search of this NAT, we decided to delete each of the known NATs'genes or putative NATs'genes, respectively, by Red recombination technology. We succeeded in deletion of rimI, rimJ, rimL and yjaB, but not yhhY, yjhQ and yjgM. Another Tα1-Gly-L12 fusion protein, named as A22, was constructed with only one Asn-Gly peptide bond, for the purpose of specific cleavage with an asparaginyl endopeptidase or hydroxylamine. When expressed in the rimJ::kan mutant, only the unacetylated form of recombinant A22 fusion protein was found, but in the original strain JM109(DE3) and its other three mutants, both acetylated and unacetylated forms of it were found. The results stated that rimJ gene is associated with the acetylation of recombinat A22 fusion protein.The relationship of them was further investigated by gene rescue. The rimJ gene was cloned into plasmid pACYCDeut-1 to get pACYC-rimJ for expresing RimJ under control of T7 promoter. The fully acetylated recombinant Tα1-fused protein was obtained by co-expressing with rimJ in the rimJ::kan mutant. Furthermore, to demonstrate that RimJ itself is an enzyme active on Tα1-fused protein, we purified the unacetylated A22 and RimJ bearing a His-tag, respectively, and showed that RimJ can acetylate the unacetylated form of A22 using acetyl-coenzyme A as acetyl donor in vitro, while A22 could not acetylate itself with acetyl-coenzyme A alone. Unlike eukaryotic NATs that consist of two or more dissimilar subunits, prokaryotic NATs appear to have identical subunits without any auxiliary subunit.Altogether, our results argue that N-terminal acetylation of recombinant Tα1-fused protein in E. coli is catalyzed by RimJ, and that fully acetylated Tα1 can be obtained by co-expressing with RimJ. This is the first description that an ectopic protein acetylation in bacterial expression systems is catalyzed by RimJ, a known prokaryotic NAT.Tα1 is hard to be directly expressed in the cytoplasm of Escherichia coli. The fusion protein shoud be cleavaged to release Tα1. According to the strutural characteristics of Tα1, for releasing it there are three choices: the first is the specific chemical cleavage of X-Cys peptide bond with CDAP, the second is the cleavage of Asn-Gly using an asparaginyl endopeptidase, and the third is of intein-mediated N-terminal cleavage. Since the cleaving efficiency of CDAP is low and asparaginyl endopeptidases are not available, this study exploring the intein-mediated N-termianl cleavage method to acquire Tα1. Tα1-fused proteins with three inteins, viz. SplDnaX Intein ( 136 amino acids, from Spirulina platensis ), PhoPol II Intein ( 166 amino acids, from Pyrococcus horikoshii ), and MxeGyrA Intein ( 198 amino acids, from Mycobacterium xenopi ), were constructed, and named as fusion proteins AS, AP and AM, respectively. And also each one had a His6-tag on the C-terminus for easy purification. The three expressing vectors were named as pET-AS, pET-AP, and pET-AM, respectively. After co-transformed with pACYC-rimJ into BL21(DE3) strain, the three recombinant strains were cultured in an autoinducible medium FML at 37°C, 250 rpm for 12 h. The cell pellets were collected and disrupted by sonication. The supernatant loaded onto a Chelating Sepharose Fast Flow column for purification. The purified intein fusion proteins AS, AP and AM were incubated at 4°C, 30°C, 37°C, 42°C and 55°C for 12 h, and analyzed by 17% SDS-PAGE. AS and AP were able to hydrolyze at above 37°C, but AM was not for its easy denaturation. At 55°C, the cleavage efficiency of AS and AP was more than 50%. However, the intein fusion protein AM was more easy to be thiolyzed than AS and AP, especially at low temperature. As for its small molecular mass, the intein fusion protein AS was chosen for preparation Tα1 by intein-mediated cleavage. After incubated with 100 mmol/L DTT at 42°C for 24 h, above 90% of AS was thiolyzed, and 56 mg Tα1 with purity of 73% was acquied through anion exchange chromatography from 1-liter culture media. The whole sequence of recombinant Tα1 was indentified by tandem mass spectrometry and its N-terminal Ser was shown to be acetylated. Therefore, this method has application potential and deserves to study further.In conclusion, the results showed :1) Tα1-fused protein can be N-terminally acetylated when expressed in Escherichia coli, and this modification is catalyzed by N-terminal acetyltransferase RimJ.2) When co-expressed with RimJ, the Tα1-fused protein can be fully acetylated in Escherichia coli.3) Preparation of Tα1 by intein-mediated N-terminal cleavage is of high potential.
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