Maris Hartmanis

Fusion proteins in biotechnology

Gene fusion techniques allow the production of recombinant proteins featuring the combined characteristics of the parental products. Originally, these techniques were used to probe transcriptional and translational activity, to translocate proteins across cell membranes, and to facilitate the recovery of proteins. Recently, new applications have emerged in areas such as protein refolding, immunology, drug targeting and protein display. A slightly modified version of this review is also published in Current Opinion in Structural Biology 1992, 2:569-575.

Uptake and activation of acetate and butyrate in Clostridium acetobutylicum

SummaryThe pathway for uptake of acids during the solvent formation phase of an acetone-butanol fermentation by Clostridium acetobutylicum ATCC 824 was studied. 13C NMR investigations on actively metabolizing cells showed that butyrate can be taken up from the medium and quantitatively converted to butanol without accumulation of intermediates. The activities of acetate phosphotransacetylase, acetate kinase and phosphate butyryltransferase rapidly decreased to very low levels when the organism began to form solvents. This indicates that the uptake of acids does not occur via a reversal of these acid forming enzymes. No short-chain acyl-CoA synthetase activity or butyryl phosphate reducing activity could be detected. Based on our results and a critical analysis of literature data on acetone-butanol fermentations, it is suggested that an acetoacetyl-CoA: acetate (butyrate) CoA-transferase is solely responsible for uptake and activation of acetate and butyrate in C. acetobutylicum. The transferase exhibits a broad carboxylic acid specificity. The key enzyme in the uptake is acetoacetate decarboxylase, which is induced late in the fermentation and pulls the transferase reaction towards formation of acetoacetate. The major implication is that it is not feasible to obtain a batch-wise butanol fermentation without acetone formation and retention of a good yield of butanol.

Fusion proteins in biotechnology and structural biology

Abstract Gene fusion techniques allow the production of recombinant proteins featuring the combined characteristics of the parental products. Originally, these techniques were used to probe transcriptional and translational activity, to translocate proteins across cell membranes, and to facilitate the recovery of proteins. Recently, new applications have emerged in areas such as protein refolding, immunology, drug targeting and protein display. A slightly modified version of this review is also published in Current Opinion in Biotechnology 1992, 3 :363–369.

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.

Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly inEscherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzmes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.

Stability of solvent formation in Clostridium acetobutylicum during repeated subculturing

SummaryClostridium acetobutylicum ATCC 824 was submitted to repeated subculturing at 24-hour intervals for 218 days. The organism retained its ability to form solvents, although the fermentation slowly became increasingly acidogenic during the first 200 days. Except for the initial spore inoculum, the cultures were not subjected to heat shocking between the serial transfers. When the inoculum volume was doubled from 3.3% to 6.7% after 200 days of subculturing, the product formation pattern quickly shifted back from acids to primarily butanol. Acetone production also resumed after being undetectable for more than 50 days. The relative formation of acetate and ethanol remained nearly constant throughout the experiments, while the formation of butyrate mirrored that of butanol.

Refolding of human recombinant insulin-like growth factor II (IGF-II) in vitro, using a solubilizing affinity handle

Publisher Summary Insulin-like growth factor II (IGF-II) consists of 67 amino acid residues in a single chain. In analogy with IGF-I, there are 6 cysteine residues forming 3 disulfide bonds. The recombinant IGF-II was initially produced as a secreted fusion protein, ZZ-IGF-II, in E. coli. However, most full-length fusion protein was found in multimeric aggregates held together with disulfide bonds. A redox system, using the fusion partner as a solubilizer, was developed to transform multimeric, misfolded variants into the monomeric, correctly folded molecule. This chapter presents a comparison between IGF-II refolding as a fusion protein and as a native molecule. A hydrophilic fusion partner may act as a solubilizer of insulin-like growth factor II during refolding. The fusion partner apparently does not interfere with the refolding. Instead it seems to keep the more hydrophobic IGF-II moiety soluble, making denaturating agents or organic solvents unnecessary in the refolding buffers. The fusion partner can then subsequently be removed using site specific cleavage. This concept can be used to refold proteins that normally precipitate during refolding.

Selenomethionine-Containing Thiolase and 3-Hydroxybutyryl-CoA Dehydrogenase from Clostridium kluyveri

Publisher Summary The chapter focuses on the role Clostridium kluyveri has played in the understanding of fatty acid oxidation and synthesis. C. kluyveri is uniquely suited for the study of selenium metabolism as the organism is readily cultured in a mineral salts medium developed by Bornstein and Barker in 1948. All the selenium enzymes are involved in oxidation reduction reactions, and the selenium moiety of several of these enzymes has been identified as selenocysteine. Initially, the selenoprotein was purified to near homogeneity by a multistep procedure, and the selenium was shown to be covalently bound to the protein. Characterization of the purified enzyme revealed that it was a basic protein with an isoelectric point of 7.7. This observation led to the development of a single-step isolation procedure. Highly purified thiolase could be obtained by gradient elution from CM-Sephadex. This enzyme was later identified as NADP + - dependent 3-hydroxybutyryl-CoA dehydrogenase. Clostridium kluyveri can ferment either ethanol or acetate to butyrate and caproate, or crotonate to butyrate and acetate. Depending on the type of fermentation, thiolase has different physiological roles. The mechanisms by which selenium is utilized by living organisms remain largely unknown. Several proteins with a specific selenium requirement contain the selenium moiety as selenocysteine. The selenocysteine residues in two of these proteins—mammalian glutathione peroxidase and clostridial glycine reduciase protein A—have been localized at a specific site within the primary structure. Significant amounts of selenium can also exist in proteins as selenomethionine. This nonspecific incorporation of selenium occurs throughout the primary structure of the protein rather than at an active, catalytic center.