We observed the presence of contaminating NADH oxidation activity in maltose binding protein (MBP) fusion proteins expressed in Escherichia coli and purified using conventional amylose resin-based affinity chromatography. This contaminating NADH oxidation activity was detectable with at least four different enzymes from Cryptosporidium parvum expressed as MBP-fusion proteins (i.e., an enoyl-reductase domain from a type I fatty acid synthase, a fatty acyl-CoA binding protein, the acyl-ligase domain from a polyketide synthase, and a putative thioesterase), regardless of their NADH dependence. However, contaminating NADH oxidation activity was not present when fusion proteins were engineered to contain a His-tag and were purified using a Ni-NTA resin-based protocol. Alternatively, for proteins containing only an MBP-tag, the contaminating activity could be eliminated through the addition of 0.1% Triton X-100 and 2% glycerol to the column buffer during homogenization of bacteria and first column wash, followed by an additional wash and elution with regular column and elution buffers. Removal of the artifactual activity is very valuable in the study of enzymes using NADH as a cofactor, particularly when the native activity is low or the recombinant proteins are inactive.
Modern biological and biomedical research, as well as the biotechnology industry, relies heavily on the production of bioactive proteins. A large number of fusion systems are available for expressing recombinant proteins in prokaryotes or eukaryotes, including Escherichia coli, Saccharomyces cerevisiae, and baculovirus. Vectors based on T7 RNA polymerase (e.g., pET plasmids) and tac promoter (e.g., pMAL plasmids) are among the most popular expression systems used in E. coli(1). The majority of expression systems use certain tags to facilitate the expression and purification of recombinant proteins, including the His-tag, glutathione S-transferase (GST), maltose binding protein (MBP), N-utilization substance A (NusA), and thioredoxin (Trx). Among these, MBP fusion is a powerful system for expressing a large quantity of protein. It is able to enhance the solubility and proper folding of its fusion partners (2, 3). The purification of MBP-fused proteins is achieved by a relatively easy and simple amylose resin-based chromatography.
Our laboratory has been using various systems including the MBP fusion system and derivatives to express a large number of Cryptosporidium parvum proteins for functional analysis (4-11). MBP fusion generally performs well, particularly in expressing very large proteins that are otherwise very difficult to express using many other fusion systems. C. parvum, a protozoan pathogen infecting both humans and animals, possesses a multifunctional type I fatty acid synthase and polyketide synthase (CpFAS1 and CpPKS1) (10, 11). We have previously expressed these two megasynthases as MBP-fused protein with molecular weights up to 250 kDa. The expressed proteins contain multiple functional domains that are biochemically active as MBP-fusion proteins (5, 8).
More recently, however, in our attempts to express individual enoyl reductase (ENR) domains from CpFAS1 and CpPKS1, we have observed that MBP-fused proteins expressed in E. coli and purified using conventional amylose resin-based affinity chromatography contained NADH oxidation activity that did not originate from the fusion proteins of interest. Further experiments confirmed that the NADH oxidation activity was in fact an artifact produced from a contaminating but undefined bacterial enzyme(s). More importantly, this unwanted artifactual activity was present in most (if not all) MBP-fusion proteins purified by the conventional amylose resin-based protocol investigated so far in our laboratory, which had caused some confusion in our study of enzymes that used NADH as a cofactor and when the recombinant MBP-fusion proteins possessed very low or no enzyme activity. To overcome the problem, we have developed alternative protocols to eliminate this NADH oxidation activity.Materials and methods Cloning and expression of MBP-fusion proteins
Several C. parvum genes including CpFAS-ENR1, CpACBP1, CpPKS-AL1, and CpTE1 were cloned into the pMAL-c2x or its derivative vector as described below. CpFAS1-ENR1 is one of the 21 functional domains within a unique type I fatty acid synthase (CpFAS1) elongation module-1 (8, 11). All CpFAS1 modules containing multiple enzymatic domains have been previously cloned and expressed, in which all enzyme activities of individual domains in the recombinant proteins were functionally active (8). In this study, our original intent was to express the CpFAS-ENR1 domain to study whether this single domain was functional when expressed alone rather than expressed as part of a multidomain fusion protein. A gene fragment encoding the CpFAS-ENR1 domain was amplified from a large synthetic gene containing the entire module 1 with codons optimized for expression in E. coli using primers CpCFAS-ENR1-F1 (5'-agagaattcGGCAGTATCAGCAATCTGAGCCTG-3') (lower case represent artificially added EcoRI linker) and CpCFAS-ENR1-R1 (5'-caggtcgacCTCCTCAGGAATGCTATTGTCGCC-3') (lower case represent artificially added SalI linker). PCR amplicons were digested with EcoRI and SalI, and the released CpFAS-ENR1 insert was purified from a 1% agarose gel, ligated into the pMAL-c2x vector (New England Biolabs, Ipswich, MA, USA), and transformed into One Shot TOP10 competent E. coli cells (Invitrogen, Carlsbad, CA, USA). Plasmid DNA was isolated from positive clones and sequenced to confirm its identity. Additionally, CpFAS-ENR1 was also similarly cloned into a pMAL-c2E-derived vector (pMAL-c2E-TEV-His) between the EcoRI and SalI sites, which produces an MBP-fused protein containing a TEV cleavage site between MBP and fused partner and a His-tag fused at the C terminus of recombinant protein.